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https://openalex.org/W3133741510	https://pubs.rsc.org/en/content/articlepdf/2021/sm/d0sm02261f	English	null	Keratins determine network stress responsiveness in reconstituted actin–keratin filament systems	Soft matter	2,021	cc-by	8,774	a Peter-Debye Institute for Soft Matter Physics, Leipzig University, 04103 Leipzig, Germany b Faculty of Science, Cairo University, 12613, Giza, Egypt c Fraunhofer Institute for Cell Therapy and Immunology, 04103 Leipzig, Germany d Molecular Genetics, German Cancer Research Centre, 69120 Heidelberg, Germany e Department of Neuropathology, University Hospital Erlangen, 91054, Erlangen, Germany f Unconventional Computing Lab, Department of Computer Science and Creative Technologies, UWE, Bristol BS16 1QY, UK † Electronic supplementary information (ESI) available. See DOI: 10.1039/ d0sm02261f ‡ These authors contributed equally to this work. Keratins determine network stress responsiveness in reconstituted actin–keratin filament systems† Cite this: Soft Matter, 2021, 17, 3954 The cytoskeleton is a major determinant of cell mechanics, and alterations in the central mechanical aspects of cells are observed during many pathological situations. Therefore, it is essential to investigate the interplay between the main filament systems of the cytoskeleton in the form of composite networks. Here, we investigate the role of keratin intermediate filaments (IFs) in network strength by studying in vitro reconstituted actin and keratin 8/18 composite filament networks via bulk shear rheology. We co-polymerized these structural proteins in varying ratios and recorded how their relative content aﬀects the overall mechanical response of the various composites. For relatively small deformations, we found that all composites exhibited an intermediate linear viscoelastic behaviour compared to that of the pure networks. In stark contrast, when larger deformations were imposed the composites displayed increasing strain stiﬀening behaviour with increasing keratin content. The extent of strain stiﬀening is much more pronounced than in corresponding experiments performed with vimentin IF as a composite network partner for actin. Our results provide new insights into the mechanical interplay between actin and keratin filaments in which keratin provides reinforcement to actin. This interplay may contribute to the overall integrity of cells. Hence, the high keratin 8/18 content of mechanically stressed simple epithelial cell layers, as found in the lung and the intestine, provides an explanation for their exceptional stability. Received 24th December 2020, Accepted 4th March 2021 DOI: 10.1039/d0sm02261f rsc.li/soft-matter-journal This journal is © The Royal Society of Chemistry 2021 Soft Matter View Article Online View Journal \| View Issue Cite this: Soft Matter, 2021, 17, 3954 Received 24th December 2020, Accepted 4th March 2021 DOI: 10.1039/d0sm02261f rsc.li/soft-matter-journal Article. Published on 05 March 2021. Downloaded on 10/24/2024 6:47:53 AM. his article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 05 March 2021. Downloaded on 10/24/2024 6:47:53 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Introduction that these very same cytoskeletal components can lead to very static, stable cell conformations, which allow, for instance, the formation of stable tissue layers such as the epithelium, or highly dynamic cells during wound healing and cancer metastasis.3 Switching between these physically seemingly contradictory cell states highly depends on the ratios of the individual cytoskeletal components. This becomes especially apparent during embryo- genesis when cells need to constantly switch between stable epithelial states and motile mesenchymal states to form complex tissue structures.4 During these switching events, which are the so-called epithelial to mesenchymal transition (EMT) along with the reverse process called mesenchymal to epithelial transition (MET), the cytoskeletal components are very diﬀerently expressed. These diﬀerent compositions lead to diﬀerent mechanical and motile behaviours. Besides actin-related structures, crucial elements are keratin IFs, which, with their 37 cytoplasmic members, constitute the largest group of the IF protein-family.5,6 They are typically expressed in epithelial cells in various combinations and provide crucial cell type-specific structural support upon mechanical stresses. These diverse mechanical properties are realized by expressing various specific keratin pairs as keratins obligate heterodimeric complexes representing parallel coiled coils made from two alpha-helical molecules of two sequence- related classes each.7 They are also involved in other The complex mechanical behaviour of eukaryotic cells is largely determined by the three principal filament systems of the cytoskeleton, i.e. actin filaments (F-actin), microtubules, and intermediate filaments (IFs) as well as their regulated interplay.1 In addition to establishing a cell’s specific functional and tissue-related shape, these components and their distinct structures are also crucial for numerous general cellular processes such as stability within tissues as well as cell motility, division, and signal transduction.2 Alterations of these key constituents are the cause of numerous human diseases. It is somehow perplexing ‡ These authors contributed equally to this work. This journal is © The Royal Society of Chemistry 2021 3954 \| Soft Matter, 2021, 17, 3954–3962 Soft Matter View Article Online View Article Online Paper Soft Matter non-mechanical functions, including regulation of cell growth, migration, and protection from apoptosis.8,9 Several studies have highlighted the potential inhibitory role of keratins for cell mobility and thus the need to downregulate them for EMT to increase cellular mobility. Polymer lengths The mass per unit length (mL) for actin is 2.66 1011 g m1,23 and for K8–K18 in Tris-based assembly buffer is 3.16 1011 g m1.24 At 0.5 mg ml1 actin monomers (11.9 mM) and 0.6 mg ml1 K8–18 tetramers (11.34 mM), both actin and keratin filament networks will have similar total filament length. Using these concentrations as our boundary conditions, we can select actin–keratin mixtures with the same total polymer length per unit volume. Protein preparation and co-polymerization G-actin was prepared from rabbit muscle and stored at 80 1C in G-Buﬀer (2 mM Tris–HCl pH 7.5, 0.2 mM ATP, 0.1 mM CaCl2, 1 mM DTT, 0.01% NaN3, pH 7.8) as described previously.25 Vectors containing keratin genes (pET 24a–K8 and pET 23a– K18) were transformed into E. coli BL21 for protein expression. Recombinant K8 and K18 were isolated and purified as previously described.26 For reconstitution, purified K8 and K18 proteins were mixed in equimolar amounts and renatured by stepwise dialysis against 8 M urea, 2 mM Tris–HCl, 1 mM DTT, pH 9.0 with stepwise reduction of urea concentration (6 M, 4 M, 2 M, 1 M, and 0 M). Each dialysis step was done for 20 minutes at room temperature, then the dialysis was continued overnight against 2 mM Tris–HCl, 1 mM DTT, pH 9.0 at 4 1C. The final protein concentration was determined by measuring the absorption at 280 nm using a DU 530 UV/vis Spectrophotometer (Beckman Coulter Inc., USA). Composite networks were prepared by mixing actin monomers at 0.5 mg ml1 and K8–K18 tetramers at 0.6 mg ml1 in varying mixing ratios. Assembly of pure and mixed networks was initiated by adding 1/10 volume of 10 F-buffer (20 mM Tris–HCl, 1 M KCl, 10 mM MgCl2, 2 mM ATP, 10 mM DTT, pH 7.5) to the protein sample. When designing and interpreting such in vitro studies, it is important to choose the system parameters carefully as observed when measuring the mechanical properties of composite filament networks of actin and vimentin. Holding the molecular content constant for diﬀerent mixing ratios of actin and vimentin has yielded contradictory results for their mechanical response, namely a synergistic increase in the linear elasticity18 and formation of stiﬀer or softer networks depending on the investigation technique.19,20 In this case, however, vimentin filaments carry roughly twice as many monomers per unit length than actin filaments, i.e. total filament length is half compared to that of F-actin. Consequently, it has been shown that actin–vimentin filament composites with the same total polymer length and mesh size, can be described by a superposition of the mechanical properties of the underlying constituents.21 This journal is © The Royal Society of Chemistry 2021 Introduction For example, it was found that keratinocytes with all keratins deleted by gene-targeting are softer, more deformable, and more invasive compared to the small overall eﬀect generated after actin depolymerization.10 They are also able to migrate twice as fast as wild type cells.11 Conversely, keratin re-expression in these studies has the opposite eﬀect. This indicates a direct link between down- regulation of keratins during EMT and loss of stiﬀness with increased migration and invasion ability of tumor cells. Although F-actin networks dramatically reorganize as a prere- quisite for morphological and migratory changes during EMT and MET, it is indeed remarkable that the entire IF system is rebuilt by a switch between keratin and vimentin expressions.12 As demonstrated for numerous physiological situations, the cytoskeletal systems act synergistically.13 By coordinating their functions, they aﬀect each other’s mechanics. However, it is challenging to investigate this mechanical interplay in cells due to their inherent complexity, which makes it diﬃcult to disentangle mechanical crosstalk and regulatory biochemical interactions. In this respect, reconstituted composite cytoskeletal filament networks have been investigated in vitro by quantitative rheological measurements and theoretical modelling. The results have often revealed unexpected mechanical responses. For instance, the presence of microtubules was found to induce unexpected local compressibility14 and non-linear strain stiﬀening in actin networks.15,16 Concomitantly, actin reinforces microtubules against compressive loads.17 networks by bulk shear rheology measurements. We aim to characterize networks with similar total filament lengths. By systematically varying the relative mass ratios of actin and keratin, we investigate how their relative presence impacts the overall mechanical response of the composites. We complement these rheology measurements with confocal microscopy of the composites. Open Access Article. Published on 05 March 2021. Downloaded on 10/24/2024 6:47:53 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Shear rheology Rheology measurements were conducted using a strain- controlled ARES (TA Instruments, USA) and a MCR 502 WESP (Anton Paar, Austria) rheometer using 40 mm plate–plate geometry with 140 mm gap width at 20 1C. In all measurements, F-buffer was mixed with the protein mixture on ice, and a sample volume of 200 ml was loaded quickly between the two plates. F-Buffer with the same conditions as in the sample was distributed around the sample to prevent artefacts from inter- facial elasticity and a solvent trap was placed around the sample to prevent evaporation. The sample was given two hours to equilibrate between the rheometer plates as the filament Due to their intrinsic ability to self-assemble in vitro, the co-polymerization of actin and keratin filaments is possible without additional accessory proteins. Hence, it has been recently demonstrated that encapsulation of actin and keratin within vesicles leads to the formation of composite filament networks, where F-actin acts as steric resistance for keratin IFs preventing their collapse to bundled clusters.22 Here, we investigate the mechanical properties of in vitro reconstituted F-actin and keratin 8/18 (K8–K18) composite 3955 Soft Matter, 2021, 17, 3954–3962 \| 3955 View Article Online Soft Matter Paper Paper assembly was monitored with a dynamic time sweep for 2 hours, one data point per minute at a frequency (o) of 1 Hz and a strain (g) of 2%. The linear viscoelastic response of equilibrated networks was measured with dynamic frequency sweeps ranging from 0.01 Hz to 80 Hz at a strain of 2% and 20 points per decade. Data points plotted in Fig. 2 are the mean of at least five independent rheology measurements. A transient step rate test with a strain rate of 0.1 s1 was applied to measure the strain-dependent stress in the non-linear strain regime. The differential shear modulus (K) was determined with a self-written Python script, calculating the gradient of the smoothed stress data divided by the strain step width. The linear differential shear modulus Klin is given by the first non-negative value of the smoothed stress data, whereas negative stress is measured due to technical limitations, particularly for small strains, lacking every physical relevance. The linear frequency sweeps as well as the nonlinear step rate measurements were evaluated in the frame of the GWLC model with a self-written Python script. Shear rheology At least six independent rheology measurements were taken into account for the evaluation and comparison with the model predictions. Details about the model are presented in the ESI.† were created by adding 1/10 volume fraction of 10 F-buffer to the sample and immediately pipetting it into an experimental sample chamber to rest for 2 hours at room temperature before imaging as described in ref. 28. Images were captured using a spinning disc confocal microscope (inverted Axio Observer.Z1/ Yokogawa CSU-X1A 5000, Carl Zeiss Microscopy GmbH, Germany), 100 oil immersion objective NA 1.40 with a Hamamatsu camera at an exposure time of 100 ms. Open Access Article. Published on 05 March 2021. Downloaded on 10/24/2024 6:47:53 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Co-polymerization and filament length In order to investigate the mechanical properties of composite filament networks of F-actin and K8–K18, we chose the initial concentrations of actin monomers and keratin tetramers that resulted in comparable total filament lengths of actin and keratin as depicted in Fig. 1. p g We reconstituted composite networks of actin and keratin by co-polymerization of actin monomers and tetrameric complexes of K8 and K18. It is well known that the ionic requirements for polymerization of actin and keratin are quite diﬀerent. Keratin can assemble in vitro into networks in an unusually low ionic strength buﬀer24 and addition of ions such as magnesium (Mg2+) or potassium (K+) at physiological con- centrations is enough to induce immediate bundling, resulting in heterogeneous networks.29–31 In contrast, for actin the addition of mono- and divalent ions to physiological concentrations is essential to initiate the polymerization from globular actin (G-actin) to filamentous actin (F-actin) to form entangled networks.32 A striking difference between actin and keratin filaments is the filament persistence length, which is the length scale for the decay of the tangent–tangent correlation along the filament contour, and is proportional to the stiffness of the polymer.33 Keratins have a significantly smaller persistence length (Lp) than actin, with Lp E 0.5 mm and 10 mm, respectively.34 Here, we assessed the polymerization efficiency of keratin and actin in Protein labelling, sample preparation and imaging We have developed a new method for direct labelling of the wild type K8–K18 without mutation, unlike the techniques used in previous studies.22,27 The labelling is based on the coupling of Atto-488-NHS-ester (ATTO-TEC, Siegen, Germany) to the free amine (NH2) residues (lysine or terminus) in K8 tetramers. K8 was renatured by stepwise dialysis against the labelling buffer (2 mM sodium phosphate, pH 8.5) as described above. K8 tetramers were mixed with Atto-488-NHS-ester solubilized in DMSO at 10 mM at a molar ratio of 5 : 1. The mixture was incubated in the dark for 1 hour at room temperature. In one step, the free dye was removed, and labelled K8 was denatured into monomers by overnight dialysis against 8 M urea, 2 mM sodium phosphate buffer pH 8.5 at 4 1C. On the second day, the labelling buffer was exchanged by the dialysis buffer (8 M urea, 2 mM Tris–HCl, pH 9.0) by dialysis for 2 hours at room temperature. The concentration of labelled K8 was determined by measuring the absorbance at 280 and 500 nm using a DU 530 UV/vis Spectrophotometer (Beckman Coulter Inc., USA). Labelled K8 was then distributed to appropriate aliquots and stored at 80 1C. One day before imaging, equal amounts of 20% labelled K8 were mixed with unlabelled K8 and K18 in 8 M urea, 2 mM Tris–HCl, pH 9.0 to have a final sample labelling of 10% by concentration. This mixture was renatured into tetramers by stepwise dialysis against 2 mM Tris–HCl, pH 9.0. k i li i l b ll d d i h For network visualization, labelled K8–K18 prepared in the previous step was mixed with unlabelled K8–K18 tetramers in a ratio of 1 : 10, with a final protein concentration of 0.6 mg ml1. Fluorescently labelled actin was prepared by mixing G-actin at 5 mM with phalloidin–tetramethylrhodamine B isothio-cyanate (phalloidin–TRITC – Sigma-Aldrich Co.) in a molar ratio of 1 : 1. For composite networks, 10% labelled keratin samples were mixed with labelled actin samples in varying ratios. Networks Fig. 1 Illustration of F-actin–keratin composite filament networks with varying mixing ratios. By co-polymerizing actin monomers at 0.5 mg ml1 and keratin tetramers at 0.6 mg ml1, an intermixed network of actin (red) and keratin (green) filaments is formed. At the noted concentrations, networks with the total polymer lengths of the composite will be approximately the same. This journal is © The Royal Society of Chemistry 2021 Composite filament networks exhibit an intermediate linear viscoelastic behaviour To date, previous in vitro studies have investigated the mechanical properties of only one-component systems of either actin filaments as entangled38–40 and crosslinked28,41–44 networks or keratin single filaments27 and networks.24,29,35,36,45 Fig. 2 Linear viscoelasticity of reconstituted filemant networks. By comparison, the keratin network (A) shows more predominant elasticity with a weaker frequency dependence of both elastic G0 (filled symbols) and viscous G00 (open symbols) moduli than the F-actin network (E); the keratin network is more elastic (tan fker = 0.13) than the F-actin network (tan fact= 0.52) with no observed G0/G00 crossover, which, in contrast, is a signature of actin networks in this frequency range. Actin–keratin composite filament networks with varying mixing ratios of actin and keratin (B–D) show intermediate viscoelastic properties. With increasing actin/decreasing keratin content, the frequency dependence of G0 and G00 moduli increases gradually, and the networks become less elastic as the loss factor values increase. Consequently, the G0/G00 crossover appears at high frequency in keratin- dominated networks and shifts gradually to lower frequencies with increasing actin content. a represents the slope between 0.1 and 10 Hz of G0. Data points in all curves represent the mean of at least five independent measurements and error bars represent the standard deviation from the mean. Fig. 2 Linear viscoelasticity of reconstituted filemant networks. By comparison, the keratin network (A) shows more predominant elasticity with a weaker frequency dependence of both elastic G0 (filled symbols) and viscous G00 (open symbols) moduli than the F-actin network (E); the keratin network is more elastic (tan fker = 0.13) than the F-actin network (tan fact= 0.52) with no observed G0/G00 crossover, which, in contrast, is a signature of actin networks in this frequency range. Actin–keratin composite filament networks with varying mixing ratios of actin and keratin (B–D) show intermediate viscoelastic properties. With increasing actin/decreasing keratin content, the frequency dependence of G0 and G00 moduli increases gradually, and the networks become less elastic as the loss factor values increase. Consequently, the G0/G00 crossover appears at high frequency in keratin- dominated networks and shifts gradually to lower frequencies with increasing actin content. a represents the slope between 0.1 and 10 Hz of G0. Data points in all curves represent the mean of at least five independent measurements and error bars represent the standard deviation from the mean. Linear viscoelasticity of pure actin and keratin filament networks Using bulk shear rheology, the linear viscoelastic properties of polymer solutions can be quantified by the frequency (o) dependent complex shear modulus G*(o) = G0(o) + iG00(o), where G0 and G00 are the elastic and viscous moduli, respectively. Under the selected assembly conditions, pure K8–K18 filament networks at 0.6 mg ml1 exhibit predominantly elastic behaviour with G0 being consistently larger than G00 by nearly one order of magnitude (Fig. 2A). Accordingly, the loss factor tan(f) = G00/G0 has a small value (tan(f) at 1 Hz = 0.13), indicative of highly elastic networks. Over the entire frequency range, G0 and G00 show a very weak frequency dependence. A weak power law was obtained by a linear fit of G0 in the log–log-plot yielding a power- law exponent of a(G0) = 0.07. We observed no G00/G0 crossover in the measurable frequency range for pure keratin. This is consistent with results previously reported for K8–K18 network bulk rheological behaviour.24,35,36 By comparison, G0 and G00 of pure F-actin filament networks showed a more pronounced frequency dependence with a(G0) = 0.23, (Fig. 2E). Accordingly, the crossover between G0 and G00 regimes was observed at low frequency. Actin networks have a larger loss factor (tan(f) at 1 Hz = 0.52) and, consequently, the elastic contributions are less dominant than in pure keratin networks. These mechanical features are similar to those obtained for entangled actin networks under comparable conditions.21,37 Open Access Article. Published on 05 March 2021. Downloaded on 10/24/2024 6:47:53 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 05 March 2021. Downloaded on 10/24/2024 6:47:53 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Protein labelling, sample preparation and imaging The ability of actin to pervade the keratin networks, depending on its content, impacts the architecture and mechanics of the composites. As depicted, actin and keratin differ in their persistence length with Lp E 10 mm and 0.5 mm respectively. Fig. 1 Illustration of F-actin–keratin composite filament networks with varying mixing ratios. By co-polymerizing actin monomers at 0.5 mg ml1 and keratin tetramers at 0.6 mg ml1, an intermixed network of actin (red) and keratin (green) filaments is formed. At the noted concentrations, networks with the total polymer lengths of the composite will be approximately the same. The ability of actin to pervade the keratin networks, depending on its content, impacts the architecture and mechanics of the composites. As depicted, actin and keratin differ in their persistence length with Lp E 10 mm and 0.5 mm respectively. 956 \| Soft Matter, 2021, 17, 3954–3962 This journal is © The Royal Society of Chemistry 2021 This journal is © The Royal Society of Chemistry 2021 3956 View Article Online Paper Paper Soft Matter i l ti it f tit t d fil t t k B near viscoelasticity of reconstituted filemant networks. By com in network (A) shows more predominant elasticity with a y dependence of both elastic G0 (filled symbols) and visc different concentrations of MgCl2 (1, 0.5, 0.1 and 0 mM) by high-speed sedimentation of protein assemblies followed by SDS-PAGE of pellet and supernatant fractions (data not shown). We found that a lack of Mg2+ ions had no effect on the polymer- ization efficiency of keratin, and soluble keratin tetramers were polymerized completely into filaments, whereas for actin at least 1 mM MgCl2 was required to ensure complete polymerization. Thus, we had to adjust the polymerization buffer (see Materials and methods) to include a concentration of 1 mM MgCl2 for the co-polymerization in order to form actin–keratin filament networks. Fig. 2 Linear viscoelasticity of reconstituted filemant networks. By comparison, the keratin network (A) shows more predominant elasticity with a weaker frequency dependence of both elastic G0 (filled symbols) and viscous G00 (open symbols) moduli than the F-actin network (E); the keratin network is more elastic (tan fker = 0.13) than the F-actin network (tan fact= 0.52) with no observed G0/G00 crossover, which, in contrast, is a signature of actin networks in this frequency range. This journal is © The Royal Society of Chemistry 2021 Soft Matter, 2021, 17, 3954–3962 \| 3957 Protein labelling, sample preparation and imaging Actin–keratin composite filament networks with varying mixing ratios of actin and keratin (B–D) show intermediate viscoelastic properties. With increasing actin/decreasing keratin content, the frequency dependence of G0 and G00 moduli increases gradually, and the networks become less elastic as the loss factor values increase. Consequently, the G0/G00 crossover appears at high frequency in keratin- dominated networks and shifts gradually to lower frequencies with increasing actin content. a represents the slope between 0.1 and 10 Hz of G0. Data points in all curves represent the mean of at least five independent measurements and error bars represent the standard deviation from the mean. Open Access Article. Published on 05 March 2021. Downloaded on 10/24/2024 6 This article is licensed under a Creative Commons Attribution 3.0 U where ln = L/n is the half wavelength of eigenmodes with mode number n and Nn = ln/L 1 the number of interactions per length ln. Mode relaxation times with ln 4 L, where L describes a characteristic interaction length, are stretched.46 For the interested reader, we would like to refer to the ESI† for a more detailed description of this model. Recently, we demonstrated that this e can be interpreted as a polymer-specific stickiness and we showed that isotropic networks of K8–K18 filaments assembled in low-salt buffer are much more sticky than F-actin networks assembled in F-buffer.37 We used the GWLC model to investigate the non-specific filament–filament interactions that are compiled in this parameter by fitting the expected values obtained from the model to our experimental data (Fig. 4). Using persistence lengths and contour lengths averaged according to the polymer composition, we the predominant viscous regime, observed only in F-actin networks, started to appear in keratin-dominated networks at high frequencies. With increasing actin content, actin contributes more to the composite’s behaviour, shifting the crossover point gradually to lower frequencies. The loss factor tan(f) increased with increasing actin/decreasing keratin content, which indicates a smooth transition to less elastic networks, as shown in Fig. 3A. We used a Mann–Whitney U test to investigate the statistical significance of the loss factor values with respect to each other. We found p-values less than 0.05, indicating significant difference, for all compared distributions except for the pure networks compared to composites with a respective division of 0.75/0.25 (Table S1, ESI†). In Fig. 3B, we show that the elastic moduli G0 for all networks exhibit only minor variations. Composite filament networks exhibit an intermediate linear viscoelastic behaviour In our study, composite networks of F-actin and K8–K18 filaments with varying mixing ratios revealed an intermediate linear viscoelastic behaviour with regard to their composite- specific protein content. With increasing actin/decreasing keratin contents as shown in Fig. 2B–D, the dependence of G0 and G00 on the frequency increased gradually as indicated by the gradual increase in a values; a = 0.09 in keratin-dominated networks (0.45 mg ml1 K8–K18–0.125 mg ml1 actin), 0.16 in equal ratio-networks (0.3 mg ml1 K8–K18–0.25 mg ml1 actin) and 0.21 in F-actin-dominated networks (0.15 mg ml1 K8–K18–0.375 mg ml1 actin). Furthermore, the crossover to This journal is © The Royal Society of Chemistry 2021 Soft Matter, 2021, 17, 3954–3962 \| 3957 Soft Matter Fig. 3 Mechanical properties of pure and composite filament networks in the linear regime. (A) Mean values of the loss factor tan(f) at f = 1 Hz of composite networks showing a gradual increase in the loss factor values, i.e. the network’s viscosity, with increasing actin/decreasing keratin content. We found the distributions to be significantly diﬀerent except for the constellations of pure actin compared to 0.15 mg ml1 keratin–0.375 mg ml1 actin and pure keratin compared to 0.45 mg ml1 keratin–0.125 mg ml1 actin respectively. (B) The plateau modulus G0 = G0 (f = 1 Hz) for all networks shows only minor variations between networks in the linear regime while in (C) the slopes a show a gradual increase with actin content where we found distributions differing significantly except for the constellation of pure actin compared to 0.15 mg ml1 keratin–0.375 mg ml1 actin (p-value = 0.5). In (A)–(C) dots represent single measurements and error bars represent the standard deviation from the mean. Open Access Article. Published on 05 March 2021. Downloaded on 10/24/2024 6:47:53 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 3 Mechanical properties of pure and composite filament networks in the linear regime. (A) Mean values of the loss factor tan(f) at f = 1 Hz of composite networks showing a gradual increase in the loss factor values, i.e. the network’s viscosity, with increasing actin/decreasing keratin content. We found the distributions to be significantly diﬀerent except for the constellations of pure actin compared to 0.15 mg ml1 keratin–0.375 mg ml1 actin and pure keratin compared to 0.45 mg ml1 keratin–0.125 mg ml1 actin respectively. Filament–filament interactions constitute a fundamental cause for mechanical responses Fig. 4 Attractive filament–filament interactions (captured in the stickiness parameter e) determine the mechanical response of composite filament networks in linear rheology. Normalized storage moduli (G0) versus fre- quency for different actin/keratin compositions evaluated with the glassy wormlike chain model. Solid lines represent the measured data and dashed lines represent the fitting curves, yielding values for stickiness e as shown. For pure keratin, the model could not be fitted to the data. Inter-filament interactions are directly related to microscopic details of polymers constituting a network, which, however, are mostly neglected in theoretical approaches. Thus, we have limited our argumentations to points we could directly control in the experiments or which were predefined by the polymer type. The transition from keratin-rich to actin-rich networks involves a gradual increase of the frequency dependence of the elastic modulus as illustrated in Fig. 3C, showing the slopes of G0 between 0.1 and 10 Hz for respective composites. The weak power law behaviour exhibited in actin filament networks can be described by the glassy worm-like chain model (GWLC), where the exponent of this power law depends on the level of pre-stress and the interaction strength (e).46 This interaction strength is a phenomenological parameter that is typically neglected in models of entangled networks such as the tube model.47 The fundamental idea of the GWLC is an exponential stretching of a wormlike chain’s mode relaxation times (tn) according to: Fig. 4 Attractive filament–filament interactions (captured in the stickiness parameter e) determine the mechanical response of composite filament networks in linear rheology. Normalized storage moduli (G0) versus fre- quency for different actin/keratin compositions evaluated with the glassy wormlike chain model. Solid lines represent the measured data and dashed lines represent the fitting curves, yielding values for stickiness e as shown. For pure keratin, the model could not be fitted to the data. Fig. 4 Attractive filament–filament interactions (captured in the stickiness parameter e) determine the mechanical response of composite filament networks in linear rheology. Normalized storage moduli (G0) versus fre- quency for different actin/keratin compositions evaluated with the glassy wormlike chain model. Solid lines represent the measured data and dashed lines represent the fitting curves, yielding values for stickiness e as shown. For pure keratin, the model could not be fitted to the data. Composite filament networks exhibit an intermediate linear viscoelastic behaviour (B) The plateau modulus G0 = G0 (f = 1 Hz) for all networks shows only minor variations between networks in the linear regime while in (C) the slopes a show a gradual increase with actin content where we found distributions differing significantly except for the constellation of pure actin compared to 0.15 mg ml1 keratin–0.375 mg ml1 actin (p-value = 0.5). In (A)–(C) dots represent single measurements and error bars represent the standard deviation from the mean. 3958 \| Soft Matter, 2021, 17, 3954–3962 Open Access Article. Published on 05 March 2021. Downloaded on 10/24/2024 6:47:53 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. As the GWLC model is based on the assumption of isotropic networks, it cannot capture pure keratin systems due to the inherent bundling under the investigated conditions (Fig. S4, ESI†). Cluster-forming networks may be better described by models for crosslinked networks such as an aﬃne model.48 Recently, a detailed model for bundling of keratin was suggested based on the interplay between inter-filament electrostatic and hydrophobic interactions. This model predicts that the process of keratin bundling is determined by the electric charge of the filaments, the number of hydrophobic residues, and the exclusion of the ions from the bundle interior.49 Compared with actin, the attractive interactions between filaments in pure keratin structures are much stronger than those in entangled actin networks.37 Consequently, the viscous loss modulus in keratin networks during oscillatory shear is significantly reduced compared to the viscous dissipation in actin networks.50 Therefore, the viscous modulus G00 of actin networks was observed steeply increasing towards the elastic modulus G0, and the crossover point was observed in the linear regime. Fig. 5 Actin–keratin filament composite networks exhibit strain stiffening increasing in manifestation with increasing keratin content. This is expressed in the differential shear modulus K = ds/dg rescaled by its value in the linear regime Klin and plotted versus stress s. Solid lines are measurement curves and dashed vertical lines indicate the yield stresses. The inset shows K/Klin versus strain. of high keratin content. This becomes evident from the shift of maxima in stress–strain relations towards higher strains, translating to higher stresses where the differential modulus intersects the x-axis as illustrated in Fig. 5. The onset of the differential modulus occurs at stresses/strains that are comparable for all networks except for the pure actin, where the onset stress is shifted an order of magnitude to higher stresses. This denotes the transition from a regime dominated by filament interactions within the network to a regime dominated by single filament behaviour, which is a signature of actin networks.51 The resulting parameters for the stickiness parameter e are shown in Fig. 4. For increasing keratin content, we observed a stronger attractive interaction between individual filaments, reflected in higher e values. Network architectures Using spinning disc confocal microscopy, we examined the architecture of all filament networks (10% labeled sample) under these assembly conditions. As mentioned above, keratin is known to quickly form networks due to its high self-aﬃnity, and bundled networks if positively charged ions are present.24,31 We initiated the assembly at low temperature by mixing the protein solution with the assembly buﬀer on ice in order to slow down the assembly process. A dense, hetero- geneous network was obtained as shown in Fig. 6A. Interestingly, a bundled keratin network was also formed even at a very low protein concentration (e.g. as low as 0.1 mg ml1) (Fig. S1A, ESI†). In the absence of salts, keratin can assemble into isotropic networks even at high protein concentrations (Fig. S1B, ESI†). By contrast, actin at 0.5 mg ml1 assembled solely into highly isotropic entangled networks under selected ionic conditions (Fig. 6B). Actin filaments often associate into bundles and networks with diverse structures only in the presence of ‘‘bund- ling factors’’ such as high salt conditions or actin-binding proteins.54–56 Consequently, bundling of actin was not an issue, and actin filaments completely arranged into isotropic networks under the investigated conditions. This journal is © The Royal Society of Chemistry 2021 Filament–filament interactions constitute a fundamental cause for mechanical responses tGWLC n ¼ tWLC n if ln L tWLC n eeNn if ln 4 L; ( This journal is © The Royal Society of Chemistry 2021 3958 \| Soft Matter, 2021, 17, 3954–3962 View Article Online Fig. 5 Actin–keratin filament composite networks exhibit strain stiffening increasing in manifestation with increasing keratin content. This is expressed in the differential shear modulus K = ds/dg rescaled by its value in the linear regime Klin and plotted versus stress s. Solid lines are measurement curves and dashed vertical lines indicate the yield stresses. The inset shows K/Klin versus strain. Soft Matter Paper Soft Matter obtained good fits for every composite, but not for pure keratin filament networks. Fig. 5 Actin–keratin filament composite networks exhibit strain stiffening ncreasing in manifestation with increasing keratin content. This is expressed in the differential shear modulus K = ds/dg rescaled by its value n the linear regime Klin and plotted versus stress s. Solid lines are measurement curves and dashed vertical lines indicate the yield stresses. The inset shows K/Klin versus strain. Fitting the GWLC model to the experimental data yields values for e that indicate stronger attractive filament–filament interactions for increasing keratin contents in composite networks (Fig. 4). While actin networks are considered a model system for entangled networks, keratin networks form sticky clusters due to pronounced hydrophobic interactions36,46 and, therefore, pure keratin networks are not readily accessible to the GWLC model. Open Access Article. Published on 05 March 2021. Downloaded on 10/24/2024 6 This article is licensed under a Creative Commons Attribution 3.0 U Fig. 6 Confocal micrographs of in situ formed filmanet networks. Keratin assembles into a dense heterogeneous network immediately after the addition of F-buﬀer (A), actin assembles solely into an isotropic entangled network as no accessory proteins or crosslinking factors have been used (B). In keratin- dominated networks, a complete co-localization of F-actin and keratin filament networks culminates in a large cluster (C). In equal ratio and F-actin- dominated networks, the isotropic actin networks provide steric hindrance against tight bundling of keratin, and thus keratin appears as filaments and small clusters in these composites (D and E respectively). Scale bar for all images: 10 mm. They may likewise utilize this tunability when changing their viscoelastic properties by varying cytoskeletal network components to meet the mechanical demands in various different simple epithelia such as found in lung and small intestine. These changes are especially important for large deformations, i.e. non-linear deformations. The F-actin/K8–K18 filament compo- sites show drastic strain stiffening under larger deformations, which is induced and dominated by the keratin content. Strain stiffening can be especially used to absorb large external forces, ensuring that tissue structures such as the epithelium remain stable. This may provide insights into the mechanical interplay they might display in a physiological situation, for instance, for cells integrated into an epithelial cell layer when starting to turn into a migrating cell during and after EMT. In the latter situation, cells build up a vimentin IF system, which provides completely different mechanical properties.57–59 Hence, this change of the IF system is probably of direct importance to the process as vimentin IF provides a much softer counterpart system to F-actin with strain stiffening more than one order of magnitude lower than K8–K18 IF.21 is consistent with a previous in vitro study on actin–keratin composites encapsulated in vesicles.22 However, we observed that this steric effect became less pronounced in increasingly keratin-dominated networks (Fig. 6C). Under these conditions, actin and keratin were observed completely co-localized, but mostly appeared as a large cluster. In actin-dominated regimes as well as equal ratio networks, keratin filaments appeared as extended networks or as very small clusters surrounded by homogenous actin filament networks (Fig. 6D and E). In all composites, both keratin and actin networks did not demix, but appeared as interdependent elements. To visualize keratin networks, we developed a new method for labeling the wild type keratin without mutations (described in Materials and methods). Open Access Article. Published on 05 March 2021. Downloaded on 10/24/2024 6 This article is licensed under a Creative Commons Attribution 3.0 U As a control, we tested the bulk mechanical properties of the 10% labeled keratin samples. We found that these labeled samples behave as unlabeled keratin, indicating that the filaments and network properties were not aﬀected by the presence of 10% labeled keratin (Fig. S2, ESI†). This journal is © The Royal Society of Chemistry 2021 Keratin induces strain stiﬀening in the non-linear regime Strain stiﬀening of biopolymer networks is a feature shared by various intermediate filament proteins.52 This physical response is of particular physiological significance for keratins, which make up a major portion of structural proteins found in epithelial cells, and provide protection against large-scale deformation. Interestingly, keratin filament networks exhibit strain stiﬀening even in the absence of crosslinkers or divalent cations,24,36 while in actin networks the strain stiﬀening depends on the crosslinks.51,53 These features of keratin and actin filament assemblies are reflected in the diﬀerential shear modulus K for the diﬀerent composite networks. For networks with lower keratin content, we see weak strain-stiﬀening eﬀects comparable to that of pure actin networks. This is expressed in a maximum value K that is two orders of magnitude lower than for pure keratin and keratin-dominated networks (Fig. 5, inset) appearing at stresses shifted to values more than one order of magnitude lower (Fig. 5). Besides distinctly higher values for the diﬀerential modulus, we see a clear increase in yield stresses for networks In composite networks, actin provides steric hindrance, creating ‘‘obstacles’’ in between keratin filaments, thereby preventing or reducing their tight bundling. This observation 3959 Soft Matter, 2021, 17, 3954–3962 \| 3959 View Article Online View Article Online Soft Matter Paper Fig. 6 Confocal micrographs of in situ formed filmanet networks. Keratin assembles into a dense heterogeneous network immediately after the addition of F-buﬀer (A), actin assembles solely into an isotropic entangled network as no accessory proteins or crosslinking factors have been used (B). In keratin- dominated networks, a complete co-localization of F-actin and keratin filament networks culminates in a large cluster (C). In equal ratio and F-actin- dominated networks, the isotropic actin networks provide steric hindrance against tight bundling of keratin, and thus keratin appears as filaments and small clusters in these composites (D and E respectively). Scale bar for all images: 10 mm. Open Access Article. Published on 05 March 2021. Downloaded on 10/24/2024 6:47:53 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Conclusions Here, our rheological measurements are in the same line with the concept that the downregulation of K8–K18 may contribute to the loss of cell stiffness. This is supported by several knockout experiments showing that cells lacking keratins are more deformable and invasive,10 migrating faster than wild type cells.11 These effects are also much more pronounced than the softening effects resulting from actin depolymerization.10 In particular, loss of K8–K18 in epithelial cancer cells was found to increase collective cell migration.60 We investigated the bulk rheology of composite networks made from F-actin and K8–K18 filaments at varying relative concen- trations. We show that for small deformations, i.e., the linear deformation regime, the composites revealed an intermediate linear viscoelastic behaviour compared to that of the pure networks. This provides important new information about the mechanical coupling between networks of these two structural proteins within the cell environment. Similar to mixed F-actin/ vimentin IF networks,21 the mechanics of composite F-actin/ K8–K18 filament networks can be described from their respective substructures as a superposition in the frame of the GWLC model.21 This suggests that cells can tune their network mechanics by varying the relative ratios of actin and keratin. To study how actin and keratins aﬀect each other’s organi- zation, we have developed a suitable labelling procedure for K8–K18, which enabled us to find that actin sterically hinders and effectively reduces the tight bundling of keratin in some composites. This observation highlights the supportive role of actin within the cell as it enables keratin networks to extend in 3960 \| Soft Matter, 2021, 17, 3954–3962 This journal is © The Royal Society of Chemistry 2021 3960 View Article Online Paper 11 K. Seltmann, W. Roth, C. Kroger, F. Loschke, M. Lederer, S. Huttelmaier and T. M. Magin, J. Invest. Dermatol., 2013, 133, 181–190. order to provide protection to the entire cell. Previous reports demonstrate that the downregulation of keratins can provide space in the cell periphery to enable F-actin networks to reorganize more freely to form protrusions for migration, while the presence of an intact keratin network in the cell periphery, however, slows down actin reorganization.61,62 These results support our observation that this hindrance effect provided by actin diminishes in keratin-dominated networks. 12 B. O. Sun, Y. Fang, Z. Li, Z. Chen and J. Xiang, Biomed. Rep., 2015, 3, 603–610. 13 M. Schoumacher, R. D. Goldman, D. Louvard and D. M. Vignjevic, J. There are no conflicts to declare. 23 U. Aebi, R. Millonig, H. Salvo and A. Engel, Ann. N. Y. Acad. Sci., 1986, 483, 100–119. 24 P. Pawelzyk, H. Herrmann and N. Willenbacher, Soft Matter, 2013, 9, 8871. Access Article. Published on 05 March 2021. Downloaded on 10/24/2024 6:47:53 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. The behaviour of composite cytoskeletal networks in cells is also highly aﬀected by diﬀerent crosslinkers. For example, many actin crosslinkers mediate the formation of actin filament bundles in cells63 and induce strain stiﬀening in in vitro reconstituted actin filament networks.51,53 In the case of keratin, depletion of keratin-associated plectin, which is able to cross- bridge individual IFs and to connect them to other cytoskeletal components, alters the organization and dynamics of keratin, but does not aﬀect the overall mechanical properties of the cell.64,65 Further studies centering on authentic cellular cytolinkers such as plectin that crossbridge keratin and actin filaments are necessary to begin to understand the complex and dynamic behaviour these composite networks exhibit in a living cell. Open Access Article. Published on 05 March 2021. Downloaded on 10/24/2024 6:47:53 AM. 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https://openalex.org/W4296305853	https://zenodo.org/records/7090896/files/IRJMETS40900028078-Sep.pdf	English	null	THE TRANSITION TIME FROM GAVAGE FEEDING TO FULL ORAL FEEDING IN PRETERM INFANTS AT THE CHILDREN'S HOSPITAL LAHORE	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	4,134	International Research Journal of Modernization in Engineering Technology and Science ( Peer-Reviewed, Open Access, Fully Refereed International Journal ) Volume:04/Issue:09/September-2022 Impact Factor- 6.752 www.irjmets.com International Research Journal of Modernization in Engineering Technology a Of Science In Nursing, Department Of Lahore School Of Nursing, The University Of Lahore, Lahore, South Asia, Pakistan 1Master Of Science In Nursing, Department Of Lahore School Of Nursing, The University Of Lahore, Lahore, South Asia, Pakistan 2Assistant Professor Paediatrics, The Children's Hospital, University of Child Health Lahore, South Asia, Pakistan. 3PhD Scholar in Nursing, Department of Lahore School of Nursing, The University of Lahore, South Asia, Pakistan. ABSTRACT It is difficult for preterm newborns to achieve oral feeding success (OFS, the capacity to swallow 100% of the specified volume by mouth), which frequently necessitates prolonged tube feeding. There is not enough research on the connection between OFS and tube feeding duration. The study's main goal is to measure the typical length of time preterm infants spend receiving tube feeding and OFS during their initial hospitalisation at The Children Hospital University of Child health Lahore. In the transition from first to complete oral feeding, we predicted that preterm infants who received tube feeding for a longer period of time would have a lower OFS. Key findings include: • The mean transition time from gavage feeding to oral feeding was 36.4 weeks of gestation among preterm infants who born between the 29 to 32 weeks of gestation. • The mean transition time from gavage feeding to oral feeding was 36.4 weeks of gestation among preterm infants who born between the 29 to 32 weeks of gestation. • Infant’s sex was not evenly distributed in male and female. The frequency of feeding difficulties was higher in male sex than female, report shows the frequency in male gender was 70% and in female gender was 30%. Keywords: Preterm Infants Oral Feeding Gavage Feed Transition Time • Infant’s sex was not evenly distributed in male and female. The frequency of feeding difficulties was higher in male sex than female, report shows the frequency in male gender was 70% and in female gender was 30%. Keywords: Preterm Infants, Oral Feeding, Gavage Feed, Transition Time. e-ISSN: 2582-5208 International Research Journal of Modernization in Engineering Technology and Science ( Peer-Reviewed, Open Access, Fully Refereed International Journal ) Volume:04/Issue:09/September-2022 Impact Factor- 6.752 www.irjmets.com THE TRANSITION TIME FROM GAVAGE FEEDING TO FULL ORAL FEEDING IN PRETERM INFANTS AT THE CHILDREN'S HOSPITAL LAHORE Samina Naz1, Waseem Humayoun2, Muhammad Afzal3 1Master Of Science In Nursing, Department Of Lahore School Of Nursing, The University Of Lahore, Lahore, South Asia, Pakistan 2Assistant Professor Paediatrics, The Children's Hospital, University of Child Health Lahore, South Asia, Pakistan. 3PhD Scholar in Nursing, Department of Lahore School of Nursing, The University of Lahore, South Asia, Pakistan. ABSTRACT e-ISSN: 2582-5208 International Research Journal of Modernization in Engineering Technology and Science ( Peer-Reviewed, Open Access, Fully Refereed International Journal ) Volume:04/Issue:09/September-2022 Impact Factor- 6.752 www.irjmets.com THE TRANSITION TIME FROM GAVAGE FEEDING TO FULL ORAL FEEDING IN PRETERM INFANTS AT THE CHILDREN'S HOSPITAL LAHORE Samina Naz1, Waseem Humayoun2, Muhammad Afzal3 1Master Of Science In Nursing, Department Of Lahore School Of Nursing, The University Of Lahore, Lahore, South Asia, Pakistan 2Assistant Professor Paediatrics, The Children's Hospital, University of Child Health Lahore, South Asia, Pakistan. *3PhD Scholar in Nursing, Department of Lahore School of Nursing, The University of Lahore, South Asia, Pakistan. ABSTRACT II. METHODOLOGY Study Design: A Retrospective cohort research design is being used used to collect data Study Design: A Retrospective cohort research design is being used used to collect data Settings: The study is conducted at Out Petients Department(OPD) and The Neonatal Nursery Department of Study Design: A Retrospective cohort research design is being used used to collect data Settings: The study is conducted at Out Petients Department(OPD) and The Neonatal Nursery Department of The Children Hospital University of Child Health Lahore. ettings: The study is conducted at Out Petients Department(OPD) and The Neonatal Nursery Dep he Children Hospital University of Child Health Lahore. Transition time from gavage to orall feeding is measured by the preterm infants gestational age at birth and the gestational age when the infant consume 100% of prescribed volume orally (100% per oral intake feeding) e-ISSN: 2582-5208 International Research Journal of Modernization in Engineering Technology and Science ( Peer-Reviewed, Open Access, Fully Refereed International Journal ) Volume:04/Issue:09/September-2022 Impact Factor- 6.752 www.irjmets.com predict oral feeding transition ((Wahyuni et al., 2022) However, one of the study shows hemodynamically stable preterm infants can take oral feed successfully at age of 32 to 34 weeks of gestation ((Morag et al., 2019) II. METHODOLOGY Study Design: A Retrospective cohort research design is being used used to collect data Settings: The study is conducted at Out Petients Department(OPD) and The Neonatal Nursery Department of The Children Hospital University of Child Health Lahore. Sample Size: 40 Preterm infants Sampling Technique: Non probability convenient sampling technique is used. Inclusion Criteria: Preterm infants 29 to 32 weeks of gestation. Types of data: Information for this report is sourced from primary source. Data Collection procedure: The data is collected from the follow up preterm infants visiting at Neonatology OPD, medical record of dischare patients, and through phone calls of discharge petients. Measures: Infant characteristics Infant characteristics, including GA, sex, is collected from the medical record at enrollment and discharge. Transition time from gavage to orall feeding Transition time from gavage to orall feeding is measured by the preterm infants gestational age at birth and the gestational age when the infant consume 100% of prescribed volume orally (100% per oral intake feeding) Analysis of Data Data is entered and analyzed by using SPSS version 21. Table and graph is used to present the quantitative data. III. RESULTS AND DISCUSSION TABLE 1: Report Summary S# Gastational age at birth (weeks) Transition time from Gavage to full oral feed (weeks) 1 32 36+2 2 29+5 37 3 31+5 36+4 4 29+6 36+4 5 32 36+4 6 32 36 7 32 37 8 31 36 9 30 36 10 29 38 11 32 36 12 30 37 13 30 37+2 14 31 36+4 e-ISSN: 2582-5208 International Research Journal of Modernization in Engineering Technology and Science ( Peer-Reviewed, Open Access, Fully Refereed International Journal ) Volume:04/Issue:09/September-2022 Impact Factor- 6.752 www.irjmets.com i j @I i l R h J l f M d i i i E i i T h l d S i predict oral feeding transition ((Wahyuni et al., 2022) However, one of the study shows hemodynamically stable preterm infants can take oral feed successfully at age of 32 to 34 weeks of gestation ((Morag et al., 2019) II. METHODOLOGY Study Design: A Retrospective cohort research design is being used used to collect data Settings: The study is conducted at Out Petients Department(OPD) and The Neonatal Nursery Department of The Children Hospital University of Child Health Lahore. Sample Size: 40 Preterm infants Sampling Technique: Non probability convenient sampling technique is used. Inclusion Criteria: Preterm infants 29 to 32 weeks of gestation. Types of data: Information for this report is sourced from primary source. Data Collection procedure: The data is collected from the follow up preterm infants visiting at Neonatology OPD, medical record of dischare patients, and through phone calls of discharge petients. Measures: Infant characteristics Infant characteristics, including GA, sex, is collected from the medical record at enrollment and discharge. Transition time from gavage to orall feeding Transition time from gavage to orall feeding is measured by the preterm infants gestational age at birth and the gestational age when the infant consume 100% of prescribed volume orally (100% per oral intake feeding) Analysis of Data Data is entered and analyzed by using SPSS version 21. Table and graph is used to present the quantitative data. III. RESULTS AND DISCUSSION TABLE 1: Report Summary S# Gastational age at birth (weeks) Transition time from Gavage to full oral feed (weeks) 1 32 36+2 2 29+5 37 3 31+5 36+4 4 29+6 36+4 5 32 36+4 6 32 36 7 32 37 8 31 36 9 30 36 10 29 38 11 32 36 12 30 37 13 30 37+2 14 31 36+4 15 29+5 36+5 16 30 38 17 32 36+4 e-ISSN: 2582-5208 International Research Journal of Modernization in Engineering Technology and Science ( Peer-Reviewed, Open Access, Fully Refereed International Journal ) Volume:04/Issue:09/September-2022 Impact Factor- 6.752 www.irjmets.com predict oral feeding transition ((Wahyuni et al., 2022) However, one of the study shows hemodynamically stable preterm infants can take oral feed successfully at age of 32 to 34 weeks of gestation ((Morag et al., 2019) e SSN 58 5 08 International Research Journal of Modernization in Engineering Technology and Science ( Peer-Reviewed, Open Access, Fully Refereed International Journal ) Volume:04/Issue:09/September-2022 Impact Factor- 6.752 www.irjmets.com predict oral feeding transition ((Wahyuni et al., 2022) However, one of the study shows hemodynamically stable preterm infants can take oral feed successfully at age of 32 to 34 weeks of gestation ((Morag et al., 2019) International Research Journal of Modernization in Engineering Technology and Science ( Peer-Reviewed, Open Access, Fully Refereed International Journal ) Volume:04/Issue:09/September-2022 Impact Factor- 6.752 www.irjmets.com predict oral feeding transition ((Wahyuni et al., 2022) However, one of the study shows hemodynamically stable preterm infants can take oral feed successfully at age of 32 to 34 weeks of gestation ((Morag et al., 2019) I. INTRODUCTION The Preterm birth complications, which account for 35% of the 3.1 million newborn mortality worldwide each year, are the leading cause of neonatal mortality ((Hashmi et al., 2021). 15 million preterm births take place annually throughout the world, with those who are born at fewer than 32 weeks' gestation having the highest risk of morbidity and mortality((Chawanpaiboon et al., 2019). Preterm infants that are undernourished suffer grave complications include higher mortality rates and chronic growth, metabolic, and neurodevelopmental abnormalities ((Chung et al., 2020) Poor nutrition has a significant impact on the brain, which causes slow neurodevelopment and poor brain growth ((Mayerl et al., 2019). Regardless of preterm level, early postnatal growth (i.e., during hospitalisation) has been linked to neurological and cognitive outcomes in infancy and preschool (Chung et al., 2020). Preterm infants are more likely to experience nutrient deficiencies because their stores are insufficient and their digestive systems are still developing. However, with proper nutrition, preterm infants should grow as normally growing foetuses of the same gestational age would. In addition to being a top priority for parents of preterm infants in the intensive neonatal care unit (NICU), the ability to feed orally via bottle or breast is also a requirement for hospital discharge ((Pighetti et al., 2022). Due to the combined effects of immature body functions and medical complications, very preterm infants (those born between 29 and 32 weeks of gestation) typically require a period of gavage feeding and oral feeding training before they are able to be fed exclusively by mouth((Mayerl et al., 2019). The postmenstrual age (PMA) in starting and finishing oral feeding as well as the transition time to full oral feeding increases when gestational age and weight at birth decreases and medical problems rise. One study has indicated that Several factors can be used to predict prolonged oral feeding transition in preterm infants, such as younger GA and a higher morbidity score In preterm infants, male sex was a significant biological risk factor for poor cognitive and motor development when compared to female sex, thus sex may @International Research Journal of Modernization in Engineering, Technology and Science [1158] www.irjmets.com @International Research Journal of Modernization in Engineering, Technology and Science Analysis of Data ta is entered and analyzed by using SPSS version 21. Table and graph is used to present the quantitative dat III. RESULTS AND DISCUSSION TABLE 1: Report Summary S# Gastational age at birth (weeks) Transition time from Gavage to full oral feed (weeks) 1 32 36+2 2 29+5 37 3 31+5 36+4 4 29+6 36+4 5 32 36+4 6 32 36 7 32 37 8 31 36 9 30 36 10 29 38 11 32 36 12 30 37 13 30 37+2 14 31 36+4 15 29+5 36+5 16 30 38 17 32 36+4 @International Research Journal of Modernization in Engineering, Technology and Scienc www.irjmets.com [1159] e-ISSN: 2582-520 ternational Research Journal of Modernization in Engineering Technology and Scienc ( Peer-Reviewed, Open Access, Fully Refereed International Journal ) olume:04/Issue:09/September-2022 Impact Factor- 6.752 www.irjmets.co 18 30 35+6 19 32 36 20 30 35+5 21 32 36 22 31+3 35+5 23 32 37 24 31+4 36+4 25 32 37 26 31 36+4 27 32 37 28 32 36+5 29 29 36+4 30 30 36 31 30 37 32 30 36 33 29 36 34 31 37 35 30 37 36 29 36 37 30 36 38 32 36 39 32 37 40 29 36 Total N 40 40 Table 2: Descriptive Statistics for the sample characteristics N Minimum Maximum Mean Std. Deviation Gestational age at birth 40 29.00 32.00 30.7450 1.12500 Transition time from gavage to oral feed 40 35.50 38.00 36.4550 .59137 Table 3: Correlations Between Duration of tube feeding and infant’s characteristics via pearson’s correlation Gestational age at birth Transition time from gavage to oral feed Gestational age at birth Pearson Correlation 1 -0.06 Sig. (2-tailed) 0.713 N 40 40 Table 3: Correlations Between Duration of tube feeding and infant’s characteristics via pearson’s correlation Gestational age at birth Transition time from gavage to oral feed Gestational age at birth Pearson Correlation 1 -0.06 Sig. (2-tailed) 0.713 N 40 40 www.irjmets.com [1161] e-ISSN: 2582-5208 International Research Journal of Modernization in Engineering Technology and Science ( Peer-Reviewed, Open Access, Fully Refereed International Journal ) Volume:04/Issue:09/September-2022 Impact Factor- 6.752 www.irjmets.com The results Descriptive Statistics for Sample Characteristics: The analysis was completed with 40 infants. Sample report summary depicted in Table 1. Notably, preterm infants were secondary referred cases from different hospitals. Infants were born at the mean GA of 30.74 weeks (SD = 1.12 & range 29-32 weeks), with a mean transition time from gavage feeding to full oral feeding 36.4 weeks (SD=0.5& range 36 to 37). Infants’ sex was not evenly distributed male (70%) and female (30%). During hospitalization, the mean total duration of tube feeding was40 days (SD = 16.65 & range 13- 62). Descriptive Statistics for Outcome Variables Descriptive statistics for the outcome variables are presented in table. Infants had an average of 14 days (SD = 6.98 & range 3-39 days) for the transition time from first to full oral feeding. From the first day of oral feeding attempts to the first day of full oral feeding, out of an average of 109 feedings (SD = 54.44 & range 31-311), infants had a mean number of 100% PO intake feedings of 25 (SD = 11.72 & range 10-72), thus yielding a mean OFS of 0.28 (SD = 0.15 & range 0.05-0.62). The mean post-menstrual age at the oral feeding evaluations, was 36.4 weeks (SD = 0.5 & range 35-38 weeks). Bivariate Analyses: We observed significant differences in the mean total duration of tube feeding and duration of exclusive tube feeding between infants with different different gestational age (Table 3). Significant negative correlations between total duration of tube feeding and GA (Table 3). Multiple Regression Analyses: After adjusting for GA, and sex in the preliminary multiple regression models, a significant correlation between total duration of tube feeding and OFS (β = −1.21, P = 0.002, Ꙍ² = 0.35) was identified. A final multiple regression model was fitted, including OFS as an outcome, total duration of tube feeding as an independent variable, and birthweight as a covariate. A significant negative correlation between total duration of tube feeding (β = −1.10, P = 0.000, Ꙍ² = 0.41) and OFS was observed (Figure 4 and Table 5) IV RECOMMENDATIONS The results Descriptive Statistics for Sample Characteristics: The analysis was completed with 40 infants. Sample report summary depicted in Table 1. Notably, preterm infants were secondary referred cases from different hospitals. Infants were born at the mean GA of 30.74 weeks (SD = 1.12 & range 29-32 weeks), with a mean transition time from gavage feeding to full oral feeding 36.4 weeks (SD=0.5& range 36 to 37). Infants’ sex was not evenly distributed male (70%) and female (30%). During hospitalization, the mean total duration of tube feeding was40 days (SD = 16.65 & range 13- 62). Descriptive Statistics for Outcome Variables Descriptive statistics for the outcome variables are presented in table. Infants had an average of 14 days (SD = 6.98 & range 3-39 days) for the transition time from first to full oral feeding. From the first day of oral feeding attempts to the first day of full oral feeding, out of an average of 109 feedings (SD = 54.44 & range 31-311), infants had a mean number of 100% PO intake feedings of 25 (SD = 11.72 & range 10-72), thus yielding a mean OFS of 0.28 (SD = 0.15 & range 0.05-0.62). The mean post-menstrual age at the oral feeding evaluations, was 36.4 weeks (SD = 0.5 & range 35-38 weeks). Bivariate Analyses: We observed significant differences in the mean total duration of tube feeding and duration of exclusive tube feeding between infants with different different gestational age (Table 3). Significant negative correlations between total duration of tube feeding and GA (Table 3). Multiple Regression Analyses: After adjusting for GA, and sex in the preliminary multiple regression models, a significant correlation between total duration of tube feeding and OFS (β = −1.21, P = 0.002, Ꙍ² = 0.35) was identified. A final multiple regression model was fitted, including OFS as an outcome, total duration of tube feeding as an independent variable, and birthweight as a covariate. A significant negative correlation between total duration of tube feeding (β = −1.10, P = 0.000, Ꙍ² = 0.41) and OFS was observed (Figure 4 and Table 5) e-ISSN: 2582-5208 International Research Journal of Modernization in Engineering Technology and Science ( Peer-Reviewed, Open Access, Fully Refereed International Journal ) Volume:04/Issue:09/September-2022 Impact Factor- 6.752 www.irjmets.com Transition time from gavage to oral feed Pearson Correlation -0.06 1 Sig. (2-tailed) 0.713 N 40 40 Table 5: Gender Frequency Gender Frequency Gender Frequency Percent Valid Percent Cumulative Percent Female 12 30 30 30 Male 28 70 70 100 Total 40 100 100 Table 6 Table 4: Model Summary and Parameter Estimates Dependent Variable: Transition time from gavage to oral feed Equation Model Summary Parameter Estimates R Square F df1 df2 Sig. Constant b1 b2 b3 Linear 0.004 0.138 1 38 0.713 37.426 -0.032 Logarithmic 0.004 0.14 1 38 0.71 39.803 -0.978 Quadratic 0.006 0.113 2 37 0.894 69.075 -2.098 0.034 Cubic 0.006 0.115 2 37 0.892 59.097 -1.093 0 0 Compound 0.003 0.124 1 38 0.726 37.379 0.999 Power 0.003 0.127 1 38 0.724 39.756 -0.025 S 0.003 0.129 1 38 0.721 3.57 0.784 Growth 0.003 0.124 1 38 0.726 3.621 -0.001 Exponential 0.003 0.124 1 38 0.726 37.379 -0.001 Logistic 0.003 0.124 1 38 0.726 0.027 1.001 Table 4: Model Summary and Parameter Estimates Dependent Variable: Transition time from gavage to oral feed Equation Model Summary Parameter Estimates R Square F df1 df2 Sig. Constant b1 b2 b3 Linear 0.004 0.138 1 38 0.713 37.426 -0.032 Logarithmic 0.004 0.14 1 38 0.71 39.803 -0.978 Quadratic 0.006 0.113 2 37 0.894 69.075 -2.098 0.034 Cubic 0.006 0.115 2 37 0.892 59.097 -1.093 0 0 Compound 0.003 0.124 1 38 0.726 37.379 0.999 Power 0.003 0.127 1 38 0.724 39.756 -0.025 S 0.003 0.129 1 38 0.721 3.57 0.784 Growth 0.003 0.124 1 38 0.726 3.621 -0.001 Exponential 0.003 0.124 1 38 0.726 37.379 -0.001 Logistic 0.003 0.124 1 38 0.726 0.027 1.001 Table 4: Model Summary and Parameter Estimates Table 5: Gender Frequency Gender Frequency Gender Frequency Percent Valid Percent Cumulative Percent Female 12 30 30 30 Male 28 70 70 100 Total 40 100 100 Table 6 Table 5: Gender Frequency @International Research Journal of Modernization in Engineering, Technology and Science [1161] www.irjmets.com @International Research Journal of Modernization in Engineering, Technology and Science [1161] www.irjmets.com e-ISSN: 2582-5208 International Research Journal of Modernization in Engineering Technology and Science ( Peer-Reviewed, Open Access, Fully Refereed International Journal ) Volume:04/Issue:09/September-2022 Impact Factor- 6.752 www.irjmets.com implement infant-directed feeding, allowing preterm infants to feed orally as early and frequently as they exhibit signs of oral feeding readiness. implement infant-directed feeding, allowing preterm infants to feed orally as early and frequently as they exhibit signs of oral feeding readiness. V. CONCLUSION The study's results give researchers a fresh method of identifying preterm babies who are at risk for delayed OFS. This study lays the groundwork for future investigations into early evaluation and intervention strategies that promote the switch from tube to oral feeding and aid with OFS. Infants born preterm who are expected to receive tube feedings for longer periods of time could experience delayed OFS. An accurate evaluation of the infants' capacity for safe and effective oral feeding is essential to facilitating their OFS. Our data could serve as the foundation for future studies. There is a requirement for uniform OFS measurements. To fully assess OFS, it may also be helpful to include other measures of oral feeding readiness, oral feeding readiness, caregiver assessment, and oral feeding experience. For preterm infants who are expected to undergo prolonged tube feedings, this information will help in the creation and execution of assessments and early treatments. Such thorough evaluation and interventions have the potential to facilitate OFS and prevent or lessen the avoidable negative consequences of protracted tube feeding IV. RECOMMENDATIONS Practice: For the assessment of infants' oral feeding advancement throughout the switch from tube to oral feeding, health care practitioners do not employ an oral feeding skills calculation. Although the length of tube feeding is an uncontrollable issue, preterm infants who are projected to have longer tube feedings may be at risk for delayed oral feeding abilities. Therefore, in order to enable OFS for these at-risk preterm infants, the caregivers should concentrate on other controllable factors, such as planning to offer adequate and timely assessment and treatments for the introduction and advancement of oral feeding. While assuring an appropriate assessment of the infants' ability to mouth feed safely and effectively, health care professionals have long utilised GA as a guide to commence oral feeding and should continue to do so. Supporting preterm infants during the critical phase of switching from tube to oral feeding will increase their likelihood of obtaining OFS. A self-paced system, non-nutritive sucking, swallowing exercises, oral motor stimulation, multimodal massage, posture, and other interventions should be used to facilitate the initiation of oral feeding and the switch from tube to oral feeding. In order to facilitate an individualised feeding plan and support infants during the switch from tube to oral feeding, the current recommendation for NICUs is to @International Research Journal of Modernization in Engineering, Technology and Science [1162] VI. REFERENCES [1] Chawanpaiboon, S., Vogel, J. P., Moller, A.-B., Lumbiganon, P., Petzold, M., Hogan, D., . . . Laopaiboon, M. (2019). Global, regional, and national estimates of levels of preterm birth in 2014: a systematic review and modelling analysis. The Lancet Global Health, 7(1), e37-e46. [1] Chawanpaiboon, S., Vogel, J. P., Moller, A.-B., Lumbiganon, P., Petzold, M., Hogan, D., . . . Laopaiboon, M. (2019). Global, regional, and national estimates of levels of preterm birth in 2014: a systematic review and modelling analysis. The Lancet Global Health, 7(1), e37-e46. [2] Chung, E. H., Chou, J., & Brown, K. A. (2020). Neurodevelopmental outcomes of preterm infants: a recent literature review. Translational pediatrics, 9(Suppl 1), S3. [2] Chung, E. H., Chou, J., & Brown, K. A. (2020). Neurodevelopmental outcomes of preterm infants: a recent literature review. Translational pediatrics, 9(Suppl 1), S3. [3] Hashmi, J. A., Javaid, A., Qureshi, W. A., Naqvi, A. S. A. H., & Hashmi, M. O. J. (2021). Survival rate of premature babies admitted at a tertiary care hospital of Bahawalpur, Pakistan. Rawal Medical Journal, 46(4), 854-854. [4] Mayerl, C. J., Gould, F. D., Bond, L. E., Stricklen, B. M., Buddington, R. K., & German, R. Z. (2019). Preterm birth disrupts the development of feeding and breathing coordination. Journal of Applied Physiology, 126(6), 1681-1686. [5] Morag, I., Hendel, Y., Karol, D., Geva, R., & Tzipi, S. (2019). Transition from nasogastric tube to oral feeding: the role of parental guided responsive feeding. Frontiers in pediatrics, 7, 190. [6] Pighetti, D., Hirschwald, J., & Gilheaney, O. (2022). Developmental feeding milestones in the transition from non-oral feeding to oral feeding in premature infants: a scoping review. Speech, Language and Hearing, 25(1), 82-97. [7] Wahyuni, L. K., Mangunatmadja, I., Kaban, R. K., Rachmawati, E. Z. K., Harini, M., Laksmitasari, B., & Nugraha, B. (2022). Factors Affecting Oral Feeding Ability in Indonesian Preterm Infants. Pediatric Reports, 14(2), 233-243. @International Research Journal of Modernization in Engineering, Technology and Science [1163] @International Research Journal of Modernization in Engineering, Technology and Science [1163] www.irjmets.com
https://openalex.org/W1986525733	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0097246&type=printable	English	null	An Invasive Clonal Plant Benefits from Clonal Integration More than a Co-Occurring Native Plant in Nutrient-Patchy and Competitive Environments	PloS one	2,014	cc-by	9,396	An Invasive Clonal Plant Benefits from Clonal Integration More than a Co-Occurring Native Plant in Nutrient- Patchy and Competitive Environments Wenhua You, Shufeng Fan, Dan Yu, Dong Xie, Chunhua Liu The National Field Station of Lake Ecosystem of Liangzi Lake, College of Life Science, Wuhan University, Wuhan, P.R. China Wenhua You, Shufeng Fan, Dan Yu, Dong Xie, Chunhua Liu The National Field Station of Lake Ecos stem of Liang i Lake College of Life Science W han Uni ersit W Wenhua You, Shufeng Fan, Dan Yu, Dong Xie, Chunhua Liu The National Field Station of Lake Ecosystem of Liangzi Lake, College of Life Science, Wuhan University, Wuhan, P.R. China Abstract Funding: This research was supported by the National Natural Science Foundation of China (30930011 and 31170339). 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: yudan01@public.wh.hb.cn (DY); liuchh@163.com (CL) Citation: You W, Fan S, Yu D, Xie D, Liu C (2014) An Invasive Clonal Plant Benefits from Clonal Integration More than a Co-Occurring Native Plant in Nutrient- Patchy and Competitive Environments. PLoS ONE 9(5): e97246. doi:10.1371/journal.pone.0097246 Editor: Fei-Hai Yu, Beijing Forestry University, China Received October 30, 2013; Accepted April 16, 2014; Published May 9, 2014 Received October 30, 2013; Accepted April 16, 2014; Published May 9, 2014 Copyright: 2014 You et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, use, distribution, and reproduction in any medium, provided the original author and source are credited. et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted roduction in any medium, provided the original author and source are credited. Funding: This research was supported by the National Natural Science Foundation of China (30930011 and 31170339). 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: yudan01@public.wh.hb.cn (DY); liuchh@163.com (CL) Abstract Many notorious invasive plants are clonal, however, little is known about the different roles of clonal integration effects between invasive and native plants. Here, we hypothesize that clonal integration affect growth, photosynthetic performance, biomass allocation and thus competitive ability of invasive and native clonal plants, and invasive clonal plants benefit from clonal integration more than co-occurring native plants in heterogeneous habitats. To test these hypotheses, two stoloniferous clonal plants, Alternanthera philoxeroides (invasive), Jussiaea repens (native) were studied in China. The apical parts of both species were grown either with or without neighboring vegetation and the basal parts without competitors were in nutrient- rich or -poor habitats, with stolon connections were either severed or kept intact. Competition significantly reduced growth and photosynthetic performance of the apical ramets in both species, but not the biomass of neighboring vegetation. Without competition, clonal integration greatly improved the growth and photosynthetic performance of both species, especially when the basal parts were in nutrient-rich habitats. When grown with neighboring vegetation, growth of J. repens and photosynthetic performance of both species were significantly enhanced by clonal integration with the basal parts in both nutrient-rich and -poor habitats, while growth and relative neighbor effect (RNE) of A. philoxeroides were greatly improved by clonal integration only when the basal parts were in nutrient-rich habitats. Moreover, clonal integration increased A. philoxeroides’s biomass allocation to roots without competition, but decreased it with competition, especially when the basal ramets were in nutrient-rich sections. Effects of clonal integration on biomass allocation of J. repens was similar to that of A. philoxeroides but with less significance. These results supported our hypothesis that invasive clonal plants A. philoxeroides benefits from clonal integration more than co-occurring native J. repens, suggesting that the invasiveness of A. philoxeroides may be closely related to clonal integration in heterogeneous environments. Citation: You W, Fan S, Yu D, Xie D, Liu C (2014) An Invasive Clonal Plant Benefits from Clonal Integration More than a Co-Occurring Native Plant in Nutrient- Patchy and Competitive Environments. PLoS ONE 9(5): e97246. doi:10.1371/journal.pone.0097246 Editor: Fei-Hai Yu, Beijing Forestry University, China Received October 30, 2013; Accepted April 16, 2014; Published May 9, 2014 Copyright: 2014 You 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. Citation: You W, Fan S, Yu D, Xie D, Liu C (2014) An Invasive Clonal Plant Benefits from Clonal Integration More than a Co-Occurring Native Plant in Nutrient- Patchy and Competitive Environments. PLoS ONE 9(5): e97246. doi:10.1371/journal.pone.0097246 Plant material A. philoxeroides is a serious economic and environmental clonal weed which originates from Parana River region of South America and now invades may countries in the world [36,37]. In China, A. philoxeroides has invaded varied ecosystems and caused great economic and environmental problems, and it is listed as one of the 16 worst alien invasive weeds [38]. J. repens is a rooted emergent stoloniferous clonal plants and a fast-proliferating species in wetlands, naturally distributed in central and south China [39]. In natural environments, these two species often co-exist in diverse habitats that from wet to aquatic in south China. In heterogeneous habitats consisting of a mixture of rich and poor resource patches, via clonal integration, clonal plants can alter biomass allocation and divert more biomass to shoots or roots for acquisition of more abundant resource, and exploration of more favourable space, a phenomenon called ‘division of labour’ [2,32,33]. This pattern of biomass allocation is different from that used by non-clonal plants, or clonal plants grown in homogeneous conditions [2,32]. In particular, the relationship between plant photosynthetic efficiency and clonal integration has not been widely studied [9,31]. Photosynthetic efficiency can be estimated by measuring chlorophyll fluorescence [34]. A sensitive indicator of plant photosynthetic performance derived from the parameters of chlorophyll fluorescence is the maximum quantum yield of photosystem II (Fv/Fm), which usually significantly decreases when plants are faced with environmental stress [31,35]. Environmental stress on ramets may be alleviated by clonal integration, which may markedly lower the negative effects of stress on Fv/Fm [9,11]. Moreover, photosynthetic activity, measured in terms of the effective quantum yield of PSII (Yield), is closely related to plant performance. Biomass allocation and photosynthetic efficiency can both contribute to the performance of clonal plants when exposed to competitive stress, however, our understanding of their responses to clonal integration for invasive plants remains limited [9,11]. In early May 2010, source material of A. philoxeroides and J. repens was collected from at least five locations at least 15 m apart in each of two wetlands in Liangzi Lake in the Hubei province of China (N 30u059–30u189, E 114u219–114u399). Given that genetic diversity of wetland clonal plants is relatively low [40], especially for A. philoxeroides in China [41], different populations of each species were assumed to belong to the same genet. Then plants from different locations were mixed and propagated in the greenhouse. Introduction invasive plants have the capacity for vigorous clonal propagation [17,18]. Some studies have suggested that the invasiveness of alien clonal plants may be closely correlated to clonal integration [4,19,20]. However, to our best of knowledge, few studies have focused on how clonal integration affects invasion of alien invasive clonal plants to native plant communities, but see [9,21–24]. Therefore, a better understanding of different clonal integration effects between alien invasive and native clonal plants when competing with each other is both scientific and practical interests. Clonal integration, through which connected ramets of clonal plants can share water, carbohydrates,nutrients and other substances such as pollutants, diseases, etc. [1–3], may improve plants’ exploitation of ubiquitous heterogeneous resources, help plants invade new environments and facilitate plants’ spatial occupation of new habitats at a local scale [4]. Previous studies have shown that clonal integration may facilitate the colonization and growth of the ramets in heterogeneous habitats with stressful conditions [5,6], help genets to survive and to recover after severe environmental change [7,8] and allow for occupation of new space [9–11]. These positive effects of clonal integration may increase the performance of clonal plants over non-clonal plants or other clonal plants with little integration [12]. Therefore, increases in performance of clonal plants by clonal integration may affect the growth and reproduction of their co-existence species, and thus influence community structure and ecosystem function [13,14]. Previous studies about the effects of clonal integration on performance of clonal plants when competing with neighbors were with inconsistent results [9]. Clonal integration had no significant effects on competitive ability of several terrestrial or amphibious plants [9,14,21,25], but it did increase growth of several salt marsh plants for below-ground resources, the competitive ability of Solidago canadensis against interspecific neighbors and the invasion of smooth brome clones to northern fescue prairies [26–28]. However, none of these studies had investigated how clonal integration affected the growth of the neighboring vegetation (competitors), but see [9]. In the study of Pennings and Callaway Plant invasions pose a great threat to biodiversity and global ecosystem stability [15,16]. Introduction Many of the most notorious alien May 2014 \| Volume 9 \| Issue 5 \| e97246 PLOS ONE \| www.plosone.org 1 An Invasive Plant Benefits from Clonal Integration (2000), the results showed that physiological integration played different roles in six salt marsh clonal plants with recipient ramets in different microhabitats or competing with co-existing neighbors in different status (clipped or kept intact). Moreover, several studies have addressed the effects of clonal integration on intra- or interspecific competition of recipient ramets [9,29,30]. Unfortu- nately, all these studies ignored the status of other connected ramets (donor ramets) and the environments which in, as clonal plants often experience small-scale spatial heterogeneity duo to large clonal systems [31]. For instance, clonal integration may increase the competitive ability of ramets when other connected ones were in resource-rich patches, because more subsidy could be supplied by donor ramets in non-limiting resource environments, which may facilitate the invasion of the clonal plants to neighboring communities. But to our knowledge, no studies have tested this. Furthermore, in a field experiment, Peltzer (2002) found that clonal integration did not alter the effects of competition from neighboring vegetation for Populus tremuloides, however, competition greatly improved the survivorship of Populus ramets after 2 years. Therefore, the importance of clonal integration in competition urgently needed for further research [21]. severe, 3) that clonal integration will enhance the photosynthetic performance and buffer the decrease in Fv/Fm of the two plants in competitive environments, especially when donor ramets are in nutrient-rich habitats, 4) that A. philoxeroides will benefit from clonal integration more than J. repens, in terms of competitive ability, photosynthetic performance and capacity of labor division, and 5) that clonal integration of these two clonal species will suppress the growth of neighboring vegetation due to competition with apical ramets. Plant material After two weeks of adaptive culture, about 600 tip cuttings of each plant were collected and planted vertically into 12 plots (30 cm diameter 615 cm height) with soil (TN 2.94 mg/g, TP 0.13 mg/ g) from the lake side of Liangzi Lake. Ten days later, a homogeneous subset of 480 vigorously growing plants of each species were selected for this experiment. Ethics Statement Plant material used in this experiment was collected from natural plant populations at the National Field Station of Freshwater Ecosystem of Liangzi Lake (N 30u059–30u189, E 114u219–114u399). Both of the plant species were common and naturally distributed in this area. No specific permissions were required for these locations. This study did not involve endangered or protected species. Therefore, to test different effects of clonal integration on one exotic invasive and one native clonal plants, we conducted a greenhouse experiment to investigate responses of growth, biomass allocation, photosynthetic performance and relative neighbor effects (RNE, used to indicate the plants’ competitive ability) of two stoloniferous clonal plants, Alternanthera philoxeroides (invasive), Jussiaea repens (native) to clonal integration when competing with competitors (neighboring vegetation) in China. We used a factorial design with resource availability, stolon severing and competition with neighboring vegetation as factors. Specifically, we hypothe- size 1) that clonal integration will improve growth and competitive ability of the two clonal plants, especially when donor ramets are in nutrient-rich habitats, 2) that clonal integration will modify biomass allocation of the two plants grown with competitors. Based on the theory of labour division [32], we predict that, through clonal integration, plants will allocate more biomass to leaves if the belowground competition is more severe and will allocate more biomass to roots if aboveground competition is more Experimental design Three integration treatments were used as follows: severed (stolon connections severed by the scissors), intact (stolon connections kept intact) and nutrient (stolon connections kept intact and with basal ramets in fertilized habitats). d i 10 1371/j l 0097246 001 On July 5th 2011, 24 clonal fragments of each species were horizontally positioned in 24 containers (12 with and 12 without competitive vegetation in apical sections), the remaining 4 containers with competitive vegetation were used as a control for plant population growth without competition. For each clonal fragment, three ramets of basal part were placed within basal section of a container and the other two ramets and apex of the apical part were within the apical section of the same container. The stolon of the apical ramets was anchored to the soil surface to facilitating rooting. Six days later, when the clonal fragments were successfully rooted, the stolon connections between the apical and basal parts were severed in 8 containers, while the other 16 ones were kept intact (see Fig. 1). The experiment was ended on September 10th 2011. The experimental units were randomly repositioned every two weeks to avoid the effects of possible environmental heterogeneity (such as light), and watered every other day to maintain the soil in the containers at wet condition. The mean light intensity in the greenhouse was 800–1200 mmol m22 s21, and the mean air temperature was 20–28uC during the experimental period. one termed as ‘basal part’ consisting of three relatively old ramets (close to the mother ramets) and the other as ‘apical part’ consisting two relatively young ramets (distal to the mother ramets) and a stolon apex. There were 28 plastic containers (50650625 cm; length6width6height), each having two separated sections in this experiment for each species (see Fig. 1). The basal section was 20 cm long and the apical section was 30 cm long. Resources (nutrients and water) and roots in the two sections did not interfere with each other. All the containers in both sections were filled with a mixture of sand and lake mud at a volume ratio of 3:1. To create highly fertilized soil patches, 12 containers were filled with the same mixture and 5 g slow-release fertilizer (OsmocoteH, N–P–K: 16–8–12, 6 month) in the basal section. Experimental design The growth experiment was conducted in a glasshouse under natural sunlight (about 14/10 day/night cycle) and ambient temperature at the National Field Station of the Lake Ecosystem of Liangzi Lake, Wuhan University. The experiment was conducted with a factorial design involving competition (without or with vegetation for control or competition treatment) and integration treatments (stolon connections were severed, intact or intact and with basal ramets in nutrient-rich patches, for severed, intact or nutrient treatment) (Fig. 1). The tested plants used in this experiment were 24 similar-sized clonal fragments (tip cuttings, 14.3360.15 cm in length, 0.6260.034 g in dry mass for A. philoxeroides; 15.0260.21 cm in length, 0.7860.041 g in dry mass for L.repens; means 6 SE), each consisting of a stolon with five ramets for each species. No differences between treatments were detected in initial size of this plants (P.0.05 for both species, One- way ANOVA). Each clonal fragment was divided into two parts, May 2014 \| Volume 9 \| Issue 5 \| e97246 May 2014 \| Volume 9 \| Issue 5 \| e97246 PLOS ONE \| www.plosone.org 2 An Invasive Plant Benefits from Clonal Integration Figure 1. Schematic representation of the experimental design. Clonal fragments of the invasive plant A. philoxeroides or native plant J. repens, each consisting of three basal ramets (dark grey circles) and two apical ramets (light gray circles) with a stolon apex (horizontal arrow), were grown either with (competition) or without (control) competitive vegetation (J. repens or A. philoxeroides, spot-shadow) and with stolon connections between basal and apical ramets were either intact or severed (fork). Three integration treatments were used as follows: severed (stolon connections severed by the scissors), intact (stolon connections kept intact) and nutrient (stolon connections kept intact and with basal ramets in fertilized habitats). doi:10 1371/journal pone 0097246 g001 Figure 1. Schematic representation of the experimental design. Clonal fragments of the invasive plant A. philoxeroides or native plant J. repens, each consisting of three basal ramets (dark grey circles) and two apical ramets (light gray circles) with a stolon apex (horizontal arrow), were grown either with (competition) or without (control) competitive vegetation (J. repens or A. philoxeroides, spot-shadow) and with stolon connections between basal and apical ramets were either intact or severed (fork). An Invasive Plant Benefits from Clonal Integration Figure 2. Effects of integration treatments and competition on growth measures of the two clonal plants. Total biomass, ramet number, stolon length, leaf number and total leaf area of the invasive plant A. philoxeroides (left: A, B, C, D, E) or native plant J. repens (right: F, G, H, I, J) in the apical sections, grown either with or without competitive vegetation (J. repens or A. philoxeroides) in three integration treatments. Data indicate the means 6 SE. Bars sharing the same letter are not significantly different at P = 0.05. doi:10.1371/journal.pone.0097246.g002 Measurements One week before harvesting the plants, the minimum (F0) and the maximum (Fm) fluorescence yield were measured for a fully developed, healthy leaf on the second-youngest of the ramets in each apical plant after a dark adaptation (shaded by leaf folders) of at least 20 minutes sufficient for photosystem II (PSII) reaction centers to open by a portable chlorophyll fluorometer (DIVING- PAM, Walz, Effeltrich, Germany) with the saturation pulse method [34]. The maximum quantum yield of PSII (Fv/Fm) was calculated as (Fm – F0)/Fm. The effective quantum yield of PS II (Yield) was calculated as (Fm9–Ft)/Fm9, where Fm9 is defined as the maximal fluorescence yield reached in a pulse of saturating light after a actinic light pulse of 120 mmol m22 s21 for 10 seconds, and Ft is the fluorescence yield of the leaf at that photosynthetic photon flux density [31,35]. At harvest, the number of ramets and leaves were counted, and the total stolon length, total leaf area (Li-3100 Area Meter, Li-Cor, USA) were measured for the apical parts of all treatments. The ramets in the apical part of the two clonal species were then harvested and separated into leaves, stolons and roots, and their biomass was determined after drying at 70 uC for 72 h. Neighboring vegetation (entire plants including roots) in the apical sections of the container for each species were also harvested and their dry mass was also determined in the same way. The relative neighbor effect (RNE) was calculated to measure the competitive intensity [42]. The RNE of plant was calculated as (C - A)/max (C, A), where is A the mean biomass of plant across replicates without competition, C is biomass of plant with competition, and max (C, A) is the larger value between A and C. Usually. The values of RNE range from -1 to 0, and the greater the values are, the smaller the neighbor’s effects is [9]. So, a significantly larger RNE with than without stolon connection treatments indicates clonal integration facilitates plant’s compet- itive ability. Experimental design On June 10th 2011, the apical sections of 16 containers were planted vertically with cultured plant fragments of each species (monoculture) in the glasshouse to mimic natural plant populations (vegetated habitats), with a density of 200 plants m22 (30 plants in each apical section) for each species. The remaining 12 containers were kept with apical sections bare. May 2014 \| Volume 9 \| Issue 5 \| e97246 PLOS ONE \| www.plosone.org 3 An Invasive Plant Benefits from Clonal Integration Data analysis y All data were log transformed to meet assumptions of normality and homoscedasticity before analysis. One-way ANOVA was used to test whether total biomass of vegetation (competitors) in the apical section for each species differed among the four treatments (no competition; competition with severed stolon connection; competition with intact stolon connection; competition with intact stolon connection and basal parts in nutrient-rich sections). Two- way ANOVA was used to assess the effects of integration treatments (severed, intact and nutrient) and competition on photosynthetic performance (Fv/Fm and Yield) of the two species in the apical section. Two-way multivariate analysis of variance (MANOVA) was employed to investigate the global effects of integration treatments and competition on growth measures (total biomass, ramet number, stolon length, leaf number and leaf area) and biomass allocation pattern (biomass allocation to leaves, stolons and roots) of both species in the apical parts, and corresponding univariate analyses were also conducted. If a significant treatment effect was detected, post-hoc pair-wise comparisons of means were made to examine differences between treatments using Studentized Tukey’s HSD for multiple compar- isons. The differences of RNE values among the integration treatments were tested by one-way ANOVA followed by Duncan tests. Statistical significance was assigned at a P,0.05. All data analyses were performed using SPSS 18.0 (SPSS, Chicago, IL, USA). Figure 2. Effects of integration treatments and competition on growth measures of the two clonal plants. Total biomass, ramet number, stolon length, leaf number and total leaf area of the invasive plant A. philoxeroides (left: A, B, C, D, E) or native plant J. repens (right: F, G, H, I, J) in the apical sections, grown either with or without competitive vegetation (J. repens or A. philoxeroides) in three integration treatments. Data indicate the means 6 SE. Bars sharing the same letter are not significantly different at P = 0.05. doi:10.1371/journal.pone.0097246.g002 ects of integration treatments and competition on May 2014 \| Volume 9 \| Issue 5 \| e97246 PLOS ONE \| www.plosone.org 4 An Invasive Plant Benefits from Clonal Integration Table 1. Summary of MANOVA and univariate ANOVA for effects of integration treatments and competition on growth measures of the two clonal plants in the apical sections. Multivariate test statistics A. philoxeroides J. repens Source Wilk’s Lambda F d.f. P Wilk’s Lambda F d.f. May 2014 \| Volume 9 \| Issue 5 \| e97246 May 2014 \| Volume 9 \| Issue 5 \| e97246 Data analysis P Integration (I) 0.008 29.37 10,28 ,0.001 0.018 17.93 10,28 ,0.001 Competition (C) 0.015 180.23 5,14 ,0.001 0.037 73.37 5,14 ,0.001 I6C 0.079 7.14 10,28 ,0.001 0.403 1.61 10,28 0.16 Univariate test statistics A. philoxeroides J. repens Source Total Stolon Ramet Leaf Total Total Stolon Ramet Leaf Total biomass length number number leaf area biomass length number number leaf area Integration (I) 611.68* 101.10* 202.22* 250.56* 275.19* 288.89* 71.32* 59.50* 60.74* 47.53* Competition(C) 88.55* 146.37* 260.16* 181.63* 208.87* 404.84* 95.78* 134.55* 113.31* 52.91* I6C 67.03* 8.79 9.54 10.15 18.45*** 3.22 0.40 2.45 0.32 1.35 d.f. 5,18 5,18 5,18 5,18 5,18 5,18 5,18 5,18 5,18 5,18 Significant P-values are presented in bold. Values give F; symbols give P: * P,0.05; P,0.01; * P,0.001. doi:10.1371/journal.pone.0097246.t001 An Invasive Plant Benefits from Clonal Integration PLOS ONE \| www.plosone.org 5 May 2014 \| Volume 9 \| Issue 5 \| e97246 Table 1. Summary of MANOVA and univariate ANOVA for effects of integration treatments and competition on growth measures of the two clonal plants in the apical sectio Multivariate test statistics A. philoxeroides J. repens Source Wilk’s Lambda F d.f. P Wilk’s Lambda F d.f. P Integration (I) 0.008 29.37 10,28 ,0.001 0.018 17.93 10,28 ,0.001 Competition (C) 0.015 180.23 5,14 ,0.001 0.037 73.37 5,14 ,0.001 I6C 0.079 7.14 10,28 ,0.001 0.403 1.61 10,28 0.16 Univariate test statistics A. philoxeroides J. repens Source Total Stolon Ramet Leaf Total Total Stolon Ramet Leaf Total biomass length number number leaf area biomass length number number leaf area Integration (I) 611.68* 101.10* 202.22* 250.56* 275.19* 288.89* 71.32* 59.50* 60.74* 47.53* Competition(C) 88.55* 146.37* 260.16* 181.63* 208.87* 404.84* 95.78* 134.55* 113.31* 52.91* I6C 67.03* 8.79 9.54 10.15 18.45* 3.22 0.40 2.45 0.32 1.35 d.f. 5,18 5,18 5,18 5,18 5,18 5,18 5,18 5,18 5,18 5,18 Significant P-values are presented in bold. May 2014 \| Volume 9 \| Issue 5 \| e97246 May 2014 \| Volume 9 \| Issue 5 \| e97246 5 An Invasive Plant Benefits from Clonal Integration Figure 3. Effects of integration treatments on the relative neighbour effect (RNE) of the two clonal plants. The relative neighbour effect (RNE) of the invasive plant A. philoxeroides and native plant J. repens in the apical sections in three integration treatments. Data indicate the means 6 SE. Bars sharing the same letter are not significantly different at P = 0.05. doi:10.1371/journal.pone.0097246.g003 Figure 3. Growth and the relative neighbor effect (RNE) Integration treatments and competition had significant effects on growth of both clonal species in the apical sections, and their interaction was also significant for A. philoxeroides but not for J. repens (Table 1). Competition greatly reduced the growth measures (including total biomass, number of ramets and leaves, stolon length and total leaf area) of the two clonal species (Table 1, Fig 2). Without competition, clonal integration greatly improved the growth of both of these two species in the apical sections, especially when the basal parts of the clonal fragments were in nutrient-rich patches (Fig 2A, B, C, D, E). However, with competition, clonal integration had no significant effect on the growth of A. philoxeroides but greatly enhanced that when its basal parts were in nutrient- rich sections (Fig 2A, B, C, D, E). For J. repens, the responses of the growth to integration treatments with competition were similar to that without competition (Fig 2F, G, H, I, J). Growth of neighboring vegetation Total neighboring vegetation biomass of the two species both had no significant differences among all the treatments (F3,15 = 0.87, P = 0.39 for A. philoxeroides; F3,15 = 0.48, P = 0.70 for J. repens). Total biomass in the apical sections for each species in four treatments (no competition; competition with severed stolon connection; competition with intact stolon connection; competi- tion with intact stolon connection and basal parts in rich-patches) were 74.7262.13 g, 76.3363.21 g, 74.5461.94 g and 73.5263.17 g for vegetation of A. philoxeroides; and 89.3863.67 g, 91.8760.70 g, 93.5962.97 g and 89.8162.99 g (means 6 SE) for that of J. repens respectively. Photosynthetic performance Integration treatments and competition significantly affected the photosynthetic performance (Fv/Fm and Yield) of both of the two clonal plants, and their interaction was also significant in Fv/Fm but not in Yield (Table 3). Competition greatly reduced the value of Fv/Fm of the two species in the apical sections, especially when the stolon connections were severed (Fig. 5A, C). Clonal integration markedly increased the value of Fv/Fm of the two species, especially when their basal parts were in nutrient-rich sections (Fig. 5A, C). The Yields of the two plants were both significantly reduced by competition (Fig. 5B, D), but greatly enhanced by clonal integration, especially when their basal parts were in nutrient-rich sections (Fig. 5B, D). Integration treatments (severed, intact and nutrient) significantly affected the relative neighbor effect (RNE) of the two clonal species in the apical parts (F2,11 = 96.14, P,0.001 for A. philoxeroides; F2,11 = 6.24, P = 0.02 for J. repens). Clonal integration significantly decreased the RNE of A. philoxeroides but greatly increased that in nutrient treatment (Fig. 3). The RNE of J. repens had a decreasing trend with the stolon connection intact and an increasing trend in nutrient treatment but not significantly (Fig. 3). Data analysis Effects of integration treatments on the relative neighbour effect (RNE) of the two clonal plants. The relative neighbour effect (RNE) of the invasive plant A. philoxeroides and native plant J. repens in the apical sections in three integration treatments. Data indicate the means 6 SE. Bars sharing the same letter are not significantly different at P = 0.05. doi:10.1371/journal.pone.0097246.g003 decreased that to the roots whether when the apical parts of plants were with competition or not (Table 2, Fig. 4A, C). Biomass allocation to the stolons of both species were not affected by clonal integration but was significantly larger when the apical ramets were grown with rather than without competition (Table 2, Fig. 4B, E). Effects of integration treatments and competition on apical parts of J. repens were similar to that of A. philoxeroides, although the trend was less obvious (Table 2, Fig. 4D, F). An Invasive Plant Benefits from Clonal Integration Figure 4. Effects of integration treatments and competition on biomass allocation of the two clonal plants. Biomass allocation (LMR, leaf mass ratio; SMR, stolon mass ratio; RMR, root mass ratio) of the invasive plant A. philoxeroides (left: A, B, C) or native plant J. repens (right: E, F, G) in the apical sections, grown either with or without competitive vegetation (J. repens or A. philoxeroides) in three integration treatments. Data indicate the means 6 SE. doi:10.1371/journal.pone.0097246.g004 including terrestrial plants [43,44], amphibious plants [9] and submerged aquatic plants [10], which showed that clonal integration facilitates establishment of newly produced ramets, improves growth of adult ramets and helps genets to occupy open space. This phenomenon was more pronounced when basal ramets were in nutrient-rich patches, probably because more subsidy was provided by the basal parts via clonal integration due to source-sink relationship [31,45,46]. These observations indicate that clonal integration is critical in allowing these two clonal species to explore new open space and rapid expansion, especially in heterogeneous habitats, which is a well-known mechanism to allow stoloniferous and rhizomatous plants to forage for resources over large areas [45,46]. When competing with neighboring vegetation, growth measures of both plants were markedly suppressed by competition, suggesting that strong interspecific competition in this experiment occurred in the apical parts of both plants. Interestingly, stolon connection had no effect on growth of A. philoxeroides with competition, while it greatly increased growth of that in the nutrient treatment. Moreover, clonal integration decreased the RNE of A. philoxeroides in intact treatment and greatly increased that in nutrient treatment, suggesting that clonal integration improved the competitive ability of A. philoxeroides only when the basal parts were in nutrient-rich conditions. The reason might be that ramets of A. philoxeroides were sophisticated (selective) and relatively independent. Therefore, few ramets were placed in the habitat with severe competition (less resources available in apical sections) and more ramets were placed in relatively more favourable conditions (more resources available in bare basal sections) [47,48]. Actually, more branches and biomass were observed in the basal sections (data not shown). However, when the basal parts of the plant were in nutrient-rich sections, strong intra-competition existed because of dense plant population due to vigorous growth in nutrient-rich habitats at the end of experiment. Nutrient-rich habitats may have equal or even less suitability compared to poor habitats because overcrowding reduces suitability [31]. Discussion Clonal integration improved the growth and photosynthetic performance, modified biomass allocation of both the introduced, invasive species A. philoxeroides and the co-occurring native species J. repens in nutrient-patchy and competitive environments. These results suggest that clonal integration is very important for both species when faced with competition in heterogeneous habitats [9]. However, some differences were observed in these two stolonif- erous clonal plants. Effects of clonal integration on growth and competitive ability Without competition, clonal integration significantly improved the growth of both clonal species in the apical sections, especially when their basal parts were in nutrient-rich sections. This result occurred most likely because the well-established ramets in the basal sections supported the growth of the interconnected young apical sections and facilitated the production of new tissue due to acropetal (from basal ramets to apical ramets) translocation of carbohydrates via clonal integration [9]. The results agree with those obtained in previous studies on several other clonal plants An Invasive Plant Benefits from Clonal Integration To avoid self-shading, clonal integration enhanced the competitive ability and facilitated the invasion of the apical ramets of A. philoxeroides into neighboring vegetation [49]. For J. repens, with competition of A. philoxeroides vegetation, clonal integration promoted the growth of apical parts of plant in both stolon connection treatments (intact and nutrient treatments). In addition, clonal integration had no significant effects on the RNE of the apical ramets whether in intact treatment or in nutrient treatment. These results suggest that native J. repens may be more dependent on physiological integration and may share resources to a higher degree [50]. Thus, when faced with severed competition, growth and spread of the invasive A. philoxeroides would in general benefit more from clonal integration than native co-occurring J. repens, because: 1) in relatively poor habitats, clonal integration may preferentially allow the ramets of A. philoxeroides to escape from competitive stress and explore other open space to rapid expansion [9]; 2) in resource-rich patchy habitats, clonal integration may enhance the its competitive ability and facilitate the invasion of A. philoxeroides to neighboring vegetation [22]. Figure 4. Effects of integration treatments and competition on biomass allocation of the two clonal plants. Biomass allocation (LMR, leaf mass ratio; SMR, stolon mass ratio; RMR, root mass ratio) of the invasive plant A. philoxeroides (left: A, B, C) or native plant J. repens (right: E, F, G) in the apical sections, grown either with or without competitive vegetation (J. repens or A. philoxeroides) in three integration treatments. Data indicate the means 6 SE. doi:10.1371/journal.pone.0097246.g004 Biomass allocation pattern Integration treatments, competition and their interaction significantly affected the biomass allocation of both species in the apical sections (Table 2). Clonal integration significantly increased biomass allocation of A. philoxeroides to the roots and decreased that to the leaves without competition, whereas it decreased biomass allocation to the roots and increased that to the leaves with competition (Table 2, Fig. 4A, C). However, when the basal parts of A. philoxeroides were in nutrient-rich sections, clonal integration increased biomass allocation to the leaves and May 2014 \| Volume 9 \| Issue 5 \| e97246 PLOS ONE \| www.plosone.org 6 An Invasive Plant Benefits from Clonal Integration Effects of clonal integration on biomass allocation pattern Biomass allocations of both plants were significantly influenced by clonal integration which is consistent with previous findings for many other clonal plants [9,32,51]. Without competition, clonal integration increased biomass allocation of A. philoxeroides to roots at the expense of that to leaves, however, when its basal parts were in nutrient-rich sections, clonal integration reversed this trend of May 2014 \| Volume 9 \| Issue 5 \| e97246 PLOS ONE \| www.plosone.org 7 An Invasive Plant Benefits from Clonal Integration Table 2. Summary of MANOVA and univariate ANOVA for effects of integration treatments and competition on biomass allocation of the two clonal plants in the apical sections. Multivariate test statistics A. philoxeroides J. repens Source Wilk’s Lambda F d.f. P Wilk’s Lambda F d.f. P Integration (I) 0.074 14.30 6,32 ,0.001 0.414 2.95 6,32 0.021 Competition (C) 0.302 12.35 3,16 ,0.001 0.472 5.96 3,16 0.006 I6C 0.080 13.50 6,32 ,0.001 0.457 1.61 6,32 0.039 Univariate test statistics A. philoxeroides J. repens Source LMR SMR RMR LMR SMR RMR Integration (I) 50.54* 1.98 47.00* 3.17 1.87 11.23 Competition(C) 0.10 14.17 41.07* 15.89 12.57 0.05 I6C 43.33* 2.45 39.13* 2.38 1.21 9.26** d.f. 5,18 5,18 5,18 5,18 5,18 5,18 Significant P-values are presented in bold. LMR: leaf mass ratio, SMR: stolon mass ratio, RMR: root mass ratio. Values give F; symbols give P: * P,0.05; P,0.01; * P,0.001. doi:10.1371/journal.pone.0097246.t002 Table 2. Summary of MANOVA and univariate ANOVA for effects of integration treatments and competition on biomass allocation of the two clonal plants in the apical sections. Table 2. Summary of MANOVA and univariate ANOVA for effects of integration treatments and competition on biomass allocation of the two clonal plants in the apical sections. Table 2. Summary of MANOVA and univariate ANOVA for effects of integration treatments and competition on biomass allocation of the two clonal plants in the apical sections. Table 2. Summary of MANOVA and univariate ANOVA for effects of integration treatments and competition on biomass allocation of the two clonal plants in the apical sections. Significant P-values are presented in bold. LMR: leaf mass ratio, SMR: stolon mass ratio, RMR: root mass ratio. Values give F; symbols give P: * P,0.05; P,0.01; * P,0.001. doi:10.1371/journal.pone.0097246.t002 efficiently from the basal ramets, so that relatively more biomass could be allocated to roots in order to improve the growth of the whole ramet system in the apical parts [9]. Effects of clonal integration on biomass allocation pattern This might be because under severe competition, allocating more biomass to leaves can help apical ramets to harvest relatively more abundant light (by placing above the canopy of the dense vegetation), whereas poor soil resources (due to dense roots of competitive vegetation) could be compensated by basal ramets through clonal integration [9,53], especially when basal ramets were placed in resourceful conditions. These observations suggest that biomass allocation of invasive plant A. philoxeroides in present study agrees with the theory of labor division theory in clonal plants [32]. That is, young ramets in the apical sections explore locally most abundant resource (light) and receive mineral nutrients and water from older ramets in the basal sections via xylem, while carbohydrates can be imported or produced locally and even exported [46]. In this situation, environmentally induced labor division occurred in the apical ramets, as two essential resources (light and nutrients and/or water) negatively correlated due to competition by neighboring vegetation [54]. Biomass allocation pattern of J. repens was similar to that of A. philoxeroides but with less significance, suggesting that different integration strategies may occur in these two clonal plants when faced with competition. For instance, differences in extent of integration or degree of physiological integration in these two species may exist in heterogeneous environments [45,55]. Indeed, invasive clonal plants with stoloniferous perennial growth are considered to be conferred with the ability of rapidly covering areas via changing biomass allocation through clonal integration [9,11]. However, this needs further in-depth investigation. Effects of clonal integration on neighboring vegetation Interestingly, total biomass of neighboring vegetation of both species was not affected by the presence of apical ramets, suggesting that competition treatments in present study did not suppress growth of plant populations. This is most likely because competition between apical ramets and competitive vegetation was asymmetrical because of low density of apical ramets in this experiment and their biomass was too small to influence the plant community [10]. This observation was supported by the fact that biomass of apical ramets in both plants with competition was sharply decreased to less than 30% as compared with that without competition. Therefore, even though clonal integration greatly improved the growth of plants in the apical sections under competition, their relatively small biomass contributed little to affect the plant community due to relatively short experimental duration (12 weeks). Effects of clonal integration on biomass allocation pattern repens Source Fv/Fm Yield Fv/Fm Yield Integration (I) F2,42 = 62.74* F2,42 = 55.34* F2,42 = 85.56* F2,42 = 75.09* Competition (C) F1,42 = 142.21* F1,42 = 78.63* F1,42 = 262.33* F1,42 = 64.72* I6C F2,42 = 50.24* F2,42 = 2.21 F2,42 = 50.70* F2,42 = 3.04 Values give F; symbols give P: * P,0.05; P,0.01; * P,0.001. doi:10.1371/journal.pone.0097246.t003 Values give F; symbols give P: * P,0.05; P,0.01; * P,0.001. doi:10.1371/journal.pone.0097246.t003 significantly increased plant photosynthetic performance. Previous studies [9,31] also found that clonal integration significantly alleviated the decrease in Fv/Fm of ramets grown in soils with heavy metals or with severe competition by neighboring plants. Moreover, photosynthetic capacity, measured in terms of the effective quantum yield of PS II (Yield), was significantly improved by clonal integration and nutrient addition in both plants. The responses of Yield values were closely correlated with the growth of apical ramets in response to clonal integration under competition, suggesting that clonal integration improved growth of both plants in patchy habitats when competing with competitors mainly by increasing photosynthetic efficiencies [31]. The benefit of clonal integration in terms of physiological traits (photochemical activity determined by Fv/Fm and Yield) supported our hypothesis and reinforced the capacity of division labor in these two plants, as an increase in Fv/Fm (and Yield) and a reduction of biomass allocation to the roots were observed in integration treatments, which could be interpreted as a specialization for aboveground resources. These results suggest that division of labor in stoloniferous clonal plants can happen both at morphological and physiological level [54]. However, no differences were observed in these two plants, indicating that differences in growth and division of biomass between the two species may due to different resource-sharing strategies mediated by clonal integration [45,55]. relationship [31,52], nutrient is not limiting resources and ramets in apical parts can allocate more fraction of biomass to aboveground (leaves and stolons) to rapid spread and occupation of new habitat. With competition, however, clonal integration greatly increased biomass allocation to leaves and decreased that to roots, especially when basal parts of plants were in nutrient-rich sections. Effects of clonal integration on biomass allocation pattern It can be expected that roles of clonal integration may be more important with longer experimental duration. Effects of clonal integration on photosynthetic performance In favourable conditions, the value of Fv/Fm for most plant species ranges from 0.8 to 0.84 [9,11]. Without competition, Fv/ Fm values of ramets of both plants in all clonal treatments were within the normal range of healthy plants and exhibited no significant differences among all the treatments, while growing with competitive vegetation greatly decreased Fv/Fm of both plants to the degree outside the normal range, suggesting that severe competition imposed stress on them. However, the decrease of plants’ Fv/Fm values was markedly alleviated by stolon connec- tions, especially when the donor ramets were in nutrient-rich sections, allowing the ramets to maintain Fv/Fm values within the normal range. Therefore, the results suggest that clonal integration significantly buffered plants against competitive stress and Effects of clonal integration on biomass allocation pattern Additionally, when the donor ramets were in nutrient-rich habitats, due to source-sink biomass allocation. The results occurred most likely because soil resources were relatively more limiting for expansion of the ramets in the apical sections without competition [9]. For the connected apical ramets, the required carbohydrates could be transported biomass allocation. The results occurred most likely because soil resources were relatively more limiting for expansion of the ramets in the apical sections without competition [9]. For the connected apical ramets, the required carbohydrates could be transported Figure 5. Effects of integration treatments and competition on photosynthetic performance of the two clonal plants. The maximum quantum yield of photosystem II (Fv/Fm) and the effective quantum yield of PSII (Yield) of the invasive plant A. philoxeroides (left: A, B) or native plant J. repens (right: C, D) in the apical sections, grown either with or without competitive vegetation (J. repens or A. philoxeroides) in three integration treatments. Data indicate the means 6 SE. doi:10.1371/journal.pone.0097246.g005 Figure 5. Effects of integration treatments and competition on photosynthetic performance of the two clonal plants. The maximum quantum yield of photosystem II (Fv/Fm) and the effective quantum yield of PSII (Yield) of the invasive plant A. philoxeroides (left: A, B) or native plant J. repens (right: C, D) in the apical sections, grown either with or without competitive vegetation (J. repens or A. philoxeroides) in three integration treatments. Data indicate the means 6 SE. doi:10.1371/journal.pone.0097246.g005 May 2014 \| Volume 9 \| Issue 5 \| e97246 PLOS ONE \| www.plosone.org 8 An Invasive Plant Benefits from Clonal Integration Table 3. Two-way ANOVA for effects of integration treatments and competition on the maximum quantum yield of photosystem II (Fv/Fm) and the effective quantum yield of PSII (Yield) of the two clonal plants in the apical sections. Table 3. Two-way ANOVA for effects of integration treatments and competition on the maximum quantum yield of photosystem II (Fv/Fm) and the effective quantum yield of PSII (Yield) of the two clonal plants in the apical sections. Table 3. Two-way ANOVA for effects of integration treatments and competition on the maximum quantum yield of photosystem II (Fv/Fm) and the effective quantum yield of PSII (Yield) of the two clonal plants in the apical sections. A. philoxeroides J. References Oikos 63: 410–419. 8. Moola FM, Vasseur L (2009) The importance of clonal growth to the recovery of Gaultheria procumbens L. (Ericaceae) after forest disturbance. Plant Ecol 201: 319– 337. 31. Roiloa SR, Retuerto R (2006) Small-scale heterogeneity in soil quality influences photosynthetic efficiency and habitat selection in a clonal plant. Ann Bot 98: 1043–1052. 9. Wang N, Yu FH, Li PX, He WH, Liu FH, et al. (2008) Clonal integration affects growth, photosynthetic efficiency and biomass allocation, but not the competitive ability, of the alien invasive Alternanthera philoxeroides under severe stress. Ann Bot 101: 671–678. 32. Hutchings MJ, Wijesinghe DK (1997) Patchy habitats, division of labour and growth dividends in clonal plants. Trends Ecol Evol 12: 390–394. 10. Xiao KY, Yu D, Wang LG, Han YQ (2011) Physiological integration helps a clonal macrophyte spread into competitive environments and coexist with other species. Aquat Bot 95: 249–253. 33. Ikegami M, Whigham DF, Werger MJA (2008) Optimal biomass allocation in heterogeneous environments in a clonal plant-spatial division of labor. Ecol Model 213: 156–164. 11. You WH, Yu D, Liu CH, Xie D, Xiong W (2013) Clonal integration facilitates invasiveness of the alien aquatic plant Myriophyllum aquaticum L. under heterogeneous water availability. Hydrobiologia 718: 27–39. 34. Schreiber U, Bilger W, Hormann H, Neubauer C (1998) Chlorophyll fluorescence as a diagnostic tool: basics and some aspects of practical relevance. In: Raghavendra AS (editor), Photosynthesis: a comprehensive treatise. Cambridge University Press, Cambridge, pp 320–336. 12. Herben T (2004) Physiological integration affects growth form and competitive ability in clonal plants. Evol Ecol 18: 493–520. Cambridge University Press, Cambridge, pp 320–336. 35. Bjo¨rkman O, Demmig B (1987) Photon yield of O2 evolution and chlorophyll fluorescence characteristics at 77 K among vascular plants of diverse origins. Planta 170: 489–504. 13. Wilsey B (2002) Clonal plants in a spatially heterogeneous environment: effects of integration on Serengeti grassland response to defoliation and urine-hits from grazing mammals. Plant Ecol 159: 15–22. 36. Julien MH, Skarratt B, Maywald GF (1995) Potential geographical distribution of alligator weed and its biological control by Agasicles hygrophila. J Aquat Plant Manage 33: 55–60. 14. Brˇezina S, Koubek T, Munzbergova Z, Herben T (2006) Ecological benefits of integration of Calamagrostis epigejos ramets under field conditions. Flora 201: 461– 467. 37. Gunasekera L, Bonila J (2001) Alligator weed: tasty vegetable in Australian backyards? J Aquat Plant Manage 39: 17–20. 15. References 22. Yu F, Wang N, Alpert P, He W, Dong M (2009) Physiological integration in an introduced, invasive plant increases its spread into experimental communities and modifies their structure. Am J Bot 96: 1983–1989. 1. Alpert P, Mooney HA (1986) Resource sharing among ramets in the clonal herb, Fragaria chiloensis. Oecologia 70: 227–233. 2. Stuefer JF, Kroon HD, During HJ (1996) Exploitation of environmental heterogeneity by spatial division of labor in a clonal plant. Funct Ecol 10: 328– 334. J 23. Wang N (2010) Clonal integration increased the competitive ability of invasive plant Alternanthera philoxeroides to Plantago virginica. Ecol Environ Sci 19: 2302– 2306. 3. Alpert P, Holzapfel C, Slonimski C (2003) Differences in performance between genotypes of Fragaria chiloensis with different degrees of resource sharing. J Ecol 91: 27–35. 24. Roiloa SR, Rodrı´guez-Echeverrı´a S, de la Pen˜a E, Freitas H (2010) Physiological integration increases the survival and growth of the clonal invader Carpobrotus edulis. Biological Invasions 12: 1815–1823. 4. Maurer DA, Zedler JB (2002) Differential invasion of a wetland grass explained by tests of nutrients and light availability on establishment and clonal growth. Oecologia 131: 279–288. 25. Price EAC, Hutchings MJ (1996) The effects of competition on growth and form in Glechoma hederacea. Oikos 75: 279–290. 26. Hartnett DC, Bazzaz FA (1985) The integration of neighbourhood effects by clonal genets of Solidago canadensis. J Ecol 73: 415–428. 5. Chidumayo EN (2006) Fitness implications of clonal integration and leaf dynamics in a stoloniferous herb, Nelsonia canescens (Lam.) Spreng (Nelsoniaceae). Evol Ecol 20: 59–73. 27. Pennings SC, Callaway RM (2000) The advantages of clonal integration under different ecological conditions: a community-wide test. Ecology 81: 709–716. 6. Roiloa SR, Retuerto R (2007) Responses of the clonal Fragaria vesca to microtopographic heterogeneity under different water and light conditions. Environ Exp Bot 61: 1–9. 28. Otfinowski R, Kenkel NC (2008) Clonal integration facilitates the proliferation of smooth brome clones invading northern fescue prairies. Plant Ecol 199: 235– 242. 7. Yu FH, Wang N, He WM, Chu Y, Dong M (2008) Adaptation of rhizome connections in drylands: increasing tolerance of clones to wind erosion. Ann Bot 102: 571–577. 29. Schmid B, Bazzaz F (1987) Clonal integration and population structure in perennials: effects of severing rhizome connections. Ecology 68: 2016–2022. 30. de Kroon H, Hara T, Kwant R (1992) Size hierarchies of shoots and clones in clonal herb monocultures: do clonal and non-clonal plants compete differently? Acknowledgments We thank Dr. KY Xiao for critical comments on an early version of the manuscript, Dr. LF Yu for help with the data analysis, and CM Han, DY Ma, J Chen, and YQ Han for assistance with plant harvest. Conclusions Overall, when competing with neighboring vegetation, clonal integration greatly improved growth and photosynthetic perfor- mance of both species when the connected basal ramets were in May 2014 \| Volume 9 \| Issue 5 \| e97246 PLOS ONE \| www.plosone.org 9 An Invasive Plant Benefits from Clonal Integration nutrient-rich habitats, suggesting that clonal integration is important for both species in nutrient-patchy and competitive environments. However, ramets of the invasive A. philoxeroides were more sophisticated and independent than the co-occurring native J. repens when faced with competition. Moreover, under compet- itive environments, changes in biomass allocation of A. philoxeroides through clonal integration was more significant than that of J. repens, although biomass allocation of both species well conformed to the theory of labor division, suggesting that different integration strategies may occur in these two clonal plants. These observations supported our hypothesis that invasive A. philoxeroides may benefit from clonal integration more than co-occurring native J. repens, indicating that invasiveness of A. philoxeroides may be closely related to clonal integration in heterogeneous environments. However, future comparative research is needed on additional species pairs in order to assemble conclusive evidence on the importance of integration for invasive and native species. in order to assemble conclusive evidence on the importance of integration for invasive and native species. Author Contributions Conceived and designed the experiments: WHY DY SFF CHL. Performed the experiments: WHY. Analyzed the data: WHY DX. Contributed reagents/materials/analysis tools: WHY DY. Wrote the paper: WHY DY CHL. 45. Jo´nsdo´ttir IS, Watson MA (1997) Extensive physiological integration: An adaptive trait in resource-poor environments? In - de Kroon H.; van Groenendael J (editors). The ecology and evolution of clonal plants, p. 109–136. References Mack RN, Simberloff D, Lonsdale WM, Evans H, Clout M, et al. (2000) Biotic invasions: causes, epidemiology, global consequences, and control. Ecol Appl 10: 689–710. 38. Ma R, Wang R (2005) Invasive mechanism and biological control of alligator weed, Alternanthera philoxeroides (Amaranthaceae), in China. Chin J Appl Environ Biol 11: 246–250. 16. Yurkonis KA, Meiners SJ, Wachholder BE (2005) Invasion impacts diversity through altered community dynamics. J Ecol 93: 1053–1061. 39. Li M, Zhang LJ, Tao L, Li W (2008) Ecophysiological responses of Jussiaea repens to cadmium exposure. Aquat Bot 88: 347–352. 17. Kolar CS, Lodge DM (2001) Progress in invasion biology: predicting invaders. Trends Ecol Evol 16: 199–204. 40. Sosnova´ M, van Diggelen R, Macek P, Klimesova J (2011) Dist 40. Sosnova´ M, van Diggelen R, Macek P, Klimesova J (2011) Distribution of clonal growth traits among wetland habitats. Aquat Bot 95: 88–93. 18. Liu J, Dong M, Miao S, Li Z, Song M, et al. (2006) Invasive alien plants in China: role of clonality and geographical origin. Biol Invasions 8: 1461–1470. growth traits among wetland habitats. Aquat Bot 95: 88–93 41. Wang B, Li W, Wang J (2005) Genetic diversity of Alternanthera philoxeroides in China. Aquat Bot 81: 277–283. 19. Song YB, Yu FH, Keser LH, Dawson W, Fischer M, et al. (2013) United we stand, divided we fall: a meta-analysis of experiments on clonal integration and its relationship to invasiveness. Oecologia 171: 317–327. 42. Kikvidze Z, Khetsuriani L, Kikodze D, Callaway RM (2006) Seasonal shifts in competition and facilitation in subalpine plant communities of the central Caucasus. J Veg Sci 17: 77–82. 20. Roiloa SR, Susana RE, Helena F (2013) Effect of physiological integration in self/non-self genotype recognition on the clonal invader Carpobrotus edulis. J Plant Ecol doi: 10.1093/jpe/rtt045. 43. Hartnett DC, Bazzaz FA (1983) Physiological integration among intraclonal ramets in Solidago canadensis. Ecology 64: 779–788. 21. Peltzer DA (2002) Does clonal integration improve competitive ability? A test using aspen (Populus tremuloides [Salicaceae]) invasion into prairie. Am J Bot 89: 494–499. 44. Yu F, Chen Y, Dong M (2002) Clonal integration enhances survival and performance of Potentilla anserina, suffering from partial sand burial on Ordos plateau, China. Evol Ecol 15: 303–318. PLOS ONE \| www.plosone.org May 2014 \| Volume 9 \| Issue 5 \| e97246 10 An Invasive Plant Benefits from Clonal Integration 46. D’Hertefeldt T, Jo´nsdo´ttir IS (1999) Extensive physiological integration in intact clonal systems of Carex arenaria. J Ecol 87: 258–264. y J 47. Hutchings MJ, de Kroon H (1994) Foraging in plants: the role of morphological plasticity in resources acquisition. Adv Ecol Res 25: 159–238. p y q 48. Day KJ, John EA, Hutchings MJ (2003) The effects of spatially heterogeneous nutrient supply on yield, intensity of competition and root placement patterns in Briza media and Festuca ovina. Funct Ecol 17: 454–463. 53. Nilsson J, D’Hertefeldt T (2008) Origin matters for level of resource sharing in the clonal herb Aegopodium podagraria. Evol Ecol 22: 437–448. 50. Watson MA (1986) Integrated physiological units in plants. Trends Ecol Evol 1: 119–123. 55. D’Hertefeldt T, Falkengren-Grerup U (2002) Extensive physiological integration in Carex arenaria and Carex disticha in relation to potassium and water availability. New Phytol 156: 469–477. 49. Birch CPD, Hutchings MJ (1994) Exploitation of patchily distributed soil resources by the clonal herb glechoma-hederacea. J Ecol 82 (3): 653–664. An Invasive Plant Benefits from Clonal Integration 45. Jo´nsdo´ttir IS, Watson MA (1997) Extensive physiological integration: An adaptive trait in resource-poor environments? In - de Kroon H.; van Groenendael J (editors). The ecology and evolution of clonal plants, p. 109–136. 51. Roiloa SR, Alpert P, Tharayil N, Hancock G, Bhowmik PC (2007) Greater capacity for division of labour in clones of Fragaria chiloensis from patchier habitats. J Ecol 95: 397–405. J ( ) gy p p 46. D’Hertefeldt T, Jo´nsdo´ttir IS (1999) Extensive physiological integration in intact clonal systems of Carex arenaria. J Ecol 87: 258–264. 52. de Kroon H, Kreulen R, van Rheenen JWA, van Dijk A (1998). The interaction between water and nitrogen translocation in a rhizomatous sedge (Carex flacca). Oecologia 116: 38–49. y J 47. Hutchings MJ, de Kroon H (1994) Foraging in plants: the role of morphological plasticity in resources acquisition. Adv Ecol Res 25: 159–238. 53. Nilsson J, D’Hertefeldt T (2008) Origin matters for level of resource sharing in the clonal herb Aegopodium podagraria. Evol Ecol 22: 437–448. 48. Day KJ, John EA, Hutchings MJ (2003) The effects of spatially heterogeneous nutrient supply on yield, intensity of competition and root placement patterns in Briza media and Festuca ovina. Funct Ecol 17: 454–463. 54. Roiloa SR, Rodrı´guez-Echeverrı´a S, Freitas H, Retuerto R (2013) Develop- mentally-programmed division of labour in the clonal invader Carpobrotus edulis. Biol Invasions 15: 1859–1905. 49. Birch CPD, Hutchings MJ (1994) Exploitation of patchily distributed soil resources by the clonal herb glechoma-hederacea. J Ecol 82 (3): 653–664. 55. D’Hertefeldt T, Falkengren-Grerup U (2002) Extensive physiological integration in Carex arenaria and Carex disticha in relation to potassium and water availability. New Phytol 156: 469–477. 50. Watson MA (1986) Integrated physiological units in plants. Trends Ecol Evol 1: 119–123. PLOS ONE \| www.plosone.org May 2014 \| Volume 9 \| Issue 5 \| e97246 PLOS ONE \| www.plosone.org 11
https://openalex.org/W2133420977	https://europepmc.org/articles/pmc4016556?pdf=render	English	null	Is there a role for high dose chemotherapy and blood stem cell rescue in childhood hepatoblastoma presenting with lung metastases? A case report and literature review	The Italian Journal of Pediatrics/Italian journal of pediatrics	2,013	cc-by	3,738	CASE REPORT Open Access Is there a role for high dose chemotherapy and blood stem cell rescue in childhood hepatoblastoma presenting with lung metastases? A case report and literature review Massimo Provenzi1, Francesco Saettini2, Valentino Conter2, Eugenia Giraldi1, Carlo Foglia1, Laura Cavalleri1, Michele Colledan3, Lorenzo D’Antiga1, Giorgio Perilongo4 and Liviana Da Dalt4 © 2013 Provenzi et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract We report the use of high dose chemotherapy with peripheral blood stem cell rescue as a consolidation treatment for a 3-year-old child affected by metastatic hepatoblastoma, who achieved complete lung response only after conventional treatment. The patient is presently alive 27 months after high dose chemotherapy with blood stem cell rescue with no evidence of disease. The role of high dose chemotherapy with blood stem cell rescue to consolidate the complete clearing of lung disease in metastatic hepatoblastoma remains controversial; the data available in the literature and our experience seems to suggest to keep this treatment option open to further consideration in the clinical setting of high-risk patients. Correspondence: francescosaettini@yahoo.it 2Department of Pediatrics, University of Milano-Bicocca, San Gerardo Hospital, Via Pergolesi 33, 20900 Monza, Italy Full list of author information is available at the end of the article Background this subject and our personal observation may serve to provide a more comprehensive understanding of the role of this therapeutic approach in children with HB and high-risk features of treatment failure, notably me- tastases, and potentially to support further stringent clinical investigation. Hepatoblastoma (HB) is the most common malignant liver tumor in children. Current multidrug chemotherapy regimens and surgery allow to obtain event free survival (EFS) and overall survival (OS) rates of approximately 70-80% [1-4]. However, the outcome of children with metastatic tumor remains unsatisfactory, with EFS and OS of 25-38% and 27-62% range respectively [2-11]. Intensification of con- ventional chemotherapy, notably with cisplatin (CDDP), and the use of innovative combinations of drugs in a “win- dow setting”, and more precisely, irinotecan and vincristine, are the two strategies adopted respectively by the European and North American cooperative study groups on HB to improve the present treatment outcome on these latter pa- tients [2,12]. Provenzi et al. Italian Journal of Pediatrics 2013, 39:65 http://www.ijponline.net/content/39/1/65 Provenzi et al. Italian Journal of Pediatrics 2013, 39:65 http://www.ijponline.net/content/39/1/65 ITALIAN JOURNAL OF PEDIATRICS Summary of the literature The use of HDCT with BSC rescue has been used in a number of patients with HB at the time of tumor recur- rence [10,14-18], but in only 22 of them, including the one here reported, as a first line treatment in patients with metastatic disease [14,15,19,20]. These 22 patients represent the focus of this report. In 2 of them a double autologous BSC rescue instead of conventional post- operative chemotherapy was performed; one of these underwent BSC rescue with persisting lung involve- ment. Both patients relapsed, but interestingly one of them was then rescued with irinotecan alone [14]. Four- teen patients did not achieve PR of the target lesions after conventional pre-operative chemotherapy (POC) and without further specifications regarding the tumor status at the time of HDCT all were then treated with HDCT with BSC rescue. Of these patients 2 died of toxicity of the procedure and only 4 were reported alive with no evidence of disease [20]. Fearing that the late response of the metastases (i.e. at least a partial response to preoperative chemotherapy and complete remission of pulmonary metastases only after postoperative chemotherapy, regardless AFP levels) could be a dismal prognostic factor it was elected, with parental agreement, to complete therapy with a course of HDCT and peripheral BSC rescue. Autologous BSC were harvested in 2 days (total 1.3 × 106 and 3 × 106 CD34+ cells/kg), after G-CSF stimulation. As used in our center for other metastatic solid tumors, the conditio- ning regimen consisted of CARBO 500 mg/sqm/die from day −5 to day −3; etoposide 300 mg/sqm/die from day −5 to day −3; melphalan 120 mg/sqm/die on day −2. On day 0 and day +1 BSC (3 × 106 and 1.3 × 106 CD34+ cells/kg) were infused. From day +4 to day +14 G-CSF was given; neutrophil count was over 500/mm3 since day +12 while platelet engraftment (>20000/mcl, transfusion independ- ent) was documented from day +13. Toxicity was modest and represented by mucositis and fever of unknown The last subgroup of children of whom we have detailed information encompasses 6 patients who achieved at least a PR after POC, and after conventional partial hepatec- tomy and post-operative chemotherapy underwent HDCT with BSC rescue, while in complete remission (CR) of lung metastases (see Table 1). Case presentation Presently the patient remains disease-free, with normal AFP levels and no radiological evidence of disease, 27 months following HDCT with peripheral BSC rescue. Case presentation A three-year-old boy was referred to our center because of an asymptomatic abdominal mass. At the time of diagnosis, the alpha-feto protein (AFP) serum level was 192,269 ng/mL (normal value < 10 ng/mL) and the platelet count 486,000/mcl. The abdominal computed tomography (CT) showed an intrinsic hepatic tumor mass involving extensively the right hepatic lobe (segment VI, VII and VIII). The mass was classified as PRETEXT II (tumor volume: 13×12×10 cm) [13]. The surgical biopsy was in favor of an epithelial HB. The chest CT scan showed bilateral widespread metastatic lung disease. After obtaining informed consent, the patient was treated according to SIOPEL 4 protocol with CDDP 70 mg/sqm as a 24-hour continuous infusion (CI) on day 1, 7, and 14 (first dose of the first block CDDP at 80 mg/sqm as a In the literature the use of high dose chemotherapy (HDCT) with blood stem cell (BSC) rescue has been reported in children affected by metastatic HB in 21 cases. This report aiming to review the pertinent literature on Page 2 of 4 Provenzi et al. Italian Journal of Pediatrics 2013, 39:65 http://www.ijponline.net/content/39/1/65 24-hour CI) and doxorubicin (DOXO) 30 mg/sqm as a 48-hour CI on day 8 and 9; this block had to be repeated three times (omitting last CDDP) at one week interval. The abdominal CT scan performed thereafter exhibited a reduction of the extension of the hepatic mass (7.5×5.1×4.5 cm), now involving only segment VII and VIII, while the chest CT scan showed only a partial response (PR) of the lung lesions, which remained bilateral and widespread. The AFP level decreased to 588 ng/ml. The surgical resection of the lung lesions was not performed because of the multifocality and the bilaterality of the disease. The patient underwent right hepatectomy and the histology was refined as a fetal HB with a rich macrotrabecular component (80% of the mass). The micro- scopic margins were clear. The AFP value further declined to 44.3 ng/ml 13 days after surgery. Post-surgery chemo- therapy consisted of carboplatin (CARBO) 500 mg/sqm on day 1, 22 and 43, and DOXO 20 mg/sqm as a 24-hour CI on day 1, 2, 22, 23, 43, and 44. At the end of the treatment the AFP returned to normal (7.7 ng/ml) and the chest CT scan revealed no signs of residual lung disease. origin. Conclusions The experiences here reported may suggest thus that HDCT with autologous BSC rescue can be of benefit for patients with metastatic HB who achieved at least a PR after POC, as a consolidation of a CR of lung metastases otherwise achieved at the end of treatment. Moreover HDCT and BSC rescue can be feasible in patients who have already received intensive conventional chemothe- rapy but the underlying possible severe toxicity of this modality should be clearly kept in mind and parents should be closely involved in the decision process. Of course nobody knows if in these children the previous treatment, which allowed them to achieve the complete tumor response, would have been enough for the cure. However, considering that late response to chemothe- rapy is indeed a matter of concern in all tumor types, to have presented the series herewith reported could serve to keep the treatment option of using HDCT with BSC rescue of children affected by metastatic HB open to further clinical investigation. 2. Zsiros J, Maibach R, Shafford E, Brugieres L, Brock P, Czauderna P, Roebuck D, Childs M, Zimmermann A, Laithier V, Otte JB, de Camargo B, MacKinlay G, Scopinaro M, Aronson D, Plaschkes J, Perilongo G: Successful treatment of childhood high-risk hepatoblastoma with dose-intensive multiagent chemotherapy and surgery: final results of the SIOPEL 3-HR study. J Clin Oncol 2010, 28:2584–2590. 3. Trobaugh-Lotrario AD, Katzenstein HM: Chemotherapeutic approaches for newly diagnosed hepatoblastoma: past, present, and future strategies. Pediatr Blood Cancer 2012, 59:809–812. 4. Malogolowkin MH, Katzenstein HM, Krailo M, Meyers RL: Treatment of hepatoblastoma: the North American cooperative group experience. Front Biosci 2012, 4:1717–1723. 5. Maibach R, Roebuck D, Brugieres L, Capra M, Brock P, Dall’Igna P, Otte JB, De Camargo B, Zsiros J, Zimmermann A, Aronson D, Childs M, Scopinaro M, Morland B, Plaschkes J, Czauderna P, Perilongo G, et al: Prognostic stratification for children with hepatoblastoma: the SIOPEL experience. Eur J Cancer 2012, 48:1543–1549. 5. Maibach R, Roebuck D, Brugieres L, Capra M, Brock P, Dall’Igna P, Otte JB, De Camargo B, Zsiros J, Zimmermann A, Aronson D, Childs M, Scopinaro M, Morland B, Plaschkes J, Czauderna P, Perilongo G, et al: Prognostic stratification for children with hepatoblastoma: the SIOPEL experience. Eur J Cancer 2012, 48:1543–1549. 6. References 1. Perilongo G, Maibach R, Shafford E, Brugieres L, Brock P, Morland B, de Camargo B, Zsiros J, Roebuck D, Zimmermann A, Aronson D, Childs M, Widing E, Laithier V, Plaschkes J, Pritchard J, Scopinaro M, MacKinlay G, Czauderna P: Cisplatin versus cisplatin plus doxorubicin for standard risk hepatoblastoma. N Engl J Med 2009, 361:1662–1670. 1. Perilongo G, Maibach R, Shafford E, Brugieres L, Brock P, Morland B, de Camargo B, Zsiros J, Roebuck D, Zimmermann A, Aronson D, Childs M, Widing E, Laithier V, Plaschkes J, Pritchard J, Scopinaro M, MacKinlay G, Czauderna P: Cisplatin versus cisplatin plus doxorubicin for standard risk hepatoblastoma. N Engl J Med 2009, 361:1662–1670. Competing interests 10. Katzenstein HM, London WB, Douglass EC, Reynolds M, Plaschkes J, Finegold MJ, Bowman LC: Treatment of unresectable and metastatic hepatoblastoma: a pediatric oncology group phase II study. J Clin Oncol 2002, 20:3438–3444. g The authors declare they have no competing interests. Provenzi et al. Italian Journal of Pediatrics 2013, 39:65 http://www.ijponline.net/content/39/1/65 Provenzi et al. Italian Journal of Pediatrics 2013, 39:65 http://www.ijponline.net/content/39/1/65 responder to conventional chemotherapy and, as described, is alive with no evidence of disease 27 months after BSC rescue. Thus as overall five of these 6 patients had been reported to be alive with no evidence of disease with a minimum follow-up of 16 months after BSC rescue. Author details 1 Author details 1Department of Pediatrics, Ospedale Papa Giovanni XXIII, Bergamo, Italy. 2Department of Pediatrics, University of Milano-Bicocca, San Gerardo Hospital, Via Pergolesi 33, 20900 Monza, Italy. 3Department of Surgery, Ospedale Papa Giovanni XXIII, Bergamo, Italy. 4Department of Woman’s and Child’s Health, University Hospital of Padua, Padua, Italy. Author details 1Department of Pediatrics, Ospedale Papa Giovanni XXIII, Bergamo, Italy. 2Department of Pediatrics, University of Milano-Bicocca, San Gerardo Hospital, Via Pergolesi 33, 20900 Monza, Italy. 3Department of Surgery, Ospedale Papa Giovanni XXIII, Bergamo, Italy. 4Department of Woman’s and Child’s Health, University Hospital of Padua, Padua, Italy. This small series of twenty-two patients cannot provide a comprehensive view of the role of HDCT with BSC res- cue in the treatment of HB. However, the fact that 5 out of the 6 children achieved at least a PR after conventional POC and received HDCT with autologous BSC rescue while in CR of lung metastases at the end of conventional treatment seems to indicate a possible role of this treat- ment modality in the management of these patients. Received: 9 August 2013 Accepted: 18 October 2013 Published: 22 October 2013 Conclusions Pritchard J, Brown J, Shafford E, Perilongo G, Brock P, Dicks-Mireaux C, Keeling J, Phillips A, Vos A, Plaschkes J, et al: Cisplatin, doxorubicin, and delayed surgery for childhood hepatoblastoma: a successful approach – results of the first prospective study of the international society of pediatric oncology. J Clin Oncol 2000, 18:3819–3828. 6. Pritchard J, Brown J, Shafford E, Perilongo G, Brock P, Dicks-Mireaux C, Keeling J, Phillips A, Vos A, Plaschkes J, et al: Cisplatin, doxorubicin, and delayed surgery for childhood hepatoblastoma: a successful approach – results of the first prospective study of the international society of pediatric oncology. J Clin Oncol 2000, 18:3819–3828. 7. Perilongo G, Shafford E, Maibach R, Aronson D, Brugières L, Brock P, Childs M, Czauderna P, MacKinlay G, Otte JB, Pritchard J, Rondelli R, Scopinaro M, Staalman C, Plaschkes J, International Society of Paediatric Oncology-SIOPEL 2: Risk-adapted treatment for childhood hepatoblastoma: Final report of the second study for the International Society of Pediatric Oncology – SIOPEL 2. Eur J Cancer 2004, 40:411–421. Abbreviations f Abbreviations AFP: Alpha-feto protein; BSC: Blood stem cell; CARBO: Carboplatin; CDDP: Cisplatin; CI: Continuous infusion; CT: Computed tomography; CR: Complete remission; DOXO: Doxorubicin; EFS: Event free survival; HB: Hepatoblastoma; HDCT: High dose chemotherapy; OS: Overall survival; POC: Pre-operative chemtherapy; PR: Partial response. 9. Fuchs J, Rydzynski J, Von Schweinitz D, Bode U, Hecker H, Weinel P, Bürger D, Harms D, Erttmann R, Oldhafer K, Mildenberger H, Study Committee of the Cooperative Pediatric Liver Tumor Study Hb94 for the German Society for Pediatric Oncology and Hematology: Pretreatment prognostic factors and treatment results in children with hepatoblastoma: a report from the German cooperative pediatric liver tumor study HB 94. Cancer 2002, 95:172–182. 9. Fuchs J, Rydzynski J, Von Schweinitz D, Bode U, Hecker H, Weinel P, Bürger D, Harms D, Erttmann R, Oldhafer K, Mildenberger H, Study Committee of the Cooperative Pediatric Liver Tumor Study Hb94 for the German Society for Pediatric Oncology and Hematology: Pretreatment prognostic factors and treatment results in children with hepatoblastoma: a report from the German cooperative pediatric liver tumor study HB 94. Cancer 2002, 95:172–182. Consent Written informed consent was obtained from the par- ents of the patients for publication of this Case report. A copy of the written consent is available for review by the Editor-in-Chief of this journal. 8. 8. Ortega JA, Douglass EC, Feusner JH, Feusner JH, Reynolds M, Quinn JJ, Finegold MJ, Haas JE, King DR, Liu-Mares W, Sensel MG, Krailo MD: Randomized comparison of cisplatin/vincristine/fluorouracil and cisplatin/continuous infusion doxorubicin for treatment of pediatric hepatoblastoma: a report from the Children’s cancer group and the pediatric oncology group. J Clin Oncol 2000, 18:2665–2675. Acknowledgements h h d b The authors are indebted with Dr. Rupert Handgretinger for sharing with us the choice of the treatment strategy in this patient. Summary of the literature In these group of children four of them obtained complete clearance of metastases with POC but presented with persisting high serum levels of AFP after post-operative chemotherapy; three of these four patients at the time of the report were alive and disease-free 16 months after BSC rescue, with normal AFP levels, while one displayed continuously increasing AFP and ultimately died of brain metastases [19]. One case pre- senting with multiple metastatic disease that completely responded to POC is reported alive and disease-free six years from stopping therapy [15]. Our patient was a late Table 1 High dose chemotherapy with blood stem cell rescue following post-operative chemotherapy for metastatic HB patients achieving partial response after pre-operative chemotherapy Reference and number of patients Age/Sex PRETEXT Previous regimen Tumor status at the time of HDCT Conditioning regimen BSC source Outcome Matsunaga T [19]: 4 patients Unknown Any PRETEXT M+ CDDP, THP- ADR AFP↑ CARBO, VP16, MLP PB 3 NED 16 mts after BSC rescue 1 DoD Nishimura SI [15]: 1 patient 10 yr/M IV M+ CDDP, DOXO NED DOXO, VP16, CARBO, 5-FU BM NED 6 yr after BSC rescue Provenzi M 2013: 1 patient 3 yr/M II M+ CDDP, DOXO NED CARBO, VP16, MLP PB NED 27 mts after BSC rescue Abbreviations: BSC blood stem cell, M + lung metastases, CDDP cisplatin, THP-ADR THP-adriamicin, AFP alpha-feto protein, CARBO carboplatin, VP16 etoposide, MLP melphalan, PB peripheral blood, NED no evidence of disease, mts months, DoD died of disease, yr years, DOXO doxorubicin, 5-FU 5 fluorouracil, BM bone marrow. Table 1 High dose chemotherapy with blood stem cell rescue following post-operative chemother HB patients achieving partial response after pre-operative chemotherapy Abbreviations: BSC blood stem cell, M + lung metastases, CDDP cisplatin, THP-ADR THP-adriamicin, AFP alpha-feto protein, CARBO carboplatin, VP16 etoposide, MLP melphalan, PB peripheral blood, NED no evidence of disease, mts months, DoD died of disease, yr years, DOXO doxorubicin, 5-FU 5 fluorouracil, BM bone marrow. Page 3 of 4 Page 3 of 4 10. Katzenstein HM, London WB, Douglass EC, Reynolds M, Plaschkes J, Finegold MJ, Bowman LC: Treatment of unresectable and metastatic hepatoblastoma: a pediatric oncology group phase II study. J Clin Oncol 2002, 20:3438–3444. 12. Bomgaars LR, Bernstein M, Krailo M, Kadota R, Das S, Chen Z, Adamson PC, Blaney SM: Phase II trial of irinotecan in children with refractory solid Authors’ contributions 11. Haberle B, Bode U, von Schweinitz D: Differentiated treatment protocols for high- and standard-risk hepatoblastoma: An interim report of the German Liver Tumor Study HB99 [German]. Klin Padiatr 2003, 215:159–165. 11. Haberle B, Bode U, von Schweinitz D: Differentiated treatment protocols for high- and standard-risk hepatoblastoma: An interim report of the German Liver Tumor Study HB99 [German]. Klin Padiatr 2003, 215:159–165. MP, FS, VC and GP conceived the study, drafted the manuscript and evaluated all the children. EG, CF, CF and MC contributed to data acquisition and drafting of the article. LDA and LDD contributed to critical revision of the article and to final approval of the version to be published. All authors read and approved the final manuscript. 12. Bomgaars LR, Bernstein M, Krailo M, Kadota R, Das S, Chen Z, Adamson PC, Blaney SM: Phase II trial of irinotecan in children with refractory solid 12. Bomgaars LR, Bernstein M, Krailo M, Kadota R, Das S, Chen Z, Adamson PC, Blaney SM: Phase II trial of irinotecan in children with refractory solid Page 4 of 4 Page 4 of 4 tumors: a Children’s oncology group study. J Clin Oncol 2007, 25:4622–4627. 13. Aronson DC, Schnater JM, Staalman CR, Weverling GJ, Plaschkes J, Perilongo G, Brown J, Phillips A, Otte JB, Czauderna P, MacKinlay G, Vos A: Predictive value of the pretreatment extent of disease system in hepatoblastoma: results from the international society of pediatric oncology liver tumor study group SIOPEL-1 study. J Clin Oncol 2005, 23:1245–1252. 14. Katzenstein HM, Rigsby C, Shaw PH, Mitchell TL, Haut PR, Kletzel M: Novel therapeutic approaches in the treatment ofChildren with hepatoblastoma. J Pediatr Hematol Oncol 2002, 24:751–755. p 15. Nishimura S, Sato T, Fujita N, Hiyama E, Yokoyama T, Ueda K: High-dose chemotherapy in children with metastatic hepatoblastoma. Pediatr Int 2002, 44:300–305. 16. Hara J, Osugi Y, Ohta H, Matsuda Y, Nakanishi K, Takai K, Fujisaki H, Tokimasa S, Fukuzawa M, Okada A, Okada S: Double-conditioning regimens consisting of thiotepa, melphalan and busulfan with stem cell rescue for the treatment of pediatric solid tumors. Bone Marrow Transplant 1998, 22:7–12. 17. Niwa A, Umeda K, Awaya T, Matsubara H, Hiramatsu H, Watanabe K, Adachi S, Itoh T, Uemoto S, Nakahata T: Successful autologous peripheral blood stem cell transplantation with a double-conditioning regimen for recurrent hepatoblastoma after liver transplantation. Pediatr Transplantation 2009, 13:259–262. 18. Yoshinari M, Imaizumi M, Hayashi Y, Sato A, Saito T, Suzuki H, Saisho T, Abukawa D, Ogawa E, Aikawa J, Goto K, Satoh T, Ohi R, Linuma K: Peripheral blood stem cell transplantation for hepatoblastoma with microscopic residue: A therapeutic approach for incompletely resected tumor. Tohuku J Exp Med 1998, 184:247–254. 19. Matsunaga T, Sasaki F, Ohira M, Hashizume K, Hayashi A, Hayashi Y, Mugishima H, Ohnuma N, Japanese Study Group for Pediatric Liver Tumor: Analysis of treatment outcome for children with recurrent or metastatic hepatoblastoma. Pediatr Surg Int 2003, 19:142–146. 19. Matsunaga T, Sasaki F, Ohira M, Hashizume K, Hayashi A, Hayashi Y, Mugishima H, Ohnuma N, Japanese Study Group for Pediatric Liver Tumor: Analysis of treatment outcome for children with recurrent or metastatic hepatoblastoma. Pediatr Surg Int 2003, 19:142–146. 20. Hishiki T, Matsunaga T, Sasaki F, Yano M, Ida K, Horie H, Kondo S, Watanabe K, Oue T, Tajiri T, Kamimatsuse A, Ohnuma N, Hiyama E: Outcome of hepatoblastomas treated using the Japanese Study Group for Pediatric Liver Tumor (JPLT) protocol-2: report from the JPLT. Pediatr Surg Int 2011, 27:1–8. 20. Provenzi et al. Italian Journal of Pediatrics 2013, 39:65 http://www.ijponline.net/content/39/1/65 Provenzi et al. Italian Journal of Pediatrics 2013, 39:65 http://www.ijponline.net/content/39/1/65 tumors: a Children’s oncology group study. J Clin Oncol 2007, 25:4622–4627. tumors: a Children’s oncology group study. J Clin Oncol 2007, 25:4622–4627. Hishiki T, Matsunaga T, Sasaki F, Yano M, Ida K, Horie H, Kondo S, Watanabe K, Oue T, Tajiri T, Kamimatsuse A, Ohnuma N, Hiyama E: Outcome of hepatoblastomas treated using the Japanese Study Group for Pediatric Liver Tumor (JPLT) protocol-2: report from the JPLT. Pediatr Surg Int 2011, 27:1–8. doi:10.1186/1824-7288-39-65 Cite this article as: Provenzi et al.: Is there a role for high dose chemotherapy and blood stem cell rescue in childhood hepatoblastoma presenting with lung metastases? A case report and literature review. Italian Journal of Pediatrics 2013 39:65. doi:10.1186/1824-7288-39-65 Cite this article as: Provenzi et al.: Is there a role for high dose chemotherapy and blood stem cell rescue in childhood hepatoblastoma presenting with lung metastases? A case report and literature review. Italian Journal of Pediatrics 2013 39:65. 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W2098777488.txt	https://bmcneurosci.biomedcentral.com/counter/pdf/10.1186/1471-2202-12-128	en		It's not what you say but the way that you say it: an fMRI study of differential lexical and non-lexical prosodic pitch processing	BMC neuroscience	2,011	cc-by	5,848	Tracy et al. BMC Neuroscience 2011, 12:128 http://www.biomedcentral.com/1471-2202/12/128 RESEARCH ARTICLE Open Access It’s not what you say but the way that you say it: an fMRI study of differential lexical and non-lexical prosodic pitch processing Derek K Tracy1, David K Ho1, Owen O’Daly1, Panayiota Michalopoulou1, Lisa C Lloyd1, Eleanor Dimond1, Kazunori Matsumoto2 and Sukhwinder S Shergill1 Abstract Background: This study aims to identify the neural substrate involved in prosodic pitch processing. Functional magnetic resonance imaging was used to test the premise that prosody pitch processing is primarily subserved by the right cortical hemisphere. Two experimental paradigms were used, firstly pairs of spoken sentences, where the only variation was a single internal phrase pitch change, and secondly, a matched condition utilizing pitch changes within analogous tonesequence phrases. This removed the potential confounder of lexical evaluation. fMRI images were obtained using these paradigms. Results: Activation was significantly greater within the right frontal and temporal cortices during the tonesequence stimuli relative to the sentence stimuli. Conclusion: This study showed that pitch changes, stripped of lexical information, are mainly processed by the right cerebral hemisphere, whilst the processing of analogous, matched, lexical pitch change is preferentially left sided. These findings, showing hemispherical differentiation of processing based on stimulus complexity, are in accord with a ‘task dependent’ hypothesis of pitch processing. Background Non-verbal components of language, included under the collective term prosody, play a central role in human communication [1]. First defined by Monrad-Krohn in 1947 [2], prosodic elements of speech can be subdivided into the two broad categories of linguistic and emotional prosody. Linguistic prosody conveys information about semantic meaning, such as pragmatic category - e.g. determining if a sentence is a statement, a question or a command - and syntactic relation - e.g. determining clause boundaries within sentences [3,4]. Emotional prosody is the mechanism by which humans convey attitudes and emotions in speech. There has been debate about how clearly these two categories can be delineated. Initial behavioural and lesion studies implicated both right [5-9] and left [10-12] hemispheric regions, likely Correspondence: derek.1.tracy@kcl.ac.uk 1 CSI Lab, Institute of Psychiatry, King’s College London, UK Full list of author information is available at the end of the article confounded both by the inherent difficulties in comparing lesion studies [13,14] and assessing “global” prosodic function without considering specific subcomponents. PET data first suggested that prosodic content and judgement activated the prefrontal cortex bilaterally [15,16], more so on the left, and hemispheric asymmetry has been demonstrated for most regions of activation [17]. Subsequent imaging studies have implicated right superior temporal regions [16,18-21] - most recent work suggesting particularly within Brodmann’s area [22], with additional, partially bilateral responses within the frontal cortex [18,20,23-25], the anterior insula [16,23,25], amygdalae [26], and the basal ganglia [27,28]. Emotional speech produces greater cortical activation than that which is prosodically neutral [16,22,29]. Electrophysiological work has supported neuroimaging findings that the right temporal cortex displays enhanced event-related potentials to emotional stimuli [30]. Variations in results, due in no small part to different experimental paradigms, have failed to definitively clarify © 2011 Tracy et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Tracy et al. BMC Neuroscience 2011, 12:128 http://www.biomedcentral.com/1471-2202/12/128 whether cerebral regional and hemispheric activation are specific to prosodic subcomponent analysis or the functional demand of the task, known as the cue dependent [31,32] and task dependent [17,33-36] hypotheses respectively. However by far the majority of work has been on emotional prosody, and it’s unclear how well such data can be applied to linguistic prosody that, in comparison, has had a paucity of research. Furthermore, work on linguistic or semantic aspects of prosody have typically focused on psychometric measures of language conceptualisation and understanding [37,38] rather than the underlying neurobiology. Most authors have recognized the difficulties of the confounding influences of the lexical content of the stimuli and the problem of the higher level cognitive processes involved in the more global process of emotional prosody [39]. The neuroimaging data that exist for linguistic prosody typically favour hemispheric specialisation [40], with left fronto-temporal regions subserving ‘simpler’ short [41] syntactic and lexical segments of speech [42], and right hemispheric analogues processing larger suprasegmental elements at a sentence level [43], most in keeping with the task dependent hypothesis. In light of this, this study set out to utilise fMRI to examine a single crucial element of linguistic prosodic comprehension; pitch change. We specifically looked at internal pitch changes, or “emphasis shift”, as our earlier work suggested that these were more sensitive markers of subtle neurological deficits and less confounded by working memory primacy and recency phenomena [44]. As the name suggests, internal pitch changes occur within - as opposed to at the beginning or end of - a sentence. Furthermore, in an effort to try eliminate the major confounder of lexical comprehension, following the work of Patel et al [45] we introduced an analogous tone-sequence paradigm that contained a delexicalised pitch pattern. By removing the lexical content but keeping the tone sequence otherwise matched this design would also allow testing of the validity of the task dependent hypothesis as the same prosodic element, pitch, was being tested, but at different levels, with the tone sequence involving suprasegmental data analysis. We hypothesised that a) there are common cortical regions including bilateral prefrontal and temporal cortices associated with pitch processing in both speech and tone-sequence analogues; b) the more “pure” pitch processing associated with tone-sequence analogues would preferentially recruit right sided frontal and temporal cortices while more lexically loaded speech would preferentially recruit left temporal cortex; and c) increasing demands on prosodic comprehension would be associated with enhanced activation in the right frontal and temporal cortex. Page 2 of 8 Methods Subjects Twelve subjects were recruited through advertisements in a city-wide newspaper. Inclusion criteria were: males aged between 18 and 55, right-handedness, English as a first language. Exclusion criteria were: previous psychiatric or neurological illness, hearing or speech impairment and illicit drug use in the previous six months. All subjects provided written informed consent. Mean age was 31 (SD = 9.6). All subjects had completed secondary education; none had any formal training in playing musical instruments. The study had been approved by the local ethics committee. Stimuli and materials A modified version of the tone-sequence and prosody discrimination task previously described by the authors [44] was used, based on the earlier protocol of Patel et al [45]. The recorded stimuli consisted of 12 lexically identical sentence pairs, spoken by an adult female native English speaker, and their non-verbal, tone-sequence analogue pairs; and 12 sentence and tone-sequence pairs that differed prosodically in internal pitch pattern on a single word or tone (e.g. “I like blue ties on gentlemen” and “I like blue ties on gentlemen”, with the italicized word emphasised). Tone-sequence stimuli were created by digitizing each sentence at 40,000 Hz with subsequent normalization to the same amplitude into a tone sequence which corresponded with the sentence’s fundamental frequency in pitch and timing, with one levelpitch tone per syllable; a more detailed description is available in Patel et al [45]. An alternative method of low-pass filtering of the sentence pairs to remove lexical information was felt to be less satisfactory, as such filtering can leave residual phonological information, and previous studies [46] had validated this method. Procedure Subjects were trained on the prosodic discrimination task, which consisted of six counterbalanced blocks. Each block was composed of twelve trials comprising four pairs of sentences, four pairs of tone-sequences, and four null trials (a silent period equal in length to four paired stimuli) presented in random order. Each trial consisted of a pair of stimuli separated by a one second interval. The pair of stimuli differed in the pitch of an internal component in 50% of trials. As some sentences were longer than others, the duration of the stimuli varied from 3432-6134 milliseconds, with an average length of 5036 ms. Following a visual cue at the end of each trial, subjects indicated whether the paired stimuli were the same or different by using their right index finger and a button press. There was a variable intertrial interval of between 8.6-11.3 seconds before the onset of the next trial. Such a jittered Tracy et al. BMC Neuroscience 2011, 12:128 http://www.biomedcentral.com/1471-2202/12/128 design results in peristimulus distribution of MRI sampling, thus ensuring that all components of an eventrelated haemodynamic response are sampled, and avoids that bias of having stimulus presentation and data acquisition time-locked [46]. The total length of the six counterbalanced blocks was 17 minutes 39 seconds. fMRI Acquisition Gradient echo echoplanar imaging (EPI) data were acquired on a neuro-optimised GE Signa 1.5 Tesla system (General Electric, Milwaukee WI, USA) at the Maudsley Hospital, London. A quadrature birdcage headcoil was used for radio frequency transmission and reception. Foam padding was placed around the subject’s head in the coil to minimize head movement. One hundred and forty four T2*-weighted whole-brain volumes depicting blood oxygen level-dependent (BOLD) contrast were acquired at each of 24 near-axial non-contiguous planes parallel to the intercommissural (AC-PC) line (slice thickness = 5 mm; gap = 0.5 mm; TR = 2.1 seconds; echo time = 40 milliseconds; flip angle = 90°; matrix = 64 × 64). This EPI data set provided complete brain coverage. At the same session, a high-resolution gradient echo image of the whole brain was acquired in the intercommissural plane consisting of 43 slices (slice thickness = 3 mm; gap = 0.3 mm; TR = 3 seconds; flip angle = 90°; matrix = 128 × 128). Scanner noise during stimuli presentation was minimised by using a partially silent acquisition [47] during the stimuli presentation lasting 6.3 seconds while fMRI data (associated with prominent scanner noise) was collected during the following 8.4 seconds. fMRI Analysis The data were first realigned [48] to minimise motion related artefacts and smoothed using a Gaussian filter (FWHM 7.2 mm). Responses to the experimental paradigm were then detected by time-series analysis using Gamma variate functions (peak responses at 4 and 8 sec) to model the BOLD response. The analysis was implemented as follows. First, in each experimental condition, trial onsets were modelled as stick-functions which were convolved separately with the 4 and 8 sec Poisson functions to yield two regressors of the expected haemodynamic response to that condition. The weighted sum of these two convolutions that gave the best fit (least-squares) to the time series at each voxel was then computed and a goodness of fit statistic was computed at each voxel, the SSQratio. It has been shown that this permutation method gives very good type I error control with minimal distributional assumptions [49]. In order to extend inference to the group level, the observed and randomized SSQratio maps were transformed into standard space by a two stage process involving first a rigid body transformation of the fMRI Page 3 of 8 data into a high-resolution inversion recovery image of the same subject followed by an affine transformation onto a Talairach template [50]. In order to increase sensitivity and reduce the multiple comparison problem encountered in fMRI, hypothesis testing was carried out at the cluster level using the method developed by Bullmore et al. [48], shown to give excellent cluster-wise type I error control in functional fMRI analysis. All analyses were performed with < 1 false positive clusters expected per image, under the null hypothesis. We examined regions of activation common to both sentence and tone-sequence prosodic comprehension with conjunction analysis. As the levels of activation in the various experiments will vary, the statistical issue is whether the minimum level of activation in any of the tasks is significantly different from zero. In parametric analysis this is done by testing the minimum t statistic. The statistical analysis program utilized (XBAM) found which task had the smallest median level of activation and tested this median against the null distribution of the activation by estimating the SSQratio for each subject at each voxel for each task [49,51]. Then we compared prosodic comprehension between the tone-sequence and sentence stimuli to clarify the effects of lexical processing. Subsequent analyses compared identical stimuli pairs with differing stimuli pairs (same versus different stimuli pairs). During the pilot phase, volunteers subjectively reported the appraisal of identical stimuli to be more demanding. This was used to examine the effects of postulated increased demand on prosodic assessment. We employed a 2 × 2 factorial design to examine the interaction of factor condition (tone sequence, sentence) with the variables of pair type (same, different). The SSQ values were extracted from whole clusters, and plotted for regions demonstrating significant interaction effects between tone sequence and sentence processing and task demand assessed by same or differing stimuli pairs. A confounder in all fMRI studies is the intrinsic scanner noise: this is particularly the case in tasks with an auditory component such as this one. We minimized this by having the scanner at a partially silent acquisition phase [47] during the presentation of stimuli. It has been shown that handedness and gender may affect the neural structures involved in the processing of language [52] and prosody [53], as such we only examined right handed males. Behavioural data were analyzed using the statistical package SPSS. Results Behavioural data There were no significant differences in response time or accuracy rates between sentence and tone-sequence categories either overall, or when analysed in the subcategories Tracy et al. BMC Neuroscience 2011, 12:128 http://www.biomedcentral.com/1471-2202/12/128 Page 4 of 8 of same and different tasks, using a two tailed t-test (a = 0.05). The subjects were generally highly accurate (0.75 0.98 on the tone-sequence task, 0.83 - 1.00 on the sentence task), with four individuals getting 100% accuracy on the sentence task, suggestive of a possible ceiling effect. However, subjects were more accurate during same tasks (mean accuracy 0.948) than during different tasks (mean accuracy 0.866) overall. Table 1 Areas of activation shown in Figure 1 Size Talairach Coordinates Hem BA Cerebral Region X Y Z 62 -43 -33 48 L 40 Inferior Parietal Lobule 60 -54 -22 37 L 2 Postcentral Gyrus 48 51 7 -7 R 22 Superior Temporal Gyrus 43 -54 -15 9 L 41 Middle Temporal Gyrus 42 47 -48 26 R 40 Inferior Parietal Lobule Neuroimaging data 40 -54 0 -2 L 22 Superior Temporal Gyrus The conjunction analysis showed significant activation common to both sentence and tone sequence prosodic processing in the bilateral Inferior Frontal Gyri, Middle (MTG) and Superior Temporal Gyri (STG), in addition to bilateral Inferior Parietal lobule and the right Superior Frontal Gyrus (Figure 1; Table 1). Activation was significantly greater within the right frontal and temporal cortices during the tone-sequence stimuli relative to the sentence stimuli (Figure 2, bottom half; Table 2). Regions of greater activation in the sentence task relative to the tone sequence task (Figure 2, top half; Table 3) were predominantly left hemispheric, including the cingulate gyrus, left MTG, STG, inferior parietal lobule as well as the basal ganglia; with additional activation in the right precuneus, right cingulate gyrus and right lingual gyrus. A statistically significant interaction between factor condition (tone sequence, sentence) and stimulus pair type (same, different) was evident in the right Inferior and Middle Frontal Gyrus and right STG (Figures 3; Table 4). 32 -32 -26 59 L 4 Precentral Gyrus 32 -7 -81 -13 L 18 Lingual Gyrus, Occipital Lobe 30 51 15 -2 R 47 Inferior Frontal Gyrus 12 58 -37 -7 R 21 10 32 15 4 R Discussion and Conclusions As hypothesized, there was activation in bilateral MTG and STG common to prosodic pitch processing across both sentence and tone-sequence stimuli. There was more prominent right inferior frontal cortex activation, although left inferior frontal activation was also present (Figure 1; Table 1). This was in accordance with previous imaging data of prosodic comprehension [18,20,23-25]. There was a large bilateral activation in the Inferior Parietal Lobule, a region associated with storage within the Middle Temporal Gyrus Claustrum Hem = hemisphere, BA = Brodmann’s Area. working memory system [1]. Such a role is in accordance with our data, as differential activation maps fail to show differences in parietal activation between the two tasks, coinciding with a purely working memory role. Left precentral and postcentral gyral and basal ganglia activity were common to both conditions, something which would be anticipated in an experimental paradigm involving a right handed finger press. Comparison between the tone-sequence and sentence stimuli aimed to clarify the relative contribution of cortical regions associated with a purer linguistic prosodic pitch analysis (tone sequence > sentences) and those associated with greater lexical or phonological analysis (sentence > tone sequence), which has been recognised as a major confounder in such studies generally [45,54-59]. Stripped of this lexical information, the tonesequence demonstrated significant activation in the right inferior and medial frontal, and right STG compared to the sentence task (Figure 2, bottom half; Table 2). Wildgruber et al [1] suggested that at lower levels of both linguistic and emotional prosody processing, the same right hemispheric network is accessed but that the explicit judgment of linguistic aspects of speech prosody is more associated with left hemispheric language regions Figure 1 Conjuction analysis of regions of cerebral activation common to both the sentence and tone-sequence tasks. 5 ascending transverse slices, with a sagittal section to the right of the image indicating where these are taken from. Exact cluster coordinates are given in Table 1. Tracy et al. BMC Neuroscience 2011, 12:128 http://www.biomedcentral.com/1471-2202/12/128 Page 5 of 8 Figure 2 ANOVA of regions of task-dependent differential activation. Ascending transverse slices with the sagittal section to the right indicating where they are taken from. The top half displays regions of relative increased activation during the sentence task; the lower half displays those more active during the tone-sequence task. The exact cluster coordinates are provided in Table 2 and Table 3 respectively. and explicit evaluation of emotional prosody is related to bilateral orbitofrontal regions. Our data support this assertion, with evident overlap between the regions preferentially activated by the tone-sequence and those elicited during emotional prosodic tasks. Explicit analysis of linguistic aspects preferentially evoked appraisal by left hemispheric regions, fitting with other work [34,55,60-62] and this may reflect the processing of this lexical content of the stimuli. Our third hypothesis was that ‘increased demand’ would be associated with enhanced activation in the right frontal and temporal cortices. Subjects reported finding tone-sequence trials harder than sentence ones - fitting with Patel’s notion of extra ‘redundancy’ cues in the lexical trials [45,58] - and that same pairs were ‘more difficult’ than different ones. Interestingly, behavioural data conflicts with subjective perception, demonstrating that subjects more accurate in same tasks: during these subjects needed to hold the entire same trial pair in working memory, and examine these for subtle (non-existent) differences; as opposed to different pairs where participants could discard the stimuli once any pitch difference was noted. As such, same and tone-sequence may have been proxy markers for cognitive demand rather than ‘difficulty’ per se, as well as tone-sequence exploring ‘purer’ pitch processing. During the tone-sequence task there was relatively increased right STG and left precuneus activation when paired tone stimuli were the same, compared to when different. The interaction analysis (Figures 3; Table 4) looked at the effect of one factor (stimulus type: sentence or tone sequence) on another factor (trial type: same or different). Regions which can differentiate between these factors are all right sided: the STG, Inferior Frontal Table 2 Areas of activation shown in Figure 2, BOTTOM HALF 97 Size Talairach Coordinates Hem BA Cerebral Region Table 3 Areas of activation shown in Figure 2-TOP HALF Size Talairach Coordinates Hem BA Cerebral Region X Y Z -14 -48 26 58 7 -63 47 -29 -11 44 -54 L 31 Cingulate Gyrus, Limbic Lobe 31 R 7 Precuneus 9 L -19 -7 L 21 Middle Temporal Gyrus 2 Postcentral Gyrus Putamen X Y Z 71 32 30 -13 R 47 Inferior Frontal Gyrus 32 -47 -26 37 L 41 7 19 42 R 32 Cingulate Gyrus, Limbic Lobe 27 -18 -22 15 L 40 11 41 37 R 7 Medial Frontal Gyrus 26 4 -44 37 R 31 Cingulate Gyrus, Limbic Lobe 29 51 11 -2 R 22 Superior Temporal Gyrus 24 14 -78 4 R 18 Lingual Gyrus, Occipital Lobe 22 36 22 4 R 13 Insula 19 -54 -52 9 L 39 Superior Temporal Gyrus 17 14 11 53 R 6 Superior Frontal Gyrus 18 -29 -4 -13 L 10 18 -19 -18 R 28 Parahippocampal Gyrus 15 -22 -56 42 L 7 Precuneus 9 -7 4 53 L 6 Medial Frontal Gyrus 12 -40 -33 26 L 40 Inferior Parietal Lobule Hem = hemisphere, BA = Brodmann’s Area. Posterior Thalamic Nucleus Amygdala Hem = hemisphere, BA = Brodmann’s Area. Tracy et al. BMC Neuroscience 2011, 12:128 http://www.biomedcentral.com/1471-2202/12/128 Page 6 of 8 Figure 3 Interaction analysis of cerebral regions that can differentiate task (sentence/tone-sequence) and trial type (same/different). Cluster coordinates are provided in Table 4. Table 4 Areas of activation in Figure 3 Size Talairach Coordinates X Y Z Hem BA Cerebral Region 21 43 7 -13 R 38 Superior Temporal Gyrus 11 43 15 -2 R 47 Inferior Frontal Gyrus 9 36 33 20 R 46 Middle Frontal Gyrus Hem = hemisphere, BA = Brodmann’s Area. Gyrus and Middle Frontal Gyrus. Each of these discriminatory regions show increased activation in tonesequence, as opposed to sentence, tasks, and during same compared to different stimuli (Figure 4). The authors’ interpretation of our data is that it best fits with the task dependent hypothesis that the left hemisphere is hemispherically specialized for lexical and short syntactic aspects of pitch whilst the right hemisphere is superior at processing suprasegmental pitch. Subjects’ reports place tone sequence and same trials as being more difficult and the interaction analysis of activation in the right inferior frontal gyrus (Figure 4) shows increasing activation for these: in both instances subjects are processing a larger, full trial, sequence at a suprasegmental level. In conclusion, our data support the premise that prosodic pitch perception is subserved by the bifrontal and temporal cortices, specifically the Superior Temporal Gyrus, Inferior Frontal Gyrus and Middle Frontal Gyrus, with the degree of hemispheric involvement dependent upon the task. These areas were activated when both tone-sequence and sentence paradigms were used, thus confounding lexical stimuli were removed, though the former preferentially activated the right hemispheric regions, the latter the left. There was a relative increase in activation in the right frontal and temporal cortices during ‘same’ stimuli tasks, which was deemed to be more demanding, as subjectively reported by subjects, in terms of prosodic comprehension and this, in our opinion, is due to the need to analyse pitch at a broader ‘sentence level’. Our data is in agreement with the assertion [40] of hemispheric specialisation fitting with the task dependent hypothesis, which would have predicted the lateralization [63] found in this study. Language prosody processing is complex and consists of multiple components. Current understanding of it involves several competing theories, neither of which has garnered consistent support. The vast majority of the literature focuses on emotional prosody: further work is needed to provide a more coherent and distinctive conceptualization of linguistic prosodic processing. Acknowledgements SSS was supported by an Advanced Clinical Training Fellowship from the Wellcome Trust (SSS) and a Young Investigator Award from NARSAD. OOD was supported by the Psychiatry Research Trust. The authors gratefully acknowledge the provision of original stimuli by Dr. A. Patel, The Neurosciences Institute, San Diego CA 92121. Figure 4 Graphed differential activation in the right inferior frontal gyrus in the interaction analysis of Figure 3 demonstrating activation in the two task types (sentence/tonesequence) and two trial types (same/different). SSQ, a “sum of squares ratio” is a statistical indicator of activity, as described in the methods section. Author details 1 CSI Lab, Institute of Psychiatry, King’s College London, UK. 2Department of Psychiatry, Tohoku University School of Medicine, Sendai, Japan. Authors’ contributions DKT participated in the study design, recruited participants, participated in fMRI data collection & analysis, interpreted the results and drafted the Tracy et al. BMC Neuroscience 2011, 12:128 http://www.biomedcentral.com/1471-2202/12/128 manuscript. DKH contributed to the drafting of the manuscript. OOD recruited participants, participated in fMRI data analysis and contributed to the drafting of the manuscript. PM participated in fMRI data collection and interpretation of the results. KM participated in the study design and coordination. LCL participated in fMRI analysis and contributed to the drafting of the manuscript. ED participated in fMRI analysis. SSS conceived the study and participated in its design and coordination. All authors read and approved the final manuscript. 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Li X, Gandour JT, Talavage T, Wong D, Hoffa A, Lowe M, Dzemidzic M: Hemispheric asymmetries in phonological processing of tones versus segmental units. Neuroreport 2010, 21(10):690-694. doi:10.1186/1471-2202-12-128 Cite this article as: Tracy et al.: It’s not what you say but the way that you say it: an fMRI study of differential lexical and non-lexical prosodic pitch processing. BMC Neuroscience 2011 12:128. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit
https://openalex.org/W1888719121	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0137084&type=printable	English	null	Pharmacological and Genetic Evidence for Gap Junctions as Potential New Insecticide Targets in the Yellow Fever Mosquito, Aedes aegypti	PloS one	2,015	cc-by	8,864	RESEARCH ARTICLE Pharmacological and Genetic Evidence for Gap Junctions as Potential New Insecticide Targets in the Yellow Fever Mosquito, Aedes aegypti Travis L. Calkins, Peter M. Piermarini* Travis L. Calkins, Peter M. Piermarini* Department of Entomology, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, Ohio, United States of America Travis L. Calkins, Peter M. Piermarini* Department of Entomology, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, Ohio, United States of America Travis L. Calkins, Peter M. Piermarini Department of Entomology, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, Ohio, United States of America Department of Entomology, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, Ohio, United States of America * piermarini.1@osu.edu * piermarini.1@osu.edu Editor: Zach N Adelman, Virginia Tech, UNITED STATES , g , STATES Received: June 15, 2015 Accepted: August 13, 2015 Published: September 1, 2015 Copyright: © 2015 Calkins, Piermarini. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: © 2015 Calkins, Piermarini. 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. Abstract The yellow fever mosquito Aedes aegypti is an important vector of viral diseases that impact global health. Insecticides are typically used to manage mosquito populations, but the evo- lution of insecticide resistance is limiting their effectiveness. Thus, identifying new molecular and physiological targets in mosquitoes is needed to facilitate insecticide discovery and development. Here we test the hypothesis that gap junctions are valid molecular and physi- ological targets for new insecticides. Gap junctions are intercellular channels that mediate direct communication between neighboring cells and consist of evolutionarily distinct pro- teins in vertebrate (connexins) and invertebrate (innexins) animals. We show that the injec- tion of pharmacological inhibitors of gap junctions (i.e., carbenoxolone, meclofenamic acid, or mefloquine) into the hemolymph of adult female mosquitoes elicits dose-dependent toxic effects, with mefloquine showing the greatest potency. In contrast, when applied topically to the cuticle, carbenoxolone was the only inhibitor to exhibit full efficacy. In vivo urine excre- tion assays demonstrate that both carbenoxolone and mefloquine inhibit the diuretic output of adult female mosquitoes, suggesting inhibition of excretory functions as part of their mechanism of action. When added to the rearing water of 1st instar larvae, carbenoxolone and meclofenamic acid both elicit dose-dependent toxic effects, with meclofenamic acid showing the greatest potency. Injecting a double-stranded RNA cocktail against innexins into the hemolymph of adult female mosquitoes knock down whole-animal innexin mRNA expression and decreases survival of the mosquitoes. Taken together these data indicate that gap junctions may provide novel molecular and physiological targets for the develop- ment of insecticides. OPEN ACCESS Citation: Calkins TL, Piermarini PM (2015) Pharmacological and Genetic Evidence for Gap Junctions as Potential New Insecticide Targets in the Yellow Fever Mosquito, Aedes aegypti. PLoS ONE 10(9): e0137084. doi:10.1371/journal.pone.0137084 , ( ) Pharmacological and Genetic Evidence for Gap Junctions as Potential New Insecticide Targets in the Yellow Fever Mosquito, Aedes aegypti. PLoS ONE 10(9): e0137084. doi:10.1371/journal.pone.0137084 Editor: Zach N Adelman, Virginia Tech, UNITED STATES Received: June 15, 2015 Accepted: August 13, 2015 Published: September 1, 2015 Copyright: © 2015 Calkins, Piermarini. 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. Editor: Zach N Adelman, Virginia Tech, UNITED STATES Editor: Zach N Adelman, Virginia Tech, UNITED STATES Introduction Competing Interests: The authors have declared that no competing interests exist. The yellow fever mosquito, Aedes aegypti, is the most important vector of the viruses that cause yellow, dengue, and chikungunya fevers in humans. These diseases have spread around the tropical and subtropical world, facilitated by globalization of human societies and climate change [1]. In particular, chikungunya fever has most recently emerged from its native range in sub-Saharan Africa and Asia to Central America and the Caribbean in 2013–2014. As of 2014, locally acquired cases of chikungunya were reported in Florida [2,3]. Ideally, these mosquito-borne diseases could be prevented through the global use of safe, effective, and affordable vaccines. For yellow fever there is such a vaccine, however for chikun- gunya and dengue fevers there currently are no effective vaccines available [4]. An alternative strategy for controlling the spread of mosquito-borne diseases is to control populations of the mosquito vectors that transmit the pathogens. The primary control methods for reducing mos- quito numbers are sanitation (cleaning and removing larval habitats from around homes) and using insecticides [5]. Although insecticides are effective at reducing mosquito populations, insecticide resistant populations have emerged because of the overuse of a few limited active compounds, such as pyrethroids [6]. The control of resistant populations of mosquitoes can be mitigated through a variety of techniques, including the development of new insecticides with novel modes of action, which begins with the identification of new insecticidal targets. Gap junctions are potential molecular and physiological targets for the development of new insecticides. On the cellular level, gap junctions are intercellular channels that allow for the transport of small molecules and ions between adjacent cells [7]. On the molecular level, gap junctions are formed by two hemichannels from neighboring cells that dock with one another. Each hemichannel consists of six protein subunits, which are encoded by genes called connex- ins in vertebrates and innexins in invertebrates. The connexin and innexin proteins possess similar structural and functional features, but have evolved independently and thus their pri- mary structures possess little similarity to one another [8,9]. In the A. aegypti genome, 6 genes encode innexins [10]; we have demonstrated that these genes are differentially expressed throughout the mosquito life cycle and in various tissues of adult mosquitoes [10,11]. In insects, innexins are known to play key roles in embryogenesis. Evidence for Gap Junctions as New Insecticide Targets Data Availability Statement: All relevant data are within the paper. Funding: This work was supported by the National Institutes of Health, R03DK090186, http://www.nih. gov/; Mosquito Research Foundation, 2014-03, http:// www.mosquitoresearch.org/; Ohio Agricultural Research and Development Center SEEDS, 2014- 078, oardc.osu.edu/seeds; Ohio Mosquito Control Association, Grant-In-Aid, http://www.ohiomosquito. org/. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 1 / 15 PLOS ONE \| DOI:10.1371/journal.pone.0137084 September 1, 2015 PLOS ONE \| DOI:10.1371/journal.pone.0137084 September 1, 2015 Adult topical assays For topical application, all inhibitors were dissolved directly at their desired concentrations in 75% ethanol/25% H2O. Adult female mosquitoes (3–10 days post-eclosion) were immobilized on ice and a Hamilton repeating dispenser (Hamilton Company, Reno, Nevada) was used to apply 500 nl of an inhibitor to the thorax of each mosquito. For a given dose of compound, ten mosquitoes were treated and transferred to small cages (10 mosquitoes per cage) with access to a 10% sucrose solution. The cages were returned to the rearing chamber and the efficacy of a dose was assessed after 24 h, as described above in ‘Adult hemolymph injection assays’. A total of four to eight independent replicates were performed for each dose of each inhibitor. Chemicals Carbenoxolone, meclofenamic acid and mefloquine were obtained from Sigma-Aldrich (St. Louis, MO). All other chemicals were obtained from Thermo Fisher Scientific (Waltham, MA). Mosquitoes Eggs of Aedes aegypti were obtained through the Malaria Research and Reference Reagent Resource Center (MR4) as part of the BEI Resources Repository (Liverpool strain; LVP-IB12 F19, deposited by M.Q. Benedict). Mosquitoes were reared as described in Piermarini et al. [20] in an environmental chamber set at 28°C and 80% relative humidity with a 12 h:12 h light: dark cycle. Adult hemolymph injection assays For direct hemolymph injection, carbenoxolone and meclofenamic acid were dissolved in HEPES buffered saline (HBS) as 100 mM stock solutions, whereas mefloquine was dissolved into 100% dimethyl sulfoxide (DMSO). Before injection, the inhibitors were diluted to their desired concentrations in HBS. The HBS consisted of 11.9 mM HEPES, 137 mM NaCl, and 2.7 mM KCl; the pH was adjusted to 7.45 using NaOH. For dilutions of carbenoxolone and meclo- fenamic acid, DMSO was added to the HBS at a final concentration of 11% to match that found in dilutions of mefloquine. Adult female mosquitoes (3–10 days post-eclosion) were immobilized on ice prior to inject- ing their hemolymph with 69 nl of an inhibitor using a Nanoject II microinjector (Drummond Scientific Company, Broomall, PA). For a given dose of a compound, ten mosquitoes were injected and transferred to small cages (10 mosquitoes per cage) with access to a 10% sucrose solution. The cages were returned to the rearing chamber and the efficacy of a dose was assessed 24 h later, as described in Raphemot et al. [21]. In brief, the efficacy was measured as the percentage of treated mosquitoes in a cage that were incapacitated by 24 h; i.e., the collec- tive percentage of mosquitoes that were flightless or dead [21]. A total of five to ten indepen- dent replicates were performed for each dose of each inhibitor. Evidence for Gap Junctions as New Insecticide Targets female mosquitoes decreases their survival over 11 days. Taken together, our results indicate that gap junctions are promising molecular and physiological targets for the development of novel insecticides to control mosquito vectors. female mosquitoes decreases their survival over 11 days. Taken together, our results indicate that gap junctions are promising molecular and physiological targets for the development of novel insecticides to control mosquito vectors. Introduction For example, knockout of innexin 3 (inx3) in Drosophila melanogaster results in a failure of dorsal closure [12]. Moreover, in Anopheles gambiae, disruption of innexin 4 (a.k.a ‘zero-popu- lation growth’ or ‘zpg’) results in sterile males [13]. Innexins are also thought to be important in adult insect neuromuscular communication and renal function [10,14,15]. In summary, given the evolutionary distinct ancestry of innexins and connexins, as well as the consequences of innexin disruption on insect biology, we hypothesized that gap junctions may serve as valu- able targets for insecticide development. To test our hypothesis, we assessed the effects of gap junction inhibition on mosquito sur- vival and/or physiology using pharmacological and genetic tools. In particular, we used three commercially available gap junction inhibitors (carbenoxolone, meclofenamic acid and meflo- quine), which block vertebrate and invertebrate gap junctions formed by connexins and innex- ins, respectively [16–19]. Furthermore, we utilized RNA interference (RNAi) to knock down the expression of the 6 innexin mRNAs expressed in adult female A. aegypti. We find that all 3 pharmacological inhibitors are toxic to adult female mosquitoes when injected into the hemolymph, and that carbenoxolone is effective when applied topically to the cuticle. Moreover, carbenoxolone and mefloquine decrease the diuretic capacity of adult female mosquitoes, suggesting disruption of Malpighian tubule function as a potential mechanism of action for these compounds. Also, carbenoxolone and meclofenamic acid kill 1st instar larvae when added to their rearing water. Lastly, the knockdown of innexin mRNA levels in adult 2 / 15 PLOS ONE \| DOI:10.1371/journal.pone.0137084 September 1, 2015 Larval assays The toxicity of the inhibitors on 1st instar larvae was assessed using the protocol of Pridgeon et al. [22]. In brief, eggs were hatched in dH2O under vacuum at room temperature for 2 h in a 250 ml beaker. The beaker was transferred to a rearing chamber (28°C) and 1st instar larvae were collected 24 h later. For a given treatment, 5 larvae were transferred to the well of a 24 well Falcon MULTIWELL plate (Becton Dickinson Labware, Franklin Lakes, NJ) containing 995 μl of H2O with a gap junction inhibitor and 5 μl of food solution. The food solution con- sisted of 0.013 g of ground Tetramin fish food (Tetra United Pet Group, Blacksburg, VA) sus- pended in 1 ml of dH2O. Stock solutions of the inhibitors were dissolved in dH2O and diluted within wells to achieve the desired concentrations (1 ml total volume per well). Plates were returned to rearing conditions and after 24 hours the larvae were assessed. The efficacy of a concentration was measured as the percentage of treated larvae in a well that were dead by 24 h. Larvae were considered dead if they did not move when disturbed with a 10 μl pipette tip. Adult excretion assay The diuretic capacity of adult female mosquitoes was assessed using an established protocol [21]. In brief, for a given treatment, 3 mosquitoes (3–10 days post eclosion) were immobilized 3 / 15 PLOS ONE \| DOI:10.1371/journal.pone.0137084 September 1, 2015 Evidence for Gap Junctions as New Insecticide Targets on ice and injected with 900 nl of HBS into their hemolymph using a Nanoject II microinjector (Drummond Scientific). After injection, the mosquitoes were placed into a graduated, packed- cell volume tube (MidSci, St. Louis, MO) for two hours at 28°C to allow them to excrete. After two hours, the mosquitoes were removed from the tube, which was then centrifuged at 17,000 g to allow for the excreted volume to be measured visually via the graduated column at the bot- tom of the tube. At minimum, 6 replicates (3 mosquitoes per replicate) were performed for each treatment. All mosquitoes were confirmed to be alive at the end of the two hours. Mosqui- toes that were not injected with HBS served as controls. The composition of the HBS was similar to that described above in ‘Adult hemolymph injec- tion assays’ except that NaOH and DMSO were reduced to 1 mM and 2%, respectively. For a given experiment, one of the following inhibitors was added to the HBS at the indicated concen- trations: carbenoxolone (1.34 mM), mefloquine (0.5 mM), and meclofenamic acid (1.53 mM). dsRNA synthesis and injection dsRNAs were synthesized from DNA templates. To generate the DNA templates, primers were designed for each innexin cDNA using Primer3 software [23] to amplify a 300–500 base pair product (Table 1). The nucleotide sequences of the primers and expected PCR products were subjected to a Basic Local Alignment Search Tool (BLAST) against the A. aegypti genome (Vec- torbase.org) to ensure specificity. A T7 promoter sequence (TAATACGACTCACTATAGGGAGA) was added to the 5’ end of each forward and reverse primer to allow for dsRNA synthesis. The DNA templates were generated via PCR, which consisted of mixing 0.5 μl plasmid DNA (from previously cloned innexin cDNAs), 1 μl forward and reverse primer mix (10 μM each) and Table 1. dsRNA template synthesis primers. Each primer set consists of an innexin specific region for amplification of the target gene from plasmid, and the T7 promoter sequence (TAATACGACTCACTATAGGGAGA). dsRNA Template Forward dsRNA Template Reverse Inx1 TAATACGACTCACTATAGGGAGAGCGAAGCTGCAGAAGCTATT TAATACGACTCACTATAGGGAGAAAATGTTTTGTCGAGGTTCATGT Inx2 TAATACGACTCACTATAGGGAGATTTGGCGTTTGAAAAGTGTG TAATACGACTCACTATAGGGAGAATACTCCCGGCTGAGCAATA Inx3 TAATACGACTCACTATAGGGAGACGACGGTGACAGATTGACTAG TAATACGACTCACTATAGGGAGAGTTCGCTCCTGGTTGTACTC Inx4 TAATACGACTCACTATAGGGAGACATTCCTGTTCTCGTTCCCC TAATACGACTCACTATAGGGAGAAAGGCACAGGGCATCAAAGT Inx7 TAATACGACTCACTATAGGGAGACAGGGACAATCCAAAAGCATG TAATACGACTCACTATAGGGAGATCAGTTTCGTCAGCCTCATC Inx8 TAATACGACTCACTATAGGGAGATTCTGACGATACTGACGACGTT TAATACGACTCACTATAGGGAGATCATGCATCCTGTATTTCACCT eGFP TAATACGACTCACTATAGGGACGTAAACGGCCACAAGTT TAATACGACTCACTATAGGGTTGGGGTCTTTGCTCAGG doi:10.1371/journal.pone.0137084.t001 PLOS ONE \| DOI:10.1371/journal.pone.0137084 September 1, 2015 4 / 15 dsRNA Template Forward 4 / 15 Evidence for Gap Junctions as New Insecticide Targets 23.5 μl Platinum Taq DNA polymerase High Fidelity (Thermo-Fisher). The mixture was sub- jected to a thermocycling protocol consisting of an initial denaturation at 95°C (2 min) followed by 30 cycles of 95°C (1 min), 60°C (30 sec), 72°C (1 min); the protocol ended with an elongation step at 72°C (5 min). The identities of the various DNA templates generated by PCR were veri- fied by agarose gel (1%) electrophoresis and Sanger DNA sequencing at the Molecular and Cel- lular Imaging Center (MCIC) of the Ohio Agricultural Research and Development Center (OARDC) of the Ohio State University (Wooster, OH). Template DNA was then used in the T7 MEGAscript dsRNA synthesis kit (Thermo-Fisher Scientific) following the manufacturer’s protocol (20 μl total reaction Volume). The resulting dsRNA was resuspended in nuclease free water and its concentration was measured on a Nano- drop 2000 spectrophotometer (Thermo Scientific). The dsRNA for each innexin was diluted to approximately 4 μg/μl, aliquoted, and stored at -80°C to avoid repeated freeze-thaw degradation. On the day of an experiment, all six innexin dsRNAs were diluted to 2 μg/μl in a PBS solu- tion (137 mM NaCl, 2.7 mM KCl and 11.9 mM phosphates; pH 7.5). dsRNA synthesis and injection Before injection into a mosquito, equal volumes of each innexin dsRNA were mixed together to form an innexin dsRNA ‘cocktail’, resulting in a final concentration of ~333 ng/μl for each dsRNA. Adult female mosquitoes were anesthetized on ice and their hemolymph was injected with either 1 μl of the innexin dsRNA cocktail or a negative control dsRNA against enhanced green fluorescent pro- tein (eGFP; 2 μg/μl) using a Nanoject II injector (Drummond Scientific). For the eGFP and innexin dsRNAs, a total of 30 mosquitoes were injected per replicate (6 mosquitoes were dedi- cated for qPCR analysis and 24 were dedicated for survival assays). After injection all mosqui- toes were placed in small cages and returned to rearing conditions. Four biological replicates were performed for knockdown analysis and three biological replicates for the survival assay. Phenotype assessment and qPCR Mosquitoes injected with eGFP or innexin dsRNA were checked at 24 h intervals for 11 days and the number of surviving mosquitoes was recorded. Real time quantitative PCR (qPCR) was utilized to determine knockdown on days 3, 7 and 11 post-injection. RNA extraction and cDNA synthesis were performed as described in Calkins et al. [11]. Primers for qPCR were designed against the six innexins and ribosomal protein S7 (RPS7; a reference gene) using Primer3 software [23] to amplify a 90–110 base pair product (Table 2). Specificity of the result- ing PCR products was confirmed using a melt curve analysis and Sanger sequencing (MCIC, OARDC). For a given sample, each qPCR consisted of three technical replicates of 10 μl reactions each consisting of 5 μl of GoTaq Master Mix, 40 ng cDNA, 400 nM forward and reverse primers, and nuclease free water. The reactions took place in 96-well unskirted, low profile plates (Bio-Rad Table 2. qPCR primer pairs. Each set of primers was selected for innexin specificity and determined specific through melt curve analysis and sequencing (MCIC, OARDC). qPCR Forward qPCR Reverse Inx1 CACCGATAGTGCCGTATTCC CCGACATATTGTGTGGCAGT Inx2 GGAGATCCTATGGCACGAGT ACGGTAGCACACAGAGTCCA Inx3 TCGTTCGGTTACTTCATCTGC GCGATTCTCCTGATCCATGTC Inx4 TTCTGTTGGACACTGGGAAC CCATGTGCGTTCCTATTTCG Inx7 TGGGTCCCGTTTGTGTTATT CCATACGAAGACCATCCACA Inx8 GACTGCGTTCACACGAAAGA GGGTACTTCGCTACCGACTTT RPS7 CTTTGATGTGCGAGTGAACAC CATCTCCAACTCCAGGATAGC doi:10.1371/journal.pone.0137084.t002 PLOS ONE \| DOI:10.1371/journal.pone.0137084 September 1, 2015 5 / 15 Table 2. qPCR primer pairs. Each set of primers was selected for innexin specificity and determined specific through melt curve analysis and sequencing (MCIC, OARDC). qPCR Forward qPCR Reverse Inx1 CACCGATAGTGCCGTATTCC CCGACATATTGTGTGGCAGT Inx2 GGAGATCCTATGGCACGAGT ACGGTAGCACACAGAGTCCA Inx3 TCGTTCGGTTACTTCATCTGC GCGATTCTCCTGATCCATGTC Inx4 TTCTGTTGGACACTGGGAAC CCATGTGCGTTCCTATTTCG Inx7 TGGGTCCCGTTTGTGTTATT CCATACGAAGACCATCCACA Inx8 GACTGCGTTCACACGAAAGA GGGTACTTCGCTACCGACTTT RPS7 CTTTGATGTGCGAGTGAACAC CATCTCCAACTCCAGGATAGC doi:10 1371/journal pone 0137084 t002 5 / 15 Evidence for Gap Junctions as New Insecticide Targets Laboratories, Hercules, CA), sealed with TempPlate RT optical film (USA Scientific). qPCR was performed using a Bio-Rad C1000 thermocycler and CFX96 real time system (Bio-Rad Labora- tories). The thermocycler used the following protocol: an initial denaturation of 95°C (3 min) followed by 39 cycles of 95°C (10 sec) and 58°C (30 sec), ending with a melt curve cycle. Data analysis and statistics GraphPad Prism 6 software (GraphPad Software Inc., La Jolla, CA) was used in all statistical analysis. Results of the toxicology experiments with gap junction inhibitors (i.e., adult hemo- lymph injections, adult topical applications, and larval assays) were analyzed using a non-linear curve fit analysis (log [inhibitor] vs. response variable-slope) to determine effective dose or concentration 50% values (i.e., ED50 or EC50). Results from the excretion assays were analyzed with a one-way ANOVA with a Newman-Keuls post hoc analysis. Relative gene expression was determined utilizing the delta CT method by normalizing target gene expression to that of the reference gene RPS7. Relative gene expression levels in eGFP controls were analyzed with a one-way ANOVA with a Newman-Keuls post hoc analysis. Percent gene silencing was calcu- lated as in Drake et al. [24] by setting relative gene expression in eGFP injected mosquitoes to 100%. Significant knockdown was determined via Student’s t-tests comparing normalized innexin mRNA levels in eGFP dsRNA injected mosquitoes to that of innexin dsRNA injected mosquitoes. Survival between eGFP and innexin injected groups was compared by a two-way repeated measures ANOVA with Holm-Sidak’s post hoc analysis. Adult excretion assays To determine if the gap junction inhibitors disrupt the diuretic capacity of adult female mos- quitoes, we volume loaded their hemolymph with a sub-lethal dose of each inhibitor. Fig 3 shows the mean volume excreted per female in 2 h after injection for each treatment, compared to the non-injected controls (C). Mosquitoes injected with a volume load (VL) and no inhibitor excreted on average 760 ± 16 nl (Fig 3). In contrast, those injected with a VL and 1.34 mM car- benoxolone (VL + CBX) excreted a significantly lower amount of urine (8.0 ± 8 nl) that is simi- lar to non-injected control mosquitoes, which excrete 39 ± 8 nl (Fig 3). Mosquitoes injected with a VL and 0.5 mM mefloquine (VL + MEF) excrete 290 ± 93 nl, which was significantly lower than the amount excreted by the VL mosquitoes, but significantly higher than that excreted by the VL + CBX mosquitoes (Fig 3). Mosquitoes injected with a VL and 1.53 mM meclofenamic acid (VL + MFA) excrete 844 ± 14 nl, which was comparable to that of VL mos- quitoes (Fig 3). Concentrations of meclofenamic acid higher than 1.53 mM were lethal to the mosquitoes before the end of the 2 h excretion assay (data not shown). Adult hemolymph injections To determine if gap junction inhibitors are toxic to adult female mosquitoes, we injected the inhibitors directly into the hemolymph. Carbenoxolone, mefloquine and meclofenamic acid all showed dose-dependent toxic effects in adult female mosquitoes (Fig 1). Mefloquine was the most effective inhibitor, with an effective dose for 50% of the population (ED50) of 15.47 ng per Fig 1. Dose-response curves of gap junction inhibitors injected directly into the hemolymph of adult female A. aegypti mosquitoes (carbenoxolone R2 = 0.873, meclofenamic acid R2 = 0.957 and mefloquine R2 = 0.906). Efficacy (dead and flightless mosquitoes) was assessed 24 h after injection. Taking into consideration the average mass of an adult female mosquito (1.97 mg), the ED50 for carbenoxolone, meclofenamic acid and mefloquine are 127.3 ng/mg, 96.4 ng/mg and 15.47 ng/mg respectively. Values are means ± SEM. n = 5–10 replicates of ten mosquitoes per dose tested. doi:10.1371/journal.pone.0137084.g001 Fig 1. Dose-response curves of gap junction inhibitors injected directly into the hemolymph of adult female A. aegypti mosquitoes (carbenoxolone R2 = 0.873, meclofenamic acid R2 = 0.957 and mefloquine R2 = 0.906). Efficacy (dead and flightless mosquitoes) was assessed 24 h after injection. Taking into consideration the average mass of an adult female mosquito (1.97 mg), the ED50 for carbenoxolone, meclofenamic acid and mefloquine are 127.3 ng/mg, 96.4 ng/mg and 15.47 ng/mg respectively. Values are means ± SEM. n = 5–10 replicates of ten mosquitoes per dose tested. doi:10 1371/journal pone 0137084 g001 Fig 1. Dose-response curves of gap junction inhibitors injected directly into the hemolymph of adult female A. aegypti mosquitoes (carbenoxolone R2 = 0.873, meclofenamic acid R2 = 0.957 and mefloquine R2 = 0.906). Efficacy (dead and flightless mosquitoes) was assessed 24 h after injection. Taking into consideration the average mass of an adult female mosquito (1.97 mg), the ED50 for carbenoxolone, meclofenamic acid and mefloquine are 127.3 ng/mg, 96.4 ng/mg and 15.47 ng/mg respectively. Values are means ± SEM. n = 5–10 replicates of ten mosquitoes per dose tested. doi:10.1371/journal.pone.0137084.g001 6 / 15 PLOS ONE \| DOI:10.1371/journal.pone.0137084 September 1, 2015 Evidence for Gap Junctions as New Insecticide Targets mg of mosquito body weight (ng/mg) followed by meclofenamic acid (ED50 = 96.39 ng/mg) and carbenoxolone (ED50 = 127.3 ng/mg; Fig 1). Adult topical assays To determine whether the gap junction inhibitors can penetrate the cuticle, we evaluated the effi- cacy of the inhibitors in adult female mosquitoes when applied topically to the thorax. Of the three inhibitors, carbenoxolone was the only one to show a dose-dependent effect nearing 100% efficacy, with an ED50 of 8.57 μg/mg. Mefloquine showed limited dose-dependent effects, with a maximal efficacy of only ~54.5%. Meclofenamic acid showed the weakest topical efficacy (Fig 2). Larval assays We assessed the efficacy of the gap junction inhibitors as larvicides by adding them to the rear- ing water of 1st instar larvae. Carbenoxolone and meclofenamic acid both showed dose- Fig 2. Dose-response curves of gap junction inhibitors applied directly to the cuticle of adult female A. aegypti mosquitoes (carbenoxolone R2 = 0.824, meclofenamic acid R2 = 0.455 and mefloquine R2 = 0.44). Efficacy (dead and flightless mosquitoes) was assessed 24 h after application. Taking into consideration the average mass of an adult female mosquito (1.97 mg), the ED50 for carbenoxolone is 8.57 μg/mg. The ED50 values for meclofenamic acid and mefloquine are not determinable. Values are means ± SEM. n = 4–8 replicates of ten mosquitoes per dose tested. doi:10.1371/journal.pone.0137084.g002 Fig 2. Dose-response curves of gap junction inhibitors applied directly to the cuticle of adult female A. aegypti mosquitoes (carbenoxolone R2 = 0.824, meclofenamic acid R2 = 0.455 and mefloquine R2 = 0.44). Efficacy (dead and flightless mosquitoes) was assessed 24 h after application. Taking into consideration the average mass of an adult female mosquito (1.97 mg), the ED50 for carbenoxolone is 8.57 μg/mg. The ED50 values for meclofenamic acid and mefloquine are not determinable. Values are means ± SEM. n = 4–8 replicates of ten mosquitoes per dose tested. Fig 2. Dose-response curves of gap junction inhibitors applied directly to the cuticle of adult female A. aegypti mosquitoes (carbenoxolone R2 = 0.824, meclofenamic acid R2 = 0.455 and mefloquine R2 = 0.44). Efficacy (dead and flightless mosquitoes) was assessed 24 h after application. Taking into consideration the average mass of an adult female mosquito (1.97 mg), the ED50 for carbenoxolone is 8.57 μg/mg. The ED50 values for meclofenamic acid and mefloquine are not determinable. Values are means ± SEM. n = 4–8 replicates of ten mosquitoes per dose tested. doi:10.1371/journal.pone.0137084.g002 doi:10.1371/journal.pone.0137084.g002 7 / 15 PLOS ONE \| DOI:10.1371/journal.pone.0137084 September 1, 2015 Evidence for Gap Junctions as New Insecticide Targets Fig 3. Effects of gap junction inhibitors on the diuretic capacity of adult female A. aegypti mosquitoes. C = non-injected control mosquitoes. VL = volume loaded mosquitoes injected with 900 nl of HBS. VL + CBX = mosquitoes injected with 900 nl of HBS and 1.34 mM carbenoxolone. VL + MEF = mosquitoes injected with 900 nl of HBS and 0.5 mM mefloquine. VL + MFA = mosquitoes injected with 900 nl of HBS and 1.53 mM meclofenamic acid. Larval assays We were unable to test the efficacy of mefloquine in this assay, because it is not soluble in water and the amount of DMSO required to keep it in solution (>2%) was toxic to larvae. dependent toxic effects in larvae (Fig 4). Meclofenamic acid was the most effective (EC50 = 244.9 ppm) followed by carbenoxolone (EC50 = 1587 ppm). We were unable to test the efficacy of mefloquine in this assay, because it is not soluble in water and the amount of DMSO required to keep it in solution (>2%) was toxic to larvae. Larval assays Letters (A, B or C) indicate statistical differences as determined by a one-way ANOVA and Newman-Keuls post-test (P < 0.05). Values are mean volumes of urine excreted per mosquito after two hours ± SEM. n = 14 for VL, n = 14 for C, n = 8 for VL + CBX, n = 6 for VL + MEF, and n = 6 for VL + MFA. Fig 3. Effects of gap junction inhibitors on the diuretic capacity of adult female A. aegypti mosquitoes. Fig 3. Effects of gap junction inhibitors on the diuretic capacity of adult female A. aegypti mosquitoes. C = non-injected control mosquitoes. VL = volume loaded mosquitoes injected with 900 nl of HBS. VL + CBX = mosquitoes injected with 900 nl of HBS and 1.34 mM carbenoxolone. VL + MEF = mosquitoes injected with 900 nl of HBS and 0.5 mM mefloquine. VL + MFA = mosquitoes injected with 900 nl of HBS and 1.53 mM meclofenamic acid. Letters (A, B or C) indicate statistical differences as determined by a one-way ANOVA and Newman-Keuls post-test (P < 0.05). Values are mean volumes of urine excreted per mosquito after two hours ± SEM. n = 14 for VL, n = 14 for C, n = 8 for VL + CBX, n = 6 for VL + MEF, and n = 6 for VL + MFA. g g p j p y gyp q C = non-injected control mosquitoes. VL = volume loaded mosquitoes injected with 900 nl of HBS. VL + CBX = mosquitoes injected with 900 nl of HBS and 1.34 mM carbenoxolone. VL + MEF = mosquitoes injected with 900 nl of HBS and 0.5 mM mefloquine. VL + MFA = mosquitoes injected with 900 nl of HBS and 1.53 mM meclofenamic acid. Letters (A, B or C) indicate statistical differences as determined by a one-way ANOVA and Newman-Keuls post-test (P < 0.05). Values are mean volumes of urine excreted per mosquito after two hours ± SEM. n = 14 for VL, n = 14 for C, n = 8 for VL + CBX, n = 6 for VL + MEF, and n = 6 for VL + MFA. doi:10.1371/journal.pone.0137084.g003 doi:10.1371/journal.pone.0137084.g003 dependent toxic effects in larvae (Fig 4). Meclofenamic acid was the most effective (EC50 = 244.9 ppm) followed by carbenoxolone (EC50 = 1587 ppm). RNA interference (RNAi) To determine if knockdown of innexin mRNA levels affected the survival of adult female mos- quitoes, we utilized RNAi. First, we used qPCR to determine the relative expression of each Fig 4. Dose-response curves of gap junction inhibitors added to the rearing water of 1st instar larval A. aegypti mosquitoes (carbenoxolone R2 = 0.873 and meclofenamic acid R2 = 0.957). Larval mortality was assessed 24 h after adding the inhibitors. LC50 for meclofenamic acid and carbenoxolone are 0.83 mM and 2.84 mM, respectively. Values are means ± SEM. n = 4–8 replicates of five larvae per concentration tested. doi:10.1371/journal.pone.0137084.g004 7084 September 1 2015 8 / 15 Fig 4. Dose-response curves of gap junction inhibitors added to the rearing water of 1st instar larval A. aegypti mosquitoes (carbenoxolone R2 = 0.873 and meclofenamic acid R2 = 0.957). Larval mortality was assessed 24 h after adding the inhibitors. LC50 for meclofenamic acid and carbenoxolone are 0.83 mM and 2.84 mM, respectively. Values are means ± SEM. n = 4–8 replicates of five larvae per concentration tested. Fig 4. Dose-response curves of gap junction inhibitors added to the rearing water of 1st instar larval A. aegypti mosquitoes (carbenoxolone R2 = 0.873 and meclofenamic acid R2 = 0.957). Larval mortality was assessed 24 h after adding the inhibitors. LC50 for meclofenamic acid and carbenoxolone are 0.83 mM and 2.84 mM, respectively. Values are means ± SEM. n = 4–8 replicates of five larvae per concentration tested. Fig 4. Dose-response curves of gap junction inhibitors added to the rearing water of 1st instar larval A. aegypti mosquitoes (carbenoxolone R2 = 0.873 and meclofenamic acid R2 = 0.957). Larval mortality was assessed 24 h after adding the inhibitors. LC50 for meclofenamic acid and carbenoxolone are 0.83 mM and 2.84 mM, respectively. Values are means ± SEM. n = 4–8 replicates of five larvae per concentration tested. doi:10.1371/journal.pone.0137084.g004 doi:10.1371/journal.pone.0137084.g004 8 / 15 PLOS ONE \| DOI:10.1371/journal.pone.0137084 September 1, 2015 Evidence for Gap Junctions as New Insecticide Targets Fig 5. Relative innexin gene expression as normalized to RPS7 gene expression in eGFP dsRNA injected mosquitoes 3 days after injection. Values are means ± SEM. n = 4 replicates of 3 mosquitoes. Letters (A, B or C) indicate statistical differences as determined by a one-way ANOVA and Newman-Keuls post-test (P < 0.05). doi:10 1371/journal pone 0137084 g005 Fig 5. RNA interference (RNAi) Relative innexin gene expression as normalized to RPS7 gene expression in eGFP dsRNA injected mosquitoes 3 days after injection. Values are means ± SEM. n = 4 replicates of 3 mosquitoes. Letters (A, B or C) indicate statistical differences as determined by a one-way ANOVA and Newman-Keuls post-test (P < 0.05). doi:10 1371/journal pone 0137084 g005 doi:10.1371/journal.pone.0137084.g005 doi:10.1371/journal.pone.0137084.g005 innexin in mosquitoes injected with eGFP dsRNA (3 days post injection). As shown in Fig 5, Inx2 was the most abundant innexin, followed by Inx3 and Inx 4. The expression of Inx1, Inx7 and Inx8 were lower, but detectable. innexin in mosquitoes injected with eGFP dsRNA (3 days post injection). As shown in Fig 5, Inx2 was the most abundant innexin, followed by Inx3 and Inx 4. The expression of Inx1, Inx7 and Inx8 were lower, but detectable. Injection of an innexin dsRNA cocktail containing dsRNAs for each innexin (333 ng per innexin, 2 μg total) resulted in significant knockdown of Inx1 (32 ± 8%), Inx2 (69 ± 3%), Inx3 (51 ± 5%), Inx4 (71 ± 10%) and Inx7 (86 ± 2%) by 3 days after injection compared to expres- sion levels in the eGFP-injected controls (Fig 6). The expression levels of Inx8 were not signifi- cantly knocked down, but the mRNA levels were very low to begin (Fig 5). The knockdown of innexin expression in mosquitoes injected with innexin dsRNA persisted until at least day 11 (data not shown). In addition, mosquitoes injected with innexin dsRNA exhibited a signifi- cantly lower survival than those injected with eGFP dsRNA that progressed over the next 11 days, and started as early as 1 day after injection (Fig 7). Fig 6. Knockdown efficiency in innexin dsRNA injected mosquitoes 3 days after dsRNA injection. Percent knockdown is relative to eGFP dsRNA injected control mosquitoes 3 days after injection (Fig 5). Values are means ± SEM. n = 4 replicates of 3 mosquitoes. Asterisks indicate significant knockdown compared to eGFP as determined by a Student’s t-test (p < 0.05). doi:10.1371/journal.pone.0137084.g006 Fig 6. Knockdown efficiency in innexin dsRNA injected mosquitoes 3 days after dsRNA injection. Percent knockdown is relative to eGFP dsRNA injected control mosquitoes 3 days after injection (Fig 5). Values are means ± SEM. n = 4 replicates of 3 mosquitoes. Asterisks indicate significant knockdown compared to eGFP as determined by a Student’s t-test (p < 0.05). RNA interference (RNAi) PLOS ONE \| DOI:10.1371/journal.pone.0137084 September 1, 2015 9 / 15 Evidence for Gap Junctions as New Insecticide Targets Fig 7. Effect of eGFP (black squares) and innexin (red circles) dsRNA injection on mosquito survival. Values are means ± SEM. n = 3 replicates of 24 mosquitoes. Asterisks indicate a significant difference in survival between eGFP and innexin dsRNA injected mosquitoes as determined by a two-way ANOVA with a Holm-Sidak’s post-hoc test. doi:10 1371/journal pone 0137084 g007 Fig 7. Effect of eGFP (black squares) and innexin (red circles) dsRNA injection on mosquito survival. Values are means ± SEM. n = 3 replicates of 24 mosquitoes. Asterisks indicate a significant difference in survival between eGFP and innexin dsRNA injected mosquitoes as determined by a two-way ANOVA with a Holm-Sidak’s post-hoc test. Fig 7. Effect of eGFP (black squares) and innexin (red circles) dsRNA injection on mosquito survival. Values are means ± SEM. n = 3 replicates of 24 mosquitoes. Asterisks indicate a significant difference in survival between eGFP and innexin dsRNA injected mosquitoes as determined by a two-way ANOVA with a Holm-Sidak’s post-hoc test. Fig 7. Effect of eGFP (black squares) and innexin (red circles) dsRNA injection on mosquito survival. Values are means ± SEM. n = 3 replicates of 24 mosquitoes. Asterisks indicate a significant difference in survival between eGFP and innexin dsRNA injected mosquitoes as determined by a two-way ANOVA with a Holm-Sidak’s post-hoc test. doi:10.1371/journal.pone.0137084.g007 Evidence for Gap Junctions as New Insecticide Targets inhibition of innexin function may cause disruptions to the nervous, digestive, excretory, and/ or reproductive systems, leading to the impairment of flight and/or death. Further investiga- tions will be required to confirm that such wide-spread disruptions to mosquito physiology are indeed occurring. In the present study, we show that at least two of the pharmacological inhibitors (carbe- noxolone and mefloquine) perturb the functions of the excretory system, as indicated by their inhibition of the diuretic capacity of adult female mosquitoes (Fig 3). These two inhibi- tors may be acting on the Malpighian tubules, which produce urine via transepithelial fluid secretion, and/or the hindgut, which attenuates the composition of urine before expelling it from the animal via muscular contractions. In Malpighian tubules, several lines of evidence suggest that gap junctions composed of innexins occur between the epithelial cells and play important roles in intercellular communication and diuresis [15,28–30]. Furthermore, in the hindgut, we have localized the expression of Inx3 immunoreactivity to the intercellular mem- branes of epithelial cells in the ileum and rectum [11]. Thus, inhibiting the activity of gap junctions in these tissues is expected to disrupt urine production and/or expulsion. However, additional experiments such as Ramsay assays of isolated Malpighian tubules and isolated hindgut contraction assays [31,32] are needed to resolve the mechanisms by which carbenox- olone and mefloquine inhibit excretory performance. Surprisingly, we did not observe effects of a sub-lethal dose of meclofenamic acid (1.53 mM) on the diuretic capacity of mosquitoes (Fig 3), and higher doses were lethal before the 2 hr experimental period finished (Calkins, unpublished observations). These findings suggest that meclofenamic acid either does not inhibit gap junctions expressed in the excretory system of mosquitoes, or it may elicit rapid toxic effects elsewhere, such as in the nervous system before any effects on excretory function can be observed. Perhaps, meclofenamic acid is able to cross the blood-brain barrier of mosquitoes more efficiently than carbenoxolone and meflo- quine, thereby leading to a more rapid toxic effect. PLOS ONE \| DOI:10.1371/journal.pone.0137084 September 1, 2015 Discussion Our study provides the first pharmacological and molecular evidence that suggests gap junc- tions are potentially valuable targets for mosquitocide discovery and development. When injected into the hemolymph of adult female mosquitoes, three structurally unique pharmaco- logical agents that are known to inhibit gap junctions (carbenoxolone, meclofenamic acid, and mefloquine) were efficacious (Fig 1), and one of the compounds (carbenoxolone) exhibited the ability to penetrate the cuticle of adult females (Fig 2). Moreover, two of the gap junction inhib- itors (carbenoxolone and meclofenamic acid) were effective when added to the rearing water of 1st instar larvae (Fig 4). Thus, our data provide proof-of-concept that chemical inhibitors of gap junctions exhibit insecticidal properties in adult and larval mosquitoes. In support of our pharmacological data, the injection of dsRNA against all 6 innexin mRNAs reduced the survival of adult female mosquitoes over the next 11 days (Fig 7). The reduced survival was associated with the significant knockdown of mRNA levels for 5 of the targeted innexins (Fig 6), which presumably leads to reductions of innexin protein levels and higher mortality. The mRNA levels of Inx8 were not significantly affected by dsRNA injection, but this gene is expressed at nominal levels in adult female mosquitoes (Fig 5) [11]. Notably, the degree and rate at which the toxic effects manifest via RNAi are respectively weaker and slower than those of the pharmacological inhibitors. The weaker effects of RNAi are likely attributable to a combination of the incomplete knockdown of innexin mRNA expression (Fig 6) and the time required for dsRNAs to elicit an effect on protein levels, which may lag behind that of mRNA levels. In contrast, pharmacological inhibition can be complete and elicit acute effects. Other studies have also noticed weaker toxic effects of inhibition of an insecticide target via RNAi vs. pharmacological inhibition [25–27]. The toxic effects of the gap junction inhibitors and the innexin dsRNAs on mosquitoes are not surprising given that innexin mRNAs are expressed throughout the mosquito life cycle [11]. Furthermore, in adult female mosquitoes, we have shown that at least 4 innexin mRNAs are expressed in each tissue of the alimentary canal (i.e., midgut, hindgut and Malpighian tubules), the ovaries, head, and thorax/abdomen [11]. Thus, the pharmacological or genetic PLOS ONE \| DOI:10.1371/journal.pone.0137084 September 1, 2015 10 / 15 Potential for gap junction inhibitors as insecticides When applied topically to the cuticle of mosquitoes, only carbenoxolone showed insecticidal activity, whereas mefloquine and meclofenamic acid had limited and nominal activity, respec- tively. Thus, carbenoxolone appears to have the capacity to penetrate the cuticle. The structures (Fig 8) and chemical properties (Table 3) of these three gap junction inhibitors are distinct and may explain their different abilities to penetrate the cuticle. For example, when evaluating the three inhibitors based on the “Rule of 5” [33,34] for identifying insecticides (Table 3), carbe- noxolone adheres closely with a ‘clog p’ below 5, zero hydrogen bond donors, 7 hydrogen bond acceptors, only 6 rotatable bonds, and a molecular weight near 500 kDa (Table 3). Besides molecular weight, the other two gap junction inhibitors differ from carbenoxolone primarily in their number of hydrogen bond donors. Carbenoxolone is the only one of the three com- pounds that adheres to the norm of current insecticides [33] with no hydrogen bond donors. Despite nominal topical efficacy against adult female mosquitoes, meclofenamic acid was the most potent larvicide of the three compounds (Fig 4). Carbenoxolone was also effective against larvae, but over 6-fold less potent than meclofenamic acid. Given that meclofenamic acid was unable to penetrate the cuticle of adult females (Fig 2), we presume that in larvae this compound is delivered to the alimentary canal via ingestion where it may act on the midgut epithelium and/or diffuse into the hemolymph. Although meclofenamic acid and carbenoxolone were respectively the most effective com- pounds against larval and adult female (topically) mosquitoes, it is important to emphasize that these compounds are not nearly as potent as conventional insecticides, such as permethrin. PLOS ONE \| DOI:10.1371/journal.pone.0137084 September 1, 2015 11 / 15 Evidence for Gap Junctions as New Insecticide Targets For example, meclofenamic acid is 46 times less effective against 1st instar larvae of A. aegypti Fig 8. Molecular structures of the gap junction inhibitors: A) carbenoxolone, B) mefloquine, and C) meclofenamic acid. Structures as shown were modified from Sigma-Aldrich (www.sigma.com) and constructed in ChemSketch (ACD/Labs, Toronto, Ontario, Canada). doi:10.1371/journal.pone.0137084.g008 Fig 8. Molecular structures of the gap junction inhibitors: A) carbenoxolone, B) mefloquine, and C) meclofenamic acid. Structures as shown were modified from Sigma-Aldrich (www.sigma.com) and constructed in ChemSketch (ACD/Labs, Toronto, Ontario, Canada). doi:10.1371/journal.pone.0137084.g008 Fig 8. Molecular structures of the gap junction inhibitors: A) carbenoxolone, B) mefloquine, and C) meclofenamic acid. Author Contributions Conceived and designed the experiments: TLC PMP. Performed the experiments: TLC. Ana- lyzed the data: TLC PMP. Contributed reagents/materials/analysis tools: TLC PMP. Wrote the paper: TLC PMP. Acknowledgments We would like to thank Dr. Matthew Rouhier (Kenyon College) for his Assistance in the Labo- ratory, and Ms. Nuris Acosta (OSU) and Ms. Edna Alfaro (OSU) for their assistance in mos- quito rearing. Funding for this study was provided by grants to 1) PMP from the NIH (R03DK090186) and Mosquito Research Foundation (2014–03), and 2) TLC from the OARDC SEEDS program (Grant# 2014–078; oardc.osu.edu/seeds) and Ohio Mosquito Control Associa- tion Grant-In-Aid. State and Federal funds appropriated to the OARDC of the Ohio State Uni- versity also supported the study. Evidence for Gap Junctions as New Insecticide Targets mosquitocide and over 18.5 million times less effective than fipronil, the most potent adulticide against A. aegypti [22]. Additi l f b th b l d l f i id th i ffi Table 3. Molecular properties of the gap junction inhibitors (rows 2–4) as compared to the properties identified by Tice [33] for screening for novel insecticides (row 5). Inhibitor Molecular Weight cLog P Hydrogen Bond Donors Hydrogen Bond Acceptors Rotatable Bonds Carbenoxolone 570.77 4.83 0 7 6 Meﬂoquine 378.32 3.70 2 3 4 Meclofenamic Acid 296.15 4.92 2 3 3 Tice Rule of 5 < 500 < 5 0 < 10 < 12 doi:10.1371/journal.pone.0137084.t003 p junction inhibitors (rows 2–4) as compared to the properties identified by Tice [33] for screening for novel Table 3. Molecular properties of the gap junction inhibitors (rows 2–4) as compared to the properties identif insecticides (row 5). mosquitocide and over 18.5 million times less effective than fipronil, the most potent adulticide against A. aegypti [22]. mosquitocide and over 18.5 million times less effective than fipronil, the most potent adulticide against A. aegypti [22]. Additional concerns for both carbenoxolone and meclofenamic acid are their efficacy on mammalian gap junctions [35,36]. Moreover, carbenoxylone has potential off target effects in mammals, and meclofenamic acid, is currently used therapeutically as a nonsteroidal anti- inflammatory drug (NSAID) pain reliever [36,37]. Thus, our data should only be taken as proof-of-concept that gap junction inhibitors possess insecticidal properties. Additional efforts will be necessary to modify the potency and selectivity of these compounds for mosquitoes before they could be considered for use as insecticides in the field. Conclusions The present study is the first to demonstrate that three different commercially available gap junction inhibitors exhibit insecticidal activity. Moreover, we show that RNAi-based knock- down of mRNAs encoding gap junctional proteins (i.e., innexins) leads to increased mortality of adult female mosquitoes. Taken together, these results suggest that gap junctions offer new potential insecticidal targets for mosquito control. However, much progress still needs to be made in terms of discovering compounds with acceptable potency and specificity for mosquito gap junctions. The long evolutionary distance between innexins and connexins leaves us hope- ful that an innexin-specific compound can be discovered and developed. Potential for gap junction inhibitors as insecticides Structures as shown were modified from Sigma-Aldrich (www.sigma.com) and constructed in ChemSketch (ACD/Labs, Toronto, Ontario, Canada). doi:10.1371/journal.pone.0137084.g008 For example, meclofenamic acid is 46 times less effective against 1st instar larvae of A. aegypti than DNOC (Dinitro-ortho-cresol), which is considered a weak insecticide, and 874,643 times less effective than permethrin, a highly potent insecticide [22]. Additionally, our most topically active inhibitor, carbenoxolone, is 5.7 times less effective than bifenzate, a very weak 12 / 15 PLOS ONE \| DOI:10.1371/journal.pone.0137084 September 1, 2015 2. Powers AM. 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https://openalex.org/W2597931951	https://scholarhub.ui.ac.id/cgi/viewcontent.cgi?article=1214&context=paradigma	English	null	Fulla’s and Barbie’s Images in Relation to Women’s Beauty and Cultural Difference	Paradigma	2,016	cc-by-sa	5,183	Paradigma, Jurnal Kajian Budaya Paradigma, Jurnal Kajian Budaya 158 Paradigma, Jurnal Kajian Budaya Keywords Barbie, Fulla, concept of beauty, cultural differences, orientalism Abstract Most people know Barbie, a doll that conveys some values about culture and about the concept of women’s beauty. However, Barbie only represents one specific culture, which is Western culture. Different with Western culture, Eastern culture needs a doll who can represent their values. Then, Fulla appears as the representative of Eastern beauty. Fulla conveys some values about Eastern culture and about the new concept of women’s beauty which are different from Barbie. Fulla’s appearance here is thought can emphasize the cultural differences among the society, but it can give another choice to shape people’s new thought about the beauty of Eastern culture. Using Said’s orientalism will help the writer to support the arguments about Eastern culture that can be an equal competitor to Western culture nowadays. Fulla’s and Barbie’s Images in Relation to Women’s Beauty and Cultural Difference Juwita Anindya 2 Newboy FZCO first created Fulla in Dubai. It is under the UAE manufacturer. Fulla is a role model for Moslem people. Although there are many other dolls wearing hijab, Fulla is more famous than the others. Fulla’s and Barbie’s Images in Relation to Women’s Beauty, Juwita Anindya Barbie is a fashion doll that first appeared on March 9th 1959. Barbie is manufactured by Mattel, Inc1. It is popular because of its appearance and packaging that convey a concept of ideal beauty. Barbie has a boyfriend named Ken. She likes to go to parties and beaches. Unlike Barbie, Fulla is a veiled doll. It looks like Barbie, but when we look at it carefully, Fulla is different from Barbie. Fulla first appeared in late 2003. Fulla was created by a manufacturer in Dubai named Newboy FZCO2. It becomes a rolemodel for Moslem girls because Fulla wears hijab, likes to pray, and takes care of her brother and sister. A doll like Barbie or Fulla can be a role model to children. Barbie can be a trendsetter for children who like to play with it. Children can follow the way Barbie dresses or how she lives. In fact, Barbie just represents one specific culture, which is western culture. The value that Barbie brings can be unsuitable for people who deal with eastern culture. Before Fulla came, Barbie conveys one dominant concept of beauty. Beauty is like Barbie, having many beautiful dresses, shoes, bags, cars, and also having handsome boyfriend. The dominance of Barbie is because there is no equal competitor for Barbie and the impact of post-colonialism. As we know, post-colonialism leads us to the thought that western countries as the colonial are more powerful and dominant that the colonized countries. After Fulla’s coming, this can be one way to show that eastern culture is not subordinate anymore. What the writer is going to show is how Fulla can be an equal competitor for Barbie to convey the concept of ideal beauty. 1 Mattel Inc first produced Barbie in 1966. It was a product of U.S but made in Japan. The first Barbie appeared as a beautiful that very similar to a perfect young woman. Introduction The main aim of this paper is to analyze the differences between Fulla and Barbie dolls in relation to women’s beauty, and how those differences can further emphasize the cultural differences between east and west. The thought that western culture is more modern and better still exist nowadays. As we know, Fulla and Barbie are different, but actually both offer some ideas of beauty. Barbie gives the description of beauty with a thin body, long legs, blond hair, and fair skin. In contrast, Fulla shows that a girl with a veil and darker skin and hair can also be categorized as beautiful. Those dolls represent their own cultures, one representing the eastern culture and the other the western one. The writer will take a deeper look into Fulla’s and Barbie’s characteristics. Then, the writer will relate them to the thought which is influenced by post-colonialism that think western culture is better than eastern. Fulla as a representation of the eastern culture offers an equal quality of beauty that provide an alternative to Barbie’s dominant concept of beauty. This research will situate Fulla’s concept of beauty and find out if it can represent the beauty of eastern girls. 159 of western culture has the equal competitor. Children who play Barbie tend to see Barbie as a trendsetter3. They try to follow Barbie when they grow up. They want to be beautiful like Barbie. Barbie’s body becomes the ideal body for beautiful girls. Girls who play Barbie since they were kids may adopt the concept of beauty in which to be beautiful is to have skinny legs, long blonde hair, fair skin, blue eyes, small hips, and also sexy breast. For that reason, Barbie as arepresentation of western culture dominates the concept of ideal beauty in this world that makes many girls want to have a body like her. The body can be an important part of human’s beauty, and Barbie succeeds in making the concept of ideal body correspond to the concept of ideal beauty. However, this concept just represents one culture and not all people in this world can fully accept that concept. It is known that post-colonialism gives impact to the stereotype about east and west until nowadays. People often see that the west is more powerful than east. It is because mostly eastern countries are colonized by western countries. Western countries are thought as the dominant countries in this world. In this context, the writer relates that impact of post-colonialism’s impact to Barbie and Fulla that become representative of their own culture. Many people have known that Barbie has dominated the value of beauty of the world since 1959. This doll can be a role model that representsthe only western culture. The domination of Barbie shapes people’s thought that beauty is like Barbie. The writer sees that when Fulla first appeared, it gave people more choice to think about the concept of beauty. Fulla’s appearance is look like Barbie, but in fact they are different. Fulla can be the doll that breaks the stereotype of east and west. The writer thinks that Fulla has an equal power to compete with Barbie in conveying a concept of beauty. Based to Reischer and Koo (2004), many girls think that body is an important part of beauty. When those girls look at Barbie, they think that Barbie’s body is perfect. This is easier to Fulla to compete Barbie because Fulla also has a good body but covered in Abaya (Middle East Moslem clothes). 3 A social learning theories which stated that children tend to learn something by imitating adults or other media including a doll. Previous Research A doll that represents one specific culture can be a model to children who play with it. It can serve as a role model to the children by introducing and promoting the values of a specific culture. Most children in this world know Barbie. Barbie is like a rolemodel of ideal beauty, but in fact Barbie just represents one specific culture, which is western culture. Barbie does not fit in the eastern culture. According to Lind in Dixon (2011), feminist critique of Barbie is that Barbie as a doll conveys a message about a particular concept of beauty that brings western values with it. On the contrary, Fulla is made as a veiled Barbie. Fulla looks like Barbie, but apparently it is different from Barbie in terms of its characteristics. Fulla as a veiled Barbie offers a different concept of beauty that represents the eastern culture. Even though the emergence of Fulla can emphasize the cultural differences of this world, it can also offer a new and equal concept of beauty that represents the values of the eastern culture. It seems that Barbie as the representative Paradigma, Jurnal Kajian Budaya Paradigma, Jurnal Kajian Budaya 160 of western culture has the equal competitor. Theory and Method This research will be based on the theory of Orientalism by Edward Said. Said’s Orientalism is a study how western power could dominate eastern countries such as North African and Middle East. According to Said (1978), the West has created a dichotomy, between the reality of the East and the romantic notion of the “Orient”.Mc Leod(2000) also said, The Orient is frequently described in a series of negative terms that serve to buttress a sense of the West’s superiority and strength. The theory of orientalism by Edward Said was also related to theory of post- 3 A social learning theories which stated that children tend to learn something by imitating adults or other media including a doll. Fulla’s and Barbie’s Images in Relation to Women’s Beauty, Juwita Anindya 161 colonialism. People’s thought of east and west is also shaped by the stereotype that comes after colonialism. However, colonialism forms the perspective that western countries are the more powerful countries in this world because they could dominate and colonize other subordinate countries which are eastern countries. The theory of orientalism and post-colonialism become two basic theories to analyze Fulla and Barbie in relation to the difference of eastern and western culture. Fulla is an equal competitor for Barbie in conveying the new concept of beauty that represents the beauty of the eastern culture. The writer thinks that Fulla as a doll is strongly enough to represent eastern culture and convey new positive thought about ideal beauty. Fulla can conveyvalues like what Barbie has done before. The word ‘value’ here means everything about aculture that can shape people’s thought, especially the children who play with Barbie and Fulla dolls. The stereotypes about west and east still exist until nowadays. Many people still think that east is subordinate while west is more powerful and more modern. This kind of thought is also represented by Barbie. Barbie conveys many values that can shape people’s thought about western culture. Then, Fulla appears as anequal competitor to be the representative of eastern culture that is ready to show alternative values which can shape new thought in people’s minds. The method that is used by the writer is observation method. The writer will observe the materials, which are Fulla and Barbie dolls. Theory and Method Fulla and Barbie are examinedcarefully based ontheir appearances, packagings, advertisings, and also their habits.In general, Fulla’s appearances and packaging are almost the same with Barbie’s, but when we see the advertising and habit, we can see many differences that could clarify the differences between east and west. From the observation, the writer will show the comparison between the two. The writer chosed Fulla as acomparison for Barbie because Fulla nowadays becomes well-known. Fulla also has a beautiful appearance that represents eastern culture. The writer assumed that Fulla’s coming is like breaking the stereotype that eastern culture is subordinate and conveying anew concept of beauty. After that, the writer will collect secondary data about Barbie’s and Fulla’sacceptance in many countries in the world to support the result of the observation. Paradigma, Jurnal Kajian Budaya Paradigma, Jurnal Kajian Budaya 162 powerful that eastern countries. Eastern countries are thought as subordinate and less modern. Until nowadays many people still think that western countries and eastern countries are different and unequal. It is believed that people will be sure about the differences between eastern and western culture if they compare Fulla to Barbie. As the doll which conveys a concept of ideal beauty, Barbie succeed enough to represent western beauty as the concept. Based from the appearance, Barbie has long blonde hair, blue eyes, fair skin, sexy lips, sexy breasts, slim body, slim waist, and also long legs. Barbie’s face is also beautiful. Her beautiful appearance is also supported by her pretty clothes. She has many kinds of beautiful dresses, bikinis, shoes, bags, and accessories. Then, we compare it to Fulla. The writer here said Fulla as the representative of Eastern culture is because Fulla represents the Eastern beauty. Not only by her veil, but also but her appearances.As the Moslem doll, Fulla comes and conveys new idea of ideal beauty. Fulla also has long hair, but it is dark brown. Fulla has brown eyes, colored skin, slim body, and also long legs. The other differences of Fulla from Barbie are her breasts and waist. Fulla’s breasts are smaller than Barbie, but Barbie’s waist is smaller than Fulla’s. Fulla comes not as a sexy doll. Her clothes are different from Barbie. Fulla wears veil and also Abaya (traditional Moslem clothes in the Middle East). She also has Mukena (clothes for praying), Prayer Mat and also Koran. If we compare Fulla and Barbie from their daily life, we can see those differences represent their own culture and habits. In her story, Barbie has many friends and has one boyfriend named Ken. Barbie loves party and also hang out with Ken. Her habit is a common thing in western culture. Unlike Barbie, the story of Fulla tells that Fulla has one brother and one sister. She likes to stay at home, praying and taking care of her siblings. Fulla does not have any boyfriend. Fulla’s habit represents eastern culture. People can see that these differences really exist in this reality, and nowadays no one can say which one is right and wrong without strong evidences.In a stereotype about Eastern culture, it is known that most people who live in Middle East countries are Moslem. 4 Part of “Barbie Girl” lyrics. This song was released in 1997. This song became famous and then was used by Barbie’s advertisement theme songs. The lyrics represents how Barbie looks like and how Barbie lives. Analysis In this world, there are so many different cultures from different countries, but in general there are two different cultures which are eastern and western culture. Those cultures exist because they are influenced by post-colonialism. Many people noticed that long time ago there were some countries which colonized other countries. Most western culture went to other countries to find their gold, glory, and gospel. That is why they colonized other countries. Most countries that are colonized are eastern countries. That situation can influence people’s thought that Western countries are more dominant and Paradigma, Jurnal Kajian Budaya They live in Eastern culture that is thought as the opposition to Western culture. That is why Fulla’s coming is said as the equal competitor to Barbie. Even though Fulla appears different to Barbie, but those differences represent many people who are in eastern culture. The writer also takes a look at some advertisements of Fulla and Barbie. We can also see the difference of those cultures in the advertisements. Barbie’s advertisement is quite attractive. It shows Barbie’s life such as party, hang out, kiss her boyfriend, dance, and many more. It likes strengthen the stereotype of western habits which like to have fun. In one advertisement, Barbie uses the famous theme song “Barbie Girl”. We can hear the lyrics “You can brush my hair, undress me everywhere”.4 The lyrics can Fulla’s and Barbie’s Images in Relation to Women’s Beauty, Juwita Anindya 163 represent Barbie’s daily life which is only suitable for western culture. Different from Barbie, Fulla’s advertisement uses songs that represent Middle East songs that use Arabic language. Fulla is seen as the true Moslem doll. She prays and takes care of her siblings. The writer also found some themes on Fullas’ advertisements, such as Fasting Fulla, Praying Fulla, and Fulla’s wudhu (washing hands and feet before praying). Overall, those advertisements seem similar if we just take a look at the appearance of both Barbie and Fulla, but Fulla is just like the other type of Barbie. The other comparison is the logo. The logos or symbols of Barbie and Fulla are quite similar with domination of pink color. The shape of the words is also quite similar. These logos are dominated with pink color and pink flower that convey the idea of a stereotype about girls. Girls are symbolized by pink color and many girls love it. This color can support the idea of beauty that is conveyed by them. As we know, beauty is strongly related to women or girls. About this similar logo, Fulla is thought as the equal competitor for Barbie. The logos or symbol makes Fulla is easier to be accepted by people because people will notice that Fulla is another type of Barbie. People can think that Fulla is made to be another option of people especially parents who want their children to get the value that is suitable with their culture. Fulla’s and Barbie’s packagings are also quite similar. Paradigma, Jurnal Kajian Budaya 164 Barbie has become the ideal beauty of a woman.Barbie could convey the value to shape how people think about the ideal beauty. In fact, Barbie has looked like a beautiful western woman. Barbie is successful enough to show the true beauty of a woman. Many women even girls think that a beautiful creature looks like Barbie, having a beautiful face, fair skin, and the perfect body. Barbie as a symbol of woman’s beauty is actually only represent a particular culture, the culture of Western culture. For so many years, Barbie has dominated the concept of beauty and many people still buy Barbie for their children to play because there is no other choice. It is true that Barbie has many themes like President Barbie, Princess Barbie, Christmas Barbie, Holiday Barbie, Rapunzel, and many more, but it has no Muslimah Barbie yet. It becomes the good opportunity to introduce something like Barbie but in Moslem clothes and veil. Basically, Barbie can not be used as a standard concept of beauty, but since Barbie has been presented in the community through the physical and life which are almost perfect, most people consider that Barbie is the ideal figure, even though she only represents one culture. Barbie’s image can stick in many people’s minds because it is acceptable for them to see that concept of women’s beauty. Then from there came Fulla which provide resistance to Barbie. Fulla comes with a new beauty concept ala eastern culture. In fact, Fulla looks like Barbie, but there are still differences that differentiate Fulla from Barbie. Maybe many people think that Fulla is produced by the same industry like Barbie. Fulla is just like another theme of Barbie, but then we know that Fulla is produced by a different factory. Fulla came and convey the other concept of beauty. Fulla’s comingcan be reviewed further by the various aspects that have been discussed previously. Fulla which almost resembles Barbie that has many assertions that east and west are different. Fulla is not only to complement the concept of beauty of Western culture but also wanted to convey a new idea of beauty. Indirectly, Fulla appeared to win the hearts of the Moslem people in order to show that they should not follow the lifestyle and the concept of beauty from Barbie. The new concept of beauty here is from Fulla. Paradigma, Jurnal Kajian Budaya If we look at them carefully, we can see the difference, but if we just have a glance at them, we will see it almost the same. The shape and the color composition are quite similar, with pink as the dominant color. In addition, it is like the symbols of Fulla and Barbie that suitable with the packagings. Fulla also uses the similar packagings to Barbie to strengthen her position as the equal competitor to Barbie. So people will easily accept Fulla’s coming as another option for people to choose which doll can represent their culture most. Fulla Barbie Fulla Barbie Fulla Fulla Barbie Barbie Paradigma, Jurnal Kajian Budaya Fulla appears as something new, but after further review, Fulla is not really new. Fulla still uses the basic concepts of beauty of Barbie, but with little polished to resemble eastern society. Basically, they are not much different, so it does not seem to shift the concept of beauty of Barbie, it just adds a variation to a woman’s beauty. Fulla said to just add a variation of a woman’s beauty because physically, Fulla as if it wants to be the figure of Barbie with a slender body, flawless skin, long hair, legs, and beautiful eyes. Although they look different, but the real difference they have is other than physically. Their differences located in the carried values that the convey, about their daily life and habits. The life Fulla and Barbie are very different and they are actually getting an effect of the values they receive. This is where we see that as Fulla grew up in an eastern culture, but Barbie grew up in western cultures that thought to be Fulla’s and Barbie’s Images in Relation to Women’s Beauty, Juwita Anindya 165 more modern. Fulla and Barbie dress styles can also be regarded as a complement to the symbol of beauty in both. Fulla comes with a new beauty concept that introduce Moslem fashion. Through the terms of clothing, Fulla only offers another model of dress that is not inferior to Barbie’s western-style. According to an article by Zoepf (2005) “Bestseller in Mideast: Barbie With a Prayer Mat” (2005) says that now Fulla has become the answer to the most Moslem people to introduce the concept of beauty that can represent eastern culture although there are some people who do not feel special to its appearance. As a doll that like really like human beings, Fulla and Barbie indirectly can teach some value to many children who play with them. Children like to imitate what they learned from their favorite things, so many parents very concern in choosing the best toys for their children. Fulla can be the answer for many Moslem people in choosing a doll when they do not feel suitable with the value that is conveyed by Barbie. Most people realize that in this world there are two general differences of culture, eastern and western culture. Paradigma, Jurnal Kajian Budaya Paradigma, Jurnal Kajian Budaya 166 in the world. The producer also makes many innovations for Barbie, so many children in the world become addicted to collect many types or themes from Barbie. Barbie’s success is also seen from the well-known brand “Barbie” as many other products. Barbie also becomes the brand of clothes, stationary, bags, shoes, and many more. This can support Barbie’s position in the worldwide. Barbie is easy to be noticed and known. That is why Barbie also has many fans that make her become an idol. Children who make Barbie as their idol are tend to imitate what Barbie has and what Barbie does. This can make Barbie as the trendsetter in fashion or in daily life for many children. Even though Barbie only convey western value, while there is no other competitor, Barbie still dominate the people’s mind about the concept of ideal beauty that is conveyed by Barbie. Said’s Orientalism also said that there is another exotic thing in this world which exist in eastern culture. Fulla can be the one that proves it. The beauty of eastern women is now represented by Fulla doll. Fulla nowadays also becomes a famous brand, especially in Middle East countries. Brand “Fulla” becomes the brandof stationary and also food like cereals. This phenomenon can support the existence of Fulla to become the equal competitor for Barbie. Since there are many people especially Moslem can accept Fulla’s appearance, Fulla also can survive in many markets in the world to compete with Barbie. Fulla can be the equal competitor to balance Barbie’s domination before. The writer sees that even though Fulla is not as famous as Barbie, but Fulla has successfully enough to convey another concept of ideal beauty in this world, which is the beauty of eastern women. Fulla can be interpreted in many ways. Fulla can be said as an intertextuality or another form of Barbie5. Fulla also can be said to break the stereotype about western culture and eastern culture. In this topic, we can see Fulla as the representative of eastern beauty can convey new ideas of ideal beauty and also can bold the differences of eastern culture and western culture. On one side, Fulla makes the differences between western and eastern culture seems true. In the other side, Fulla can break the stereotype about which culture is more dominant and more modern. 5 The theory of intertextuality is a theory about how signs derives their meaning in a structure of a text. (Kristeva,1966). Paradigma, Jurnal Kajian Budaya Based on the theory of orientalism by Edward Said, the eastern is thought as the subordinate ones while western culture can dominate the world, but if we see from Fulla and Barbie, we can see that Fulla can be the equal competitor for Barbie. Barbie was successful to dominate the concept of ideal beauty, but the Fulla came in 2003 to convey another concept. Fulla’s concept of beauty is also accepted in many countries, especially in the Middle East.That makes Fulla can be said as the equal competitor for Barbie, so Barbie cannot dominate the world in the concept of ideal beauty that only represent one specific culture only. Fulla’s appearance also affirms that western culture and eastern culture are really different, like many people think. Besides the appearance, Fulla also affirms the differences of western culture and eastern culture from many sides, such as daily life and habits. These differences seem to be contradicting, based on how people think about it. For many western people, they can see that eastern culture is less modern, conservative, and old. If eastern people see western culture, they can think that western culture just knows to have fun and freedom. The assumption like this can appear if the difference is seen from a different point of view, but it is believed that is no right or wrong from this difference.ii Fulla’s first appearance is around 2003, which is much newer than Barbie’s first appearance. This factor can also be the reason that Barbie is more famous than Fulla. Barbie becomes well-known by most people in the world. Even though Barbie has many types of friend, her friends are also looking like so American. Here, the words “so American” means that Barbie’s friends just represent the multiculturalism in America. The writer says this because we can see from their appearances and habits that represent the western culture only. For many years, Barbie, her friends and boyfriend could dominate the world as the human-like dolls that can convey some values about life. This interesting doll is very successful in conveying some values. We can see that Barbie can survive in many markets Paradigma, Jurnal Kajian Budaya Fulla’s appearance is acceptable and it can replace Barbie in many countries in the world, especially in the Middle East. If we imagine Fulla and Barbie, it is more like an equal competitor that conveys the different concept of beauty and culture. It’s just like giving a choice which one is more suitable to the people who choose them, not which one is better. We now more notice that there are some real differences in this world that cannot be represented by only one representative. If there is only one representative that represent the culture in this world, that is not enough. Many other people can not feel 167 Fulla’s and Barbie’s Images in Relation to Women’s Beauty, Juwita Anindya being represented by that one. So, this happens to Barbie and Fulla that they can offer two general differences of cultural differences in this world equally6. People can choose the most suitable doll to be played with. 6 This is like breaking the stereotype that thought Western countries are more powerful than Eastern countries because in the past, Eastern countries were colonized by Western countries. Conclusion From this article, the writer tries to compare between Fulla and Barbie as the doll that conveys a concept of ideal beauty. Barbie, which appears earlier than Fulla could convey a concept of ideal beauty that only represents the beauty of western women. This western concept of beauty seems like dominating the world. Then Fulla appeared around 2003 and convey another concept of ideal beauty. Fulla represents the beauty of Eastern women. Manufactured by a different factory from Barbie, Fulla is the equal competitor for Barbie to convey another concept of ideal beauty. The writer then takes a deeper look on Barbie’s and Fulla’s characteristics to compare them from many aspects. Their comparison emphasizes the true differences between Western culture and Eastern culture. The differences are though as unequal differences that many people still thought that Eastern culture is subordinate while Western culture is more modern and powerful. After comparing both of the dolls, the writer found that Fulla and Barbie is not really the same, but they can convey their own cultures. Fulla as the representative of Eastern beauty is strong enough to compete with Barbie as the representative of Western beauty. Fulla also emphasizes the cultural differences in this world, but in this modern age, those differences become the choices for people to choose where they are.Fulla is also accepted in many countries especially in Middle East countries because the value that is embodied in Fulla is more suitable to the people there rather than Barbie’s value of beauty. This research is based on the analysis of Fulla’s and Barbie’s characteristics that are thought as the equal representative to her own culture in conveying the concept of women’s beauty. The writer aims this research to be useful in study about stereotype and also cultural difference. It also can give information about another doll beside Barbie that can convey some values to about culture. Then the people in this world can more notice that there are cultural differences here, but these differences are equal, which are West and East. Fulla and Barbie are now the representatives for their own culture and ready to be equal competitor for each other. Moore-Gilbert, Bart. 2000. Postcolonial Theory: Context, Practices, Politics. London: Verso. Hobsbawn, Eric and Terence Ranger (editors). 2004. The Invention of Tradition. Paradigma, Jurnal Kajian Budaya Paradigma, Jurnal Kajian Budaya 168 References Moore-Gilbert, Bart. 2000. Postcolonial Theory: Context, Practices, Politics. London: Verso. Verso. Hobsbawn, Eric and Terence Ranger (editors). 2004. The Invention Paradigma, Jurnal Kajian Budaya 168 Cambridge: Cambridge University Press. Labib, Malak. 2006. “Veiled Fulla is Arab Answer to Barbie” http://www.middle- east-online.com/english/?id=15449 Reischer, Erica and Kathryn S, Koo. “The Body Beautiful: Symbolism and Agency in the Social World”. http://www.jstor.org/stable/25064855?seq=4&Search=yes&searchText =concept&searchText=ideal&searchText=beauty&list=hide&searchUri=%2Faction%2Fd oBasicSearch%3FQuery%3Dconcept%2Bof%2Bideal%2Bbeauty%26gw%3Djtx%26acc% 3Don%26prq%3Deastern%2BAND%2Bwestern%2Bculture%26Search%3DSearch%26hp %3D25%26wc%3Don&prevSearch=&item=16&ttl=33330&returnArticleService=showFul lText&resultsServiceName=null>@73` Said, Edward W. 1978. Orientalism. United States: Random House Inc. Zoepf, Katherine. 2005. “Best Seller in Mideast: Barbie with a Prayer Mat”, http:// www.nytimes.com/2005/09/22/international/middleeast/22doll.html?ex=1285041600&en =72bb8cc089bf9435&ei=5090 Cambridge: Cambridge University Press. Labib, Malak. 2006. “Veiled Fulla is Arab Answer to Barbie” http://www.middle- east-online.com/english/?id=15449 Reischer, Erica and Kathryn S, Koo. “The Body Beautiful: Symbolism and Agency in the Social World”. http://www.jstor.org/stable/25064855?seq=4&Search=yes&searchText =concept&searchText=ideal&searchText=beauty&list=hide&searchUri=%2Faction%2Fd oBasicSearch%3FQuery%3Dconcept%2Bof%2Bideal%2Bbeauty%26gw%3Djtx%26acc% 3Don%26prq%3Deastern%2BAND%2Bwestern%2Bculture%26Search%3DSearch%26hp %3D25%26wc%3Don&prevSearch=&item=16&ttl=33330&returnArticleService=showFul lText&resultsServiceName=null>@73` Said, Edward W. 1978. Orientalism. United States: Random House Inc. Zoepf, Katherine. 2005. “Best Seller in Mideast: Barbie with a Prayer Mat”, http:// www.nytimes.com/2005/09/22/international/middleeast/22doll.html?ex=1285041600&en =72bb8cc089bf9435&ei=5090
https://openalex.org/W4388405809	https://www.medrxiv.org/content/medrxiv/early/2023/11/06/2023.11.05.23298118.full.pdf	English	null	A community-based mentoring scheme for pregnant and parenting adolescents in Sierra Leone: protocol for a hybrid pilot cluster randomised controlled trial	medRxiv (Cold Spring Harbor Laboratory)	2,023	cc-by	18,111	. CC-BY 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 6, 2023. ; https://doi.org/10.1101/2023.11.05.23298118 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 6, 2023. ; https://doi.org/10.1101/2023.11.05.23298118 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint (which was not certified by peer review) preprint The copyright holder for t this version posted November 6, 2023. ; https://doi.org/10.1101/2023.11.05.23298118 doi: medRxiv preprint 1 A community-based mentoring scheme for pregnant and parenting 2 adolescents in Sierra Leone: protocol for a hybrid pilot cluster randomised 3 controlled trial 4 5 Fernandez Turienzo C1, Kamara M2, November L1, Kamara P3, Kingsford AM4, Ridout A1 Thomas 6 S4, Seed PT1, Shennan AH1, Sandall J1#,Williams PT3# 7 8 9 1Department of Women and Children’s Health, Faculty of Life Sciences & Medicine, King’s College 10 London, London, United Kingdom 11 2University of Sierra Leone, Freetown, Sierra Leone 12 3Lifeline Nehemiah Projects, Freetown, Sierra Leone 13 4Welbodi Partnership, Freetown, Sierra Leone 14 Joint first authors 15 #Joint last authors 16 17 18 19 20 21 22 23 24 25 26 27 28 29 Corresponding author: Dr Cristina Fernandez Turienzo, Department of Women & Children’s Health, 30 School of Life Course and Population Sciences, Faculty of Life Sciences & Medicine, King’s College 31 London, Westminster Bridge Road, London, SE1 7EH, United Kingdom; 32 cristina.fernandez_turienzo@kcl.ac.uk 33 . CC-BY 4.0 International license It is made available under a perpetuity. . CC-BY 4.0 International license It is made available under a perpetuity. . CC-BY 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 6, 2023. ; https://doi.org/10.1101/2023.11.05.23298118 doi: medRxiv preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 6, 2023. ; https://doi.org/10.1101/2023.11.05.23298118 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 6, 2023. ; https://doi.org/10.1101/2023.11.05.23298118 doi: medRxiv preprint perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in was not certified by peer review) The copyright holder for this this version posted November 6, 2023. ; https://doi.org/10.1101/2023.11.05.23298118 doi: . CC-BY 4.0 International license It is made available under a pe petu ty 36 Abstract 37 Background 38 Sierra Leone has a very high maternal mortality rate, and this burden falls heavily on adolescents, a 39 particularly vulnerable group; this is usually driven by poverty, lack of education and employment 40 opportunities. In 2017, a local grassroots organisation, Lifeline Nehemiah Projects, developed a 41 community-based mentoring intervention ‘2YoungLives’ (2YLs) for adolescent girls in Eastern 42 Freetown. We aim to formally assess the feasibility and implementation of the 2YL mentorship 43 scheme in new communities in Sierra Leone. 44 45 Methods 46 A hybrid type 2 pilot cluster randomised controlled trial of the 2YL mentoring scheme in urban and 47 rural communities living around twelve peripheral health units (PHU) across five districts in Sierra 48 Leone. Clusters will be matched into pairs and randomisation will be determined by computer- 49 generated random numbers via a secure web-based system hosted by MedSciNet. All under- 50 eighteen adolescents identified as pregnant in the community and/or the PHU are included. 51 Feasibility (recruitment, retention, and attrition rates; data collection and completeness; sample 52 calculation) and primary clinical outcome data (composite of maternal deaths, stillbirths, neonatal 53 deaths) will be collected. A mixed-methods process evaluation will explore implementation 54 outcomes, mechanisms of change, contextual factors, experiences of care, and health and wellbeing. 55 A concurrent cost-consequence analysis will be undertaken. Main trial analysis will be pragmatic, by 56 intention to treat, and a complementary per protocol analysis will also be included. 57 58 Discussion 59 Improving health and wellbeing for adolescent girls (including sexual and reproductive health) 60 remains a top priority in Sierra Leone indicated by several government policies targeted to this group, 61 in which maternal and infant mortality are still persistently high. Supporting these girls and facilitating 62 their wellbeing is imperative, along with sensitisation of communities, strengthening of youth 63 friendly services and collaboration with stakeholders at all levels (government, regional, community, 64 family). We believe 2YL supports the global holistic agenda to integrate and implement interventions 65 across health, education, and social systems in order to protect, nurture, and support the health and 66 development potential of every adolescent girl, and thus become a model of good practice for 67 adolescent pregnancy to be adopted more widely in Sierra Leone and elsewhere 37 Background 38 Sierra Leone has a very high maternal mortality rate, and this burden falls heavily on adolescents, a 39 particularly vulnerable group; this is usually driven by poverty, lack of education and employment 40 opportunities. In 2017, a local grassroots organisation, Lifeline Nehemiah Projects, developed a 41 community-based mentoring intervention ‘2YoungLives’ (2YLs) for adolescent girls in Eastern 42 Freetown. We aim to formally assess the feasibility and implementation of the 2YL mentorship 43 scheme in new communities in Sierra Leone. 44 2 44 45 Methods 46 A hybrid type 2 pilot cluster randomised controlled trial of the 2YL mentoring scheme in urban and 47 rural communities living around twelve peripheral health units (PHU) across five districts in Sierra 48 Leone. Clusters will be matched into pairs and randomisation will be determined by computer- 49 generated random numbers via a secure web-based system hosted by MedSciNet. All under- 50 eighteen adolescents identified as pregnant in the community and/or the PHU are included. 51 Feasibility (recruitment, retention, and attrition rates; data collection and completeness; sample 52 calculation) and primary clinical outcome data (composite of maternal deaths, stillbirths, neonatal 53 deaths) will be collected. A mixed-methods process evaluation will explore implementation 54 outcomes, mechanisms of change, contextual factors, experiences of care, and health and wellbeing. 55 A concurrent cost-consequence analysis will be undertaken. Main trial analysis will be pragmatic, by 56 intention to treat, and a complementary per protocol analysis will also be included. 57 58 Discussion 59 Improving health and wellbeing for adolescent girls (including sexual and reproductive health) 60 remains a top priority in Sierra Leone indicated by several government policies targeted to this group, 61 in which maternal and infant mortality are still persistently high. Supporting these girls and facilitating 62 their wellbeing is imperative, along with sensitisation of communities, strengthening of youth 63 friendly services and collaboration with stakeholders at all levels (government, regional, community, 64 family). We believe 2YL supports the global holistic agenda to integrate and implement interventions 65 across health, education, and social systems in order to protect, nurture, and support the health and 66 development potential of every adolescent girl, and thus become a model of good practice for 67 adolescent pregnancy, to be adopted more widely in Sierra Leone and elsewhere. 68 69 Trial registration 70 ISRCTN registry ISRCTN32414369. Prospectively registered on 14/03/2022. 71 . CC-BY 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 6, 2023. ; https://doi.org/10.1101/2023.11.05.23298118 doi: medRxiv preprint 1 A community-based mentoring scheme for 2 adolescents in Sierra Leone: protocol for a hybri 3 controlled trial 4 5 Fernandez Turienzo C1, Kamara M2, November L1, Kamara P3, K 6 S4, Seed PT1, Shennan AH1, Sandall J1#,Williams PT3# 7 8 9 1Department of Women and Children’s Health, Faculty of Life S 10 London, London, United Kingdom 11 2University of Sierra Leone, Freetown, Sierra Leone 12 3Lifeline Nehemiah Projects, Freetown, Sierra Leone 13 4Welbodi Partnership, Freetown, Sierra Leone 14 Joint first authors 15 #Joint last authors 16 17 18 19 20 21 22 23 24 25 26 27 28 29 Corresponding author: Dr Cristina Fernandez Turienzo, Departme 30 School of Life Course and Population Sciences, Faculty of Life S 31 London, Westminster Bridge Road, London, SE 32 cristina.fernandez_turienzo@kcl.ac.uk 33 34 35 CC-BY 4.0 Internatio It is made available under a NOTE: This preprint reports new research that has not been certified by peer review a 1 A community-based mentoring scheme for pregnant and parenting 2 adolescents in Sierra Leone: protocol for a hybrid pilot cluster randomised 3 controlled trial 4 5 Fernandez Turienzo C1, Kamara M2, November L1, Kamara P3, Kingsford AM4, Ridout A1 Thomas 6 S4, Seed PT1, Shennan AH1, Sandall J1#,Williams PT3# 7 8 9 1Department of Women and Children’s Health, Faculty of Life Sciences & Medicine, King’s College 10 London, London, United Kingdom 11 2University of Sierra Leone, Freetown, Sierra Leone 12 3Lifeline Nehemiah Projects, Freetown, Sierra Leone 13 4Welbodi Partnership, Freetown, Sierra Leone 14 Joint first authors 15 #Joint last authors 16 17 18 19 20 21 22 23 24 25 26 27 28 29 Corresponding author: Dr Cristina Fernandez Turienzo, Department of Women & Children’s Health, 30 School of Life Course and Population Sciences, Faculty of Life Sciences & Medicine, King’s College 31 London, Westminster Bridge Road, London, SE1 7EH, United Kingdom; 32 cristina.fernandez turienzo@kcl.ac.uk Corresponding author: Dr Cristina Fernandez Turienzo, Department of Women & Children’s Health, School of Life Course and Population Sciences, Faculty of Life Sciences & Medicine, King’s College London, Westminster Bridge Road, London, SE1 7EH, United Kingdom; cristina.fernandez_turienzo@kcl.ac.uk 34 35 NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice. 1 1 . CC-BY 4.0 International license It is made available under a perpetuity. 45 Methods 46 A hybrid type 2 pilot cluster randomised controlled trial of the 2YL mentoring scheme in urban and 47 rural communities living around twelve peripheral health units (PHU) across five districts in Sierra 48 Leone. Clusters will be matched into pairs and randomisation will be determined by computer- 49 generated random numbers via a secure web-based system hosted by MedSciNet. All under- 50 eighteen adolescents identified as pregnant in the community and/or the PHU are included. 51 Feasibility (recruitment, retention, and attrition rates; data collection and completeness; sample 52 calculation) and primary clinical outcome data (composite of maternal deaths, stillbirths, neonatal 53 deaths) will be collected. A mixed-methods process evaluation will explore implementation 54 outcomes, mechanisms of change, contextual factors, experiences of care, and health and wellbeing. 55 A concurrent cost-consequence analysis will be undertaken. Main trial analysis will be pragmatic, by 56 intention to treat, and a complementary per protocol analysis will also be included. 57 71 2 2 . CC-BY 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 6, 2023. ; https://doi.org/10.1101/2023.11.05.23298118 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 6, 2023. ; https://doi.org/10.1101/2023.11.05.23298118 doi: medRxiv preprint perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in was not certified by peer review) The copyright holder for this this version posted November 6, 2023. ; https://doi.org/10.1101/2023.11.05.23298118 doi: . CC-BY 4.0 International license It is made available under a perpetuity. 72 73 Keywords 74 Community-based interventions; mentoring; pregnant and parenting adolescents; Sierra Leone 75 76 77 Introduction 78 Maternal mortality rate in Sierra Leone remains very high despite recent improvements (717 deaths 79 per 100 000 livebirths in 2019) [1] with adolescent pregnancy a leading cause of death for mothers; 80 adolescent mothers are 40-60% more likely to die during childbirth [2,3]. A household survey 81 conducted post-Ebola in 2015 indicated a maternal death rate of 1 in 10 for under-18-year-olds in a 82 poor suburb of Eastern Freetown. 73 Keywords In the 2YL cohort 103 of young women who had mentors there were no maternal deaths (0% vs 10%) and lower levels of 104 perinatal mortality (6% vs 16%), and infant mortality (11% vs 26%). Nearly all women receiving the 105 2YL intervention gave birth with a skilled birth attendant and breastfed for longer than 6 months; 106 and more than two thirds were using contraception by the baby’s first birthday, with no second 73 Keywords 77 Introduction 78 Maternal mortality rate in Sierra Leone remains very high despite recent improvements (717 deaths 79 per 100 000 livebirths in 2019) [1] with adolescent pregnancy a leading cause of death for mothers; 80 adolescent mothers are 40-60% more likely to die during childbirth [2,3]. A household survey 81 conducted post-Ebola in 2015 indicated a maternal death rate of 1 in 10 for under-18-year-olds in a 82 poor suburb of Eastern Freetown. A subsequent study examined the causes of this high incidence of 83 maternal death in adolescents and highlighted intersecting health and socio-economic vulnerabilities 84 [4]. Among key findings were that pregnant adolescents were often neglected by their families, 85 particularly when being cared for by a non-parental adult, often sleeping on bare ground without a 86 mosquito net, and fed once a day in exchange for heavy domestic duties such as water collection and 87 laundering, in many cases with little protection from ongoing gender-based violence. This lack of 88 adult care or support often leads to delays in care-seeking or complete lack of antenatal and delivery 89 care, putting girls at high risk of death from undetected pre-eclampsia, untreated infections, 90 anaemia, lack of birth preparation, and other common obstetric risks [4]. To achieve the Sustainable 91 Development Goals target of reducing the maternal mortality rate to less than 70 per 100 000 live 92 births by 2030, adolescent girls are a priority group [5-7]. 93 94 These findings led a grassroots organisation, Lifeline Nehemiah Projects (LNP), to develop a holistic 95 and locally designed community-based mentoring intervention, 2YoungLives (2YL), from pregnancy 96 through to one-year post-birth for adolescent girls [8]. Mentors support mentees to take up 97 antenatal care and hospital birth, re-establish family connections where this is safe and appropriate, 98 run a small business, and return to education or start vocational training. They promote and model 99 early health-seeking behaviour, and provide practical advice about childbirth, parenting and 100 contraception. The 2YL scheme was developed in 2017 in a suburb of the capital, Freetown, and 101 expanded to five new sites in 2018, 2019 and 2021. A formative evaluation compared outcomes of 102 young girls pre-intervention with those of other women post 2YL intervention [9]. 45 Methods A subsequent study examined the causes of this high incidence of 83 maternal death in adolescents and highlighted intersecting health and socio-economic vulnerabilities 84 [4]. Among key findings were that pregnant adolescents were often neglected by their families, 85 particularly when being cared for by a non-parental adult, often sleeping on bare ground without a 86 mosquito net, and fed once a day in exchange for heavy domestic duties such as water collection and 87 laundering, in many cases with little protection from ongoing gender-based violence. This lack of 88 adult care or support often leads to delays in care-seeking or complete lack of antenatal and delivery 89 care, putting girls at high risk of death from undetected pre-eclampsia, untreated infections, 90 anaemia, lack of birth preparation, and other common obstetric risks [4]. To achieve the Sustainable 91 Development Goals target of reducing the maternal mortality rate to less than 70 per 100 000 live 92 births by 2030, adolescent girls are a priority group [5-7]. 93 94 These findings led a grassroots organisation, Lifeline Nehemiah Projects (LNP), to develop a holistic 77 Introduction 94 These findings led a grassroots organisation, Lifeline Nehemiah Projects (LNP), to develop a holistic 95 and locally designed community-based mentoring intervention, 2YoungLives (2YL), from pregnancy 96 through to one-year post-birth for adolescent girls [8]. Mentors support mentees to take up 97 antenatal care and hospital birth, re-establish family connections where this is safe and appropriate, 98 run a small business, and return to education or start vocational training. They promote and model 99 early health-seeking behaviour, and provide practical advice about childbirth, parenting and 100 contraception. The 2YL scheme was developed in 2017 in a suburb of the capital, Freetown, and 101 expanded to five new sites in 2018, 2019 and 2021. A formative evaluation compared outcomes of 102 young girls pre-intervention with those of other women post 2YL intervention [9]. In the 2YL cohort 103 of young women who had mentors there were no maternal deaths (0% vs 10%) and lower levels of 104 perinatal mortality (6% vs 16%), and infant mortality (11% vs 26%). Nearly all women receiving the 105 2YL intervention gave birth with a skilled birth attendant and breastfed for longer than 6 months; 106 and more than two thirds were using contraception by the baby’s first birthday, with no second 3 . CC-BY 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 6, 2023. ; https://doi.org/10.1101/2023.11.05.23298118 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 6, 2023. ; https://doi.org/10.1101/2023.11.05.23298118 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a perpetuity. 107 pregnancies. All of the 2YL young women successfully ran a small business with support from their 108 mentor, allowing them to learn business skills, eat well throughout pregnancy and provide for their 109 babies. They reported increased self-confidence, supportive peer relationships, and a high level of 110 satisfaction with the mentoring scheme [9]. 77 Introduction The design is a pragmatic hybrid pilot cluster-randomised controlled trial 163 (cRCT) of the introduction of the 2YL mentoring adjunct to maternity care in Sierra Leone. The trial 164 structure is based on the Medical Research Council’s (MRC’s) guideline for developing and evaluating 165 complex interventions [15], and Curran and colleagues’ hybrid type 2 effectiveness-implementation 166 research design that place similar focus on assessing effectiveness of an intervention as well as how 167 best to implement it [16]. The trial will be reported according to the Consolidated Standards of 168 Reporting Trials (CONSORT) statement for randomised pilot and feasibility trials [17], the Standard 169 Protocol Items: Recommendations for Interventional Trials (SPIRIT) statement [18] (S1 SPIRIT 170 Checklist) as well as the Template for Intervention Description and Replication (TIDieR) checklist for 171 intervention description [19]. The trial has been prospectively registered in the International 172 Standard Randomised Controlled Trial Number Registry (ISRCTN32414369) and approved by ethics 173 committee at King’s College London UK (HR/DP-21/22-26320) and the Office of the Sierra Leone 174 Ethics and Scientific Review Committee. The (SPIRIT) Figure adapted for the 2YL trial is shown below 175 in Figure 1. Any protocol amendments will be notified to ethics committees and all collaborators 176 informed. 177 . CC-BY 4.0 International license It is made available under a perpetuity. s t e aut o u de , o as g a ted ed a ce se to d sp ay t e p ep t ( c as ot ce t ed by pee e e ) p ep t 143 hygiene [11]. Sexual violence and rape are common, and capacity to provide treatment to affected 144 women and girls is extremely limited (State of Emergency over rape and sexual violence was declared 145 in 2019) [12]. Sierra Leone also suffers an inadequacy of human resources for health; it has 2 skilled 146 providers (doctors, nurses and midwives) per 10,000 population which is well below WHO’s health 147 workforce targets for UHC and Sustainable Development Goal of 23 [13]. Suboptimal maternal and 148 child health care contributes to premature deaths, disability, and devastating spending in a country 149 with an incipient financial crisis exacerbated by the 2014 Ebola epidemic (and further weakened 150 during the ongoing COVID-19 epidemic and the global fuel and food instability in the wake of the war 151 in Ukraine). 77 Introduction Subsequent anecdotal evidence is also showing that the 111 community engagement strategy pursued as an integral part of the early implementation is 112 influencing widely-held ingrained attitudes towards practices which perpetuate gender inequality 113 such as school non-attendance in pregnancy and child marriage. 114 115 Based on this preliminary data, the 2YL mentoring scheme is promising [10]. A theory of change was 116 co-developed to understand how and why the 2YL intervention may work in adolescent girls in Sierra 117 Leone. 2YL has potential to improve the health and wellbeing of girls and their babies and sustainably 118 improve livelihoods. Relationship building, engagement and advocacy, educational, social, and 119 economic empowerment, and respectful community engagement and involvement are important 120 mechanisms of action to consider. However, more robust evidence is needed to understand the 121 impact and mechanisms of how 2YL can address determinants of adolescent maternal mortality, and 122 general health and wellbeing [10]. The overall aim of this study is therefore to assess the feasibility 123 and implementation of the 2YL mentorship scheme in new communities in Sierra Leone to inform 124 procedures for a subsequent fully powered cluster trial evaluating the effectiveness and social and 125 economic impact of 2YL. Specifically, the study objectives include: 126 - To assess the integrity of study protocol including: recruitment, randomization procedure, 127 data collection, retention procedures, acceptability, primary outcome measure to inform the 128 sample size calculation, refinement of the intervention and training and supervision 129 procedures. 130 - To assess pregnancy outcomes of adolescents receiving the 2YL intervention compared to 131 those receiving usual care. 132 - To compare experiences of care, mentoring, health and wellbeing, and thriving among young 133 women receiving the 2YL intervention compared to those receiving usual care. 34 - To evaluate the implementation, contextual factors and mechanisms of change of the 2YL 35 intervention in order to understand the results and its potential impact. 4 140 Sierra Leone, a low-income West African country with a population of 8.8 million, has a very high 141 maternal mortality rate and a high burden of communicable and non-communicable diseases. It 142 struggles to achieve basic universal health coverage and access to potable water, sanitation and 4 . CC-BY 4.0 International license It is made available under a perpetuity. 77 Introduction is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 6, 2023. ; https://doi.org/10.1101/2023.11.05.23298118 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 6, 2023. ; https://doi.org/10.1101/2023.11.05.23298118 doi: medRxiv preprint 143 hygiene [11]. Sexual violence and rape are common, and capacity to provide treatment to affected 144 women and girls is extremely limited (State of Emergency over rape and sexual violence was declared 145 in 2019) [12]. Sierra Leone also suffers an inadequacy of human resources for health; it has 2 skilled 146 providers (doctors, nurses and midwives) per 10,000 population which is well below WHO’s health 147 workforce targets for UHC and Sustainable Development Goal of 23 [13]. Suboptimal maternal and 148 child health care contributes to premature deaths, disability, and devastating spending in a country 149 with an incipient financial crisis exacerbated by the 2014 Ebola epidemic (and further weakened 150 during the ongoing COVID-19 epidemic and the global fuel and food instability in the wake of the war 151 in Ukraine). Despite recent improvements, adolescents account for nearly half of all maternal deaths, 152 and neonatal, infant and child mortality are also high; infants of mothers who die are up to 10 times 153 more likely to die within their first two years, while infants born to adolescent mothers are also at 154 increased risk of sickness and death [1,2]. Although healthcare services are free for pregnant women, 155 lactating mothers, and children below the age of five, antenatal attendance, access to skilled birth 156 attendants and facility deliveries are low, and quality service delivery is often poor with disparities in 157 access and availability to appropriate assessment and intervention, with delays in delivery, escalation 158 of care and emergency maternity care [14]. 159 160 The 2YL trial will run in the communities served by twelve peripheral healthcare units (PHUs); the 161 clusters, representing a range of urban and rural settings in five of the administrative regions or 162 districts of Sierra Leone. 77 Introduction is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 6, 2023. ; https://doi.org/10.1101/2023.11.05.23298118 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 6, 2023. ; https://doi.org/10.1101/2023.11.05.23298118 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a perpetuity. 179 Eligibility criteria for the clusters is not having been previously exposed to the 2YL. All adolescent girls 180 aged under 18 identified as pregnant in the community or presenting for maternity care at the 181 selected clusters within the trial time frame will be included. There will be no exclusion criteria. 182 183 Recruitment 184 Twelve clusters, (each with named communities routinely served by peripheral health units), will be 185 identified by local partners and invited to participate in the study by the local research staff. 186 Institutional-level consent and relevant local approvals from district health authorities and hospitals 187 on behalf of the cluster will be obtained at the start of the trial (time point zero), and prior to 188 intervention implementation. Participants living in the twelve clusters will be recruited by trained 189 data collectors and can be identified through community members and different registers (i.e., 190 antenatal, maternal and delivery register, referrals, outreach). In the event of changes to the PHUs 191 serving a community during the study period (e.g. existing PHUs closing) we may invite a PHU to 192 participate in the same randomised arm if it is serving the same community. 193 194 In intervention sites, pregnant adolescents can self-refer or be referred by a friend or family member 195 who has heard about the mentoring scheme from community engagement activities. As per usual 196 2YL practice, the local team coordinator will meet with eligible pregnant girls and enrol them into the 197 mentoring scheme as they come forward to a maximum of 36 (maximum number of girls that 198 mentors can support per cluster due to limited resources). This helps us to test the feasibility of the 199 intervention in a pragmatic way while avoiding selection bias. 77 Introduction Despite recent improvements, adolescents account for nearly half of all maternal deaths, 152 and neonatal, infant and child mortality are also high; infants of mothers who die are up to 10 times 153 more likely to die within their first two years, while infants born to adolescent mothers are also at 154 increased risk of sickness and death [1,2]. Although healthcare services are free for pregnant women, 155 lactating mothers, and children below the age of five, antenatal attendance, access to skilled birth 156 attendants and facility deliveries are low, and quality service delivery is often poor with disparities in 157 access and availability to appropriate assessment and intervention, with delays in delivery, escalation 158 of care and emergency maternity care [14]. 159 160 The 2YL trial will run in the communities served by twelve peripheral healthcare units (PHUs); the 161 clusters, representing a range of urban and rural settings in five of the administrative regions or 162 districts of Sierra Leone. The design is a pragmatic hybrid pilot cluster-randomised controlled trial 163 (cRCT) of the introduction of the 2YL mentoring adjunct to maternity care in Sierra Leone. The trial 164 structure is based on the Medical Research Council’s (MRC’s) guideline for developing and evaluating 165 complex interventions [15], and Curran and colleagues’ hybrid type 2 effectiveness-implementation 166 research design that place similar focus on assessing effectiveness of an intervention as well as how 167 best to implement it [16]. The trial will be reported according to the Consolidated Standards of 168 Reporting Trials (CONSORT) statement for randomised pilot and feasibility trials [17], the Standard 169 Protocol Items: Recommendations for Interventional Trials (SPIRIT) statement [18] (S1 SPIRIT 170 Checklist) as well as the Template for Intervention Description and Replication (TIDieR) checklist for 171 intervention description [19]. The trial has been prospectively registered in the International 172 Standard Randomised Controlled Trial Number Registry (ISRCTN32414369) and approved by ethics 173 committee at King’s College London UK (HR/DP-21/22-26320) and the Office of the Sierra Leone 174 Ethics and Scientific Review Committee. The (SPIRIT) Figure adapted for the 2YL trial is shown below 175 in Figure 1. Any protocol amendments will be notified to ethics committees and all collaborators 176 informed. 5 5 . CC-BY 4.0 International license It is made available under a perpetuity. 77 Introduction At their 6 weeks routine postnatal 200 appointment, girls in both arms will be given the option to individually enrol and participate in two 201 sub-studies (e.g. qualitative interviews, photovoice), and for those agreeing to participate in the sub- 202 studies, explicit written informed consent (including from parents or guardians) will be gained and 203 will be contacted around six-nine months after birth. Adolescents return to the PHU at one year for 204 their routine infant immunisations appointment. A flow chart for 2YL is presented in Figure 2. 205 206 Randomisation 207 The unit of randomisation is the trial area (or cluster), rather than the individual woman. The twelve 208 clusters will be matched into pairs accounting for cluster size (births by adolescents) and distance 209 from the referral hospitals. The clusters will be first allocated random cluster numbers and the 210 sequence generation for receiving the 2YL intervention will then be determined by computer- 179 Eligibility criteria for the clusters is not having been previously exposed to the 2YL. All adolescent girls 180 aged under 18 identified as pregnant in the community or presenting for maternity care at the 181 selected clusters within the trial time frame will be included. There will be no exclusion criteria. 182 179 Eligibility criteria for the clusters is not having been previously exposed to the 2YL. All adolescent girls 180 aged under 18 identified as pregnant in the community or presenting for maternity care at the 181 selected clusters within the trial time frame will be included. There will be no exclusion criteria. 182 183 Recruitment 184 Twelve clusters, (each with named communities routinely served by peripheral health units), will be 185 identified by local partners and invited to participate in the study by the local research staff. 186 Institutional-level consent and relevant local approvals from district health authorities and hospitals 187 on behalf of the cluster will be obtained at the start of the trial (time point zero), and prior to 188 intervention implementation. Participants living in the twelve clusters will be recruited by trained 189 data collectors and can be identified through community members and different registers (i.e., 190 antenatal, maternal and delivery register, referrals, outreach). In the event of changes to the PHUs 191 serving a community during the study period (e.g. existing PHUs closing) we may invite a PHU to 192 participate in the same randomised arm if it is serving the same community. 194 In intervention sites, pregnant adolescents can self-refer or be referred by a friend or family member 195 who has heard about the mentoring scheme from community engagement activities. As per usual 196 2YL practice, the local team coordinator will meet with eligible pregnant girls and enrol them into the 197 mentoring scheme as they come forward to a maximum of 36 (maximum number of girls that 198 mentors can support per cluster due to limited resources). This helps us to test the feasibility of the 199 intervention in a pragmatic way while avoiding selection bias. At their 6 weeks routine postnatal 200 appointment, girls in both arms will be given the option to individually enrol and participate in two 201 sub-studies (e.g. qualitative interviews, photovoice), and for those agreeing to participate in the sub- 202 studies, explicit written informed consent (including from parents or guardians) will be gained and 203 will be contacted around six-nine months after birth. Adolescents return to the PHU at one year for 204 their routine infant immunisations appointment. A flow chart for 2YL is presented in Figure 2. 205 206 Randomisation 207 The unit of randomisation is the trial area (or cluster), rather than the individual woman. The twelve 208 clusters will be matched into pairs accounting for cluster size (births by adolescents) and distance 209 from the referral hospitals. The clusters will be first allocated random cluster numbers and the 210 sequence generation for receiving the 2YL intervention will then be determined by computer- 211 generated random numbers by the trial statistician via a secure web-based randomisation and data 212 management system hosted by MedSciNet with telephone back-up available at all times. The 213 clusters will be masked until they are informed of their allocation two weeks prior to their 214 implementation start date to give sufficient time for organizing community engagement activities, 6 . CC-BY 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 6, 2023. ; https://doi.org/10.1101/2023.11.05.23298118 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 6, 2023. ; https://doi.org/10.1101/2023.11.05.23298118 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a perpetuity. 215 leading to recruitment and training of mentors and coordinators. A minimisation algorithm will also 216 be used to ensure balance between groups with respect to PHU use and distance to referral hospital. 217 The nature of the 2YL intervention is such that blinding of girls, mentors or healthcare providers 218 cannot be achieved. However, outcome assessment will be masked to the 2YL statistician and the 219 researchers who will analyse the data. 220 221 Interventions 222 Intervention: 2YL + usual maternity care 223 Clusters and participants in the intervention group will receive the different components of the 2YL 224 mentoring intervention (provided as an adjunct to usual maternity care). The structure of the 2YL 225 intervention is focused saving adolescents’ lives and promoting their health and development. 206 Randomisation 226 227 Key roles: The intervention is run on the ground by a team from Lifeline Nehemiah Projects; with 228 oversight from LNP’s Executive Director, a CEI specialist who leads on the initial community 229 engagement activities. The project manager, a gender specialist, leads on recruitment, training and 230 supervision of mentor teams, with support from a lead coordinator, and administrative and finance 231 assistance from the LNP team. 232 233 Community engagement and involvement (CEI): this is a core first component of the 2YL intervention. 234 LNP has devised a CEI strategy with three trips to each site to pave the way for the acceptance and 235 tailoring of the intervention, considering local contextual factors, understanding how communities 236 operate, and engaging those who would bring out various perspectives. Listening, discussing, and 237 connecting with communities is imperative to build trusting relationships, and more CEI activities are 238 undertaken in a bespoke fashion, depending on the specific requirements of each community. 239 240 Recruitment and training of mentors: women with a passion to support vulnerable girls are identified 241 during CEI activities and recruited as mentors in collaboration with community stakeholders based 242 on their experience, availability, knowledge of the community, commitment to improve girls’ lives 243 and for having a reputation for kindness and being trustworthy. Each team also has a local 244 coordinator who has the additional skill of literacy and record keeping allowing her to record details 245 of activities and feed back to the central LNP team. Each site is scheduled to have a team of four 246 mentors (and 1 local coordinator). Training and supervision: mentors and coordinators receive a 4- 247 day manualised training programme using discussion, role play and bespoke pictorial maternal and 248 newborn health knowledge and resources to learn and share important health messages and give 249 practical support. The training emphasises the importance of commitment and confidentiality and is 240 Recruitment and training of mentors: women with a passion to support vulnerable girls are identified 241 during CEI activities and recruited as mentors in collaboration with community stakeholders based 242 on their experience, availability, knowledge of the community, commitment to improve girls’ lives 243 and for having a reputation for kindness and being trustworthy. 206 Randomisation Each team also has a local 244 coordinator who has the additional skill of literacy and record keeping allowing her to record details 245 of activities and feed back to the central LNP team. Each site is scheduled to have a team of four 246 mentors (and 1 local coordinator). Training and supervision: mentors and coordinators receive a 4- 247 day manualised training programme using discussion, role play and bespoke pictorial maternal and 248 newborn health knowledge and resources to learn and share important health messages and give 249 practical support. The training emphasises the importance of commitment and confidentiality and is 250 provided by an experienced educationalist with experience of training community members. 7 . CC-BY 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 6, 2023. ; https://doi.org/10.1101/2023.11.05.23298118 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint (which was not certified by peer review) preprint The copyright holder for t this version posted November 6, 2023. ; https://doi.org/10.1101/2023.11.05.23298118 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 6, 2023. ; https://doi.org/10.1101/2023.11.05.23298118 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 6, 2023. ; https://doi.org/10.1101/2023.11.05.23298118 doi: medRxiv preprint 251 Ongoing support and supervision are provided to mentors by both their local coordinators and the 252 LNP’s 2YL central management team. Mentors and coordinators are all volunteers who receive a 253 monthly stipend to cover out of pocket expenses. 254 255 Matching of mentor-mentees: Adolescent girls are recruited to 2YL at any stage of pregnancy, and 256 receive the mentoring for one year after birth (regardless of pregnancy outcome). 206 Randomisation They are identified 257 by mentors in their localities, often by the girl herself hearing about the mentoring scheme from a 258 peer, and approaching the mentor, or by the mentor hearing of a teenage pregnancy and asking the 259 girl if she would like to join the scheme. Geographical proximity and shared tribal language are taken 260 in consideration for matching girls with mentors. A total of 3-4 girls are matched to one mentor who 261 will work with the adolescent for the entire one-year period unless covering or changing a mentor if 262 needed for any arising issues. 263 264 2YoungLives activities include: at least weekly face to face meetings between mentor and mentee 265 where the mentee feels comfortable and confidential conversation is possible; promotion of health 266 services uptake; reminding to attend or attending with mentee for usual antenatal care; 267 accompanying mentee to health facility when in labour or needing emergency care (or ensuring other 268 birth partner is available according to mentee’s wishes); visits from mentor to mentee’s family to 269 advocate for family support, if safe and appropriate; flexible and practical support for pregnancy and 270 parenthood dependent on mentee’s support network; discussion with mentee to consider small 271 business options and accompanying mentee to purchase first supply of goods; encouragement to 272 return to school or start vocational training; a practical session making healthy baby food; 273 encouragement to take up post-partum contraception; the importance of first line home treatment 274 and early health-seeking behaviour for their baby; and an informal ‘graduation’ celebration at the 275 end of the mentoring scheme. They also run monthly site meetings in the community with all mentors 276 and mentees for peer support, cooking, eating and group discussion. At each of these sessions, 277 visitors such as healthcare providers from local health facilities, teachers, other community members 278 are invited for discussions about health topics or educational or training opportunities. 279 280 There are some aspects of the intervention that can be tailored depending on individual needs and 281 circumstances; for example, if mentees are identified who have physical or intellectual disability, the 282 team can enhance support with more visits and provide communication cards (for example for a deaf 283 girl to use in labour) or by monitoring uptake of routine pregnancy medication (for girl with a learning 284 disability). 206 Randomisation is the author/funder, who has granted medRxiv a license to display the preprint (which was not certified by peer review) preprint The copyright holder for t this version posted November 6, 2023. ; https://doi.org/10.1101/2023.11.05.23298118 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 6, 2023. ; https://doi.org/10.1101/2023.11.05.23298118 doi: medRxiv preprint 286 Usual or routine maternity care is described below and will be the same in both intervention and 287 control groups. 288 289 Control: usual maternity care 290 The comprehensive care package will follow local and national guidelines for maternity care in Sierra 291 Leone [20, 21]. Intervention packages prioritised under antenatal care are aligned to the 2016 WHO 292 Antenatal model that recommends a minimum of eight antenatal contacts, with an overarching aim 293 of providing pregnant adolescents or women with quality, respectful, individualised, person-centred 294 care [22]. The latest Sierra Leone National Reproductive, Maternal, Newborn, Child and Adolescent 295 Health Strategy outlines 1) interventions to be delivered under the antenatal care for positive 296 pregnancy across the different service delivery levels, and 2) the priority for skilled birth attendance 297 and essential newborn care all packaged under emergency obstetric and newborn care (EmONC) 298 [20]. 299 300 301 Outcomes 302 303 Primary feasibility outcomes: 304 Eligibility, recruitment, retention and attrition rates 305 Data collection and data completeness 306 Selection of most appropriate primary outcome measure and sample size calculation 307 308 Primary clinical outcome: 309 The primary clinical outcome is a composite of maternal death (all-cause, occurring during 310 pregnancy, labour, or within 42 days of birth), stillbirth (born with no signs of life at or after 28 weeks 311 of pregnancy, but before or during birth) and neonatal death (deaths among live births during the 312 first 28 days). We will report the effect of the 2YL intervention on both the composite and its 313 components for all woman/baby deaths in the study; those who experience any one of the 314 components will be considered to have experienced the composite outcome. 289 Control: usual maternity care 309 The primary clinical outcome is a composite of maternal death (all-cause, occurring during 310 pregnancy, labour, or within 42 days of birth), stillbirth (born with no signs of life at or after 28 weeks 311 of pregnancy, but before or during birth) and neonatal death (deaths among live births during the 312 first 28 days). We will report the effect of the 2YL intervention on both the composite and its 313 components for all woman/baby deaths in the study; those who experience any one of the 314 components will be considered to have experienced the composite outcome. Evaluating the data 315 collection strategy and tool is an important element of testing the feasibility of a larger trial; for 316 example, whether collecting outcome data up to six weeks postnatally is possible due to the mobility 317 of this population. 318 206 Randomisation The whole team will always follow LNP’s safeguarding policies. 285 264 2YoungLives activities include: at least weekly face to face meetings between mentor and mentee 265 where the mentee feels comfortable and confidential conversation is possible; promotion of health 266 services uptake; reminding to attend or attending with mentee for usual antenatal care; 267 accompanying mentee to health facility when in labour or needing emergency care (or ensuring other 268 birth partner is available according to mentee’s wishes); visits from mentor to mentee’s family to 269 advocate for family support, if safe and appropriate; flexible and practical support for pregnancy and 270 parenthood dependent on mentee’s support network; discussion with mentee to consider small 271 business options and accompanying mentee to purchase first supply of goods; encouragement to 272 return to school or start vocational training; a practical session making healthy baby food; 273 encouragement to take up post-partum contraception; the importance of first line home treatment 274 and early health-seeking behaviour for their baby; and an informal ‘graduation’ celebration at the 275 end of the mentoring scheme. They also run monthly site meetings in the community with all mentors 276 and mentees for peer support, cooking, eating and group discussion. At each of these sessions, 277 visitors such as healthcare providers from local health facilities, teachers, other community members 278 are invited for discussions about health topics or educational or training opportunities. 279 280 There are some aspects of the intervention that can be tailored depending on individual needs and 281 circumstances; for example, if mentees are identified who have physical or intellectual disability, the 282 team can enhance support with more visits and provide communication cards (for example for a deaf 283 girl to use in labour) or by monitoring uptake of routine pregnancy medication (for girl with a learning 284 disability). The whole team will always follow LNP’s safeguarding policies. 285 8 8 . CC-BY 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 6, 2023. ; https://doi.org/10.1101/2023.11.05.23298118 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a perpetuity. 206 Randomisation Evaluating the data 315 collection strategy and tool is an important element of testing the feasibility of a larger trial; for 316 example, whether collecting outcome data up to six weeks postnatally is possible due to the mobility 317 286 Usual or routine maternity care is described below and will be the same in both intervention and 287 control groups. 319 Secondary clinical and process outcomes 320 Secondary maternal outcomes will include intermittent preventive therapy for malaria, anaemia, 321 tetanus toxoid immunizations, caesarean sections, blood transfusions, birth at facility, sepsis, 9 . CC-BY 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 6, 2023. ; https://doi.org/10.1101/2023.11.05.23298118 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 6, 2023. ; https://doi.org/10.1101/2023.11.05.23298118 doi: medRxiv preprint 322 postpartum haemorrhage, place of birth and attendant, and uptake of contraception. Secondary 323 perinatal outcomes will include; gestational age, birthweight, Apgar score, resuscitation, immediate 324 breastfeeding, Kangaroo mother care, six weeks postnatal immunisation uptake, infant mortality. 325 326 Other secondary process and implementation outcomes will include service use, processes and 327 implementation outcomes including; antenatal checks, antenatal checks with blood pressure 328 measurement, referrals, postnatal check within a week of birth; measures of fidelity (e.g., mentors 329 and team coordinators recruited, training packages and workshops, weekly one to one meetings, 330 monthly social gatherings), acceptability (e.g. satisfaction with different components of the 331 intervention among young girls, mentors, healthcare providers and local stakeholders), reach, 332 adoption, women’s experiences of mentoring and care (e.g., respectful care access, trust, system 333 responsiveness, quality and safety, attitude to and uptake of contraception in the postnatal year) and 334 economic and social progress/wellbeing measures (e.g., return to education, experiences of running 335 small businesses, perspectives of thriving), mechanisms of change (e.g., access to care, advocacy, 336 support, respectful care, engagement, referral and escalation, health promotion, empowerment, 337 relationships) and for social and economic wellbeing (e.g., social and economic empowerment, self- 338 confidence, sense of agency, trustworthy adult and peers, respectful community engagement with 339 families). 340 341 Sample size 342 Sample size estimation has been provided by the 2YL trial statistician. 319 Secondary clinical and process outcomes Based on experience with 343 some of the proposed trial areas and global maternal health literature relevant to these settings [4- 344 7], we estimate the risk to adolescent pregnant women in Sierra Leone of experiencing a primary 345 outcome (one of maternal death or stillbirth or neonatal death with no double counting) to be 346 approximately 20% of all adolescent women. We also estimate a modest intra-cluster correlation 347 (ICC) of 0.02 (sites similar but not identical, and a simple exchangeable autocorrelation structure 348 based on previous experience in Sierra Leone) [23]. 349 350 Prior to full proposal development, the 2YL team visited communities served by a typical PHU similar 351 in size and distance to referral hospital to trial sites. At least 16% of all deliveries among teens had 352 resulted in maternal death, stillbirth, or early neonatal death. It was not possible to include late 353 neonatal deaths up to 28 days. In addition, girls who were referred to a referral hospital in an 354 emergency were not included in these numbers. We believe that this data could match the indicative 355 estimate of 20% of girls experiencing a primary outcome. 356 . CC-BY 4.0 International license It is made available under a 322 postpartum haemorrhage, place of birth and attendant, and uptake of contraception. Secondary 323 perinatal outcomes will include; gestational age, birthweight, Apgar score, resuscitation, immediate 324 breastfeeding, Kangaroo mother care, six weeks postnatal immunisation uptake, infant mortality. 325 326 Other secondary process and implementation outcomes will include service use, processes and 327 implementation outcomes including; antenatal checks, antenatal checks with blood pressure 328 measurement, referrals, postnatal check within a week of birth; measures of fidelity (e.g., mentors 329 and team coordinators recruited, training packages and workshops, weekly one to one meetings, 330 monthly social gatherings), acceptability (e.g. 319 Secondary clinical and process outcomes satisfaction with different components of the 331 intervention among young girls, mentors, healthcare providers and local stakeholders), reach, 332 adoption, women’s experiences of mentoring and care (e.g., respectful care access, trust, system 333 responsiveness, quality and safety, attitude to and uptake of contraception in the postnatal year) and 334 economic and social progress/wellbeing measures (e.g., return to education, experiences of running 335 small businesses, perspectives of thriving), mechanisms of change (e.g., access to care, advocacy, 336 support, respectful care, engagement, referral and escalation, health promotion, empowerment, 337 relationships) and for social and economic wellbeing (e.g., social and economic empowerment, self- 338 confidence, sense of agency, trustworthy adult and peers, respectful community engagement with 339 families). 10 10 . CC-BY 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 6, 2023. ; https://doi.org/10.1101/2023.11.05.23298118 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 6, 2023. ; https://doi.org/10.1101/2023.11.05.23298118 doi: medRxiv preprint 357 This study is planned as a pilot, with the main stated aim to demonstrate the feasibility of a fully 358 powered study, and to help with its design. But for the purpose of the power calculation (intention 359 to treat and not per protocol), an assumption of at least 42 deliveries per site has been made, with 360 20% having primary outcome composite events during the trial duration. These numbers will provide 361 84% power to detect a 52% relative risk reduction (RRR) of the primary outcome (from 20% to 9%) 362 assuming a models ICC 0.02. 363 364 Data collection 365 Primary and secondary pregnancy outcome 366 Primary and secondary pregnancy outcome data will be recorded over the study period and will be 367 collected at an individual level from study-specific and routine data collection tools (e.g., antenatal 368 attendance register, birth register, referral and outreach registers, 6-week postnatal check 369 appointment book, hospital registers). At some PHUs, an additional ‘adolescent book’ is in use to 370 record ANC registration of under-18-year-olds and associated birth outcomes. 319 Secondary clinical and process outcomes Data collection at both 371 intervention and control clusters will be done by local data collectors who will enter the data 372 anonymised onto MedSciNet, the study-specific management system. 373 374 Process Evaluation: experiences and implementation 375 In line with the current best practice in implementation research, the team will undertake a process 376 evaluation to understand variations in the impact of the intervention on outcomes of interest and to 377 contextualise findings. This will draw on the six core elements considered in the updated NIHR/MRC 378 framework for evaluating complex interventions which include: interactions between intervention 379 and context, theorising how the intervention may work, diversity of stakeholder’s perspectives in 380 research, uncertainties, refinement of the intervention and resource and outcome consequences of 381 the intervention [15]. A mixed methods approach will be used to assess implementation outcomes 382 and identification of mechanisms and contextual factors that may influence variation in outcomes 383 and experiences across multiple stakeholder groups, and at different stages of implementation. This 384 offers a 360-degree evaluation, which considers needs and perspectives that typically differ between 385 stakeholder groups and can vary over time. 386 387 Both quantitative and qualitative data on processes, experiences and implementation will be 388 collected from monthly checklists, separate focus groups with a purposive sample of mentors, PHU 389 healthcare providers and community stakeholders, and semi-structured interviews with some HCPs, 390 community leaders, regional and national stakeholders, and adolescent girls in control and 391 intervention sites. If appropriate, a family member or a friend will also be invited to participate in an 392 interview. Girls will be offered the opportunity to be one-to-one (interviewer and participant) or one- . CC-BY 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprin (which was not certified by peer review) preprint 357 This study is planned as a pilot, with the main stated aim to demonstrate the feasibility of a fully 358 powered study, and to help with its design. But for the purpose of the power calculation (intention 359 to treat and not per protocol), an assumption of at least 42 deliveries per site has been made, with 360 20% having primary outcome composite events during the trial duration. 319 Secondary clinical and process outcomes These numbers will provide 361 84% power to detect a 52% relative risk reduction (RRR) of the primary outcome (from 20% to 9%) 362 assuming a models ICC 0.02. 11 . CC-BY 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 6, 2023. ; https://doi.org/10.1101/2023.11.05.23298118 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 6, 2023. ; https://doi.org/10.1101/2023.11.05.23298118 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a perpetuity. 393 to-two (interviewer and participant with chosen family members/friend). Thus, if appropriate, the 394 family member/friend can be invited to the interviews, as social support is culturally patterned and 395 could play an important part in the mechanisms of the intervention, particularly in women’s 396 engagement with care. Wellbeing measures will include, for example, the adolescent flourishing scale 397 [24] and a girl’s empowerment tool based on existing guides for impact evaluations [25]. A small 398 sub-group of girls will also be invited to take part in the photovoice project, a participatory research 399 methodology developed by Wang and Burrit [26] that partners with participants to use photography 400 to help them to record, reflect upon, critically dialogue and share knowledge about their perspectives 401 and priorities to reach policy makers, foster social change and strengthen partnerships [27]. 402 403 Statistical analysis 404 We will undertake two statistical analyses: the main pragmatic, intention to treat analysis (ITT) to 405 compare intervention and control clusters that include all adolescent girls as originally allocated after 406 randomisation; and a complementary per protocol (PP) analysis to compare intervention and control 407 clusters that includes only those girls who received the intervention originally allocated. The 408 CONSORT guidelines for reporting of parallel group RCTs recommend that both ITT and PP analyses 409 should be reported for all planned outcomes to allow readers to interpret the effect of an 410 intervention. It is not possible to blind clusters to interventions they receive, but outcome 411 assessments will be blinded. 442 Data management and monitoring 443 Anonymised data will be collected by the local data collectors, under the supervision of the trial 444 manager. All participants will be given a unique identifier and no personal information will be entered 445 onto the secure, online trial database (MedSciNet). Where possible, all anonymised data will be 446 collected directly onto MedSciNet, but if exceptional circumstances make this not possible (e.g. no 447 internet and very remote PHUs), a paper-based data collection tool will be used and stored 448 anonymously in secure areas of each health facility and of the implementation partners. All data 449 uploaded on the MedSciNet database will be automatically stored and backed up. Data collection 450 and storage will be governed by the UK Data Protection Act 1998. All MedSciNet data is stored on 451 high-capacity servers kept in highly secure monitoring and surveillance systems. 452 453 The MedSciNet database will be accessible for at least one year following the end of the trial, and a 454 copy of this will then be kept on the KCL Server for 20 years, in accordance with the KCL Data 455 retention schedule. Informed consent forms for qualitative components of the trial will be kept in 456 files in secure areas of the management team (Lifeline Nehemiah Projects). Only the research 457 assistant and the local and UK based managers will have access to these. All paper forms will be 458 stored securely and kept in confidence in compliance with the UK Data Protection Act 1998. 459 460 The local research team will work in collaboration with the local community workforce and current 461 local initiatives, to avoid missing primary outcome events. A flexible approach will be used to 462 individualize our methodology according to local capacity and community healthcare workforce 463 infrastructure. Consistency and quality of source data will be monitored by a research assistant and 464 the Chief Investigators. MedSciNet allows for extensive monitoring and query processing features, as 443 Anonymised data will be collected by the local data collectors, under the supervision of the trial 444 manager. All participants will be given a unique identifier and no personal information will be entered 445 onto the secure, online trial database (MedSciNet). Where possible, all anonymised data will be 446 collected directly onto MedSciNet, but if exceptional circumstances make this not possible (e.g. 319 Secondary clinical and process outcomes Thus, the focus of 435 health economics in this pilot trial will be limited to developing or refining service use schedules and 436 other measures of outcomes. We will explore the resource use associated with the mentoring 437 scheme and savings or other service use impacts and will also estimate costs associated with the 438 delivery of the mentoring scheme. Cost consequences analyses will be used to present the results 439 from this pilot study, as the costs and effects observed can also be used in value of information 440 modelling to determine whether the cost of a definitive trial is worthwhile. 441 429 secondary grounded theory analysis will be undertaken to generate theory to explore and explain 430 the phenomenon of adolescent pregnancy in Sierra Leone [33]. 319 Secondary clinical and process outcomes is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 6, 2023. ; https://doi.org/10.1101/2023.11.05.23298118 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 6, 2023. ; https://doi.org/10.1101/2023.11.05.23298118 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for th this version posted November 6, 2023. ; https://doi.org/10.1101/2023.11.05.23298118 doi: medRxiv preprint 429 secondary grounded theory analysis will be undertaken to generate theory to explore and explain 430 the phenomenon of adolescent pregnancy in Sierra Leone [33]. 431 432 Implementation costs are important in process evaluations, and we will assess the feasibility of health 433 economics for 2YL. It would not be appropriate to conduct a full economic evaluation for a pilot study 434 designed to estimate the parameters needed to design a future definitive trial. Thus, the focus of 435 health economics in this pilot trial will be limited to developing or refining service use schedules and 436 other measures of outcomes. We will explore the resource use associated with the mentoring 437 scheme and savings or other service use impacts and will also estimate costs associated with the 438 delivery of the mentoring scheme. Cost consequences analyses will be used to present the results 439 from this pilot study, as the costs and effects observed can also be used in value of information 440 modelling to determine whether the cost of a definitive trial is worthwhile. 441 429 secondary grounded theory analysis will be undertaken to generate theory to explore and explain 430 the phenomenon of adolescent pregnancy in Sierra Leone [33]. 431 432 Implementation costs are important in process evaluations, and we will assess the feasibility of health 433 economics for 2YL. It would not be appropriate to conduct a full economic evaluation for a pilot study 434 designed to estimate the parameters needed to design a future definitive trial. 319 Secondary clinical and process outcomes 412 413 The main analysis will use a permutation test designed for stepped-wedge trials [28] to adjust the 414 standard errors, confidence intervals and significance tests for clustering. This gives equal weight to 415 each woman included in the study. We will adjust for important baseline differences that might be 416 related to outcomes (e.g. parity, gestation at first antenatal visits). As individual-specific data will be 417 used there is a potential for missing data; but the main impact of missing data would be to invalidate 418 the randomised comparison if there was unequal dropout between the two arms, leading to 419 potential bias. Multiple regression, as described, would correct for any such bias. 420 421 Qualitative data from interviews and focus group discussions will be mainly analyzed using thematic 422 analysis [29]. In brief, this includes familiarisation with the data, generation of initial codes, the 423 searching for and review of themes, naming and offering explanations for each theme, and lastly 424 producing a report. This practical analytic approach involves inductive coding practices which are 425 both consultative and initially open, and thus helpful to explore the perspectives of different research 426 participants, highlighting similarities and differences, and generating unanticipated insights [29]. 427 Implementation and contextual factors (barriers and facilitators) will be further assessed using the 428 Consolidated Framework for Implementation Research [30] and Proctor’s framework [31,32]. A 393 to-two (interviewer and participant with chosen family members/friend). Thus, if appropriate, the 394 family member/friend can be invited to the interviews, as social support is culturally patterned and 395 could play an important part in the mechanisms of the intervention, particularly in women’s 396 engagement with care. Wellbeing measures will include, for example, the adolescent flourishing scale 397 [24] and a girl’s empowerment tool based on existing guides for impact evaluations [25]. A small 398 sub-group of girls will also be invited to take part in the photovoice project, a participatory research 399 methodology developed by Wang and Burrit [26] that partners with participants to use photography 400 to help them to record, reflect upon, critically dialogue and share knowledge about their perspectives 401 and priorities to reach policy makers, foster social change and strengthen partnerships [27]. 402 12 . CC-BY 4.0 International license It is made available under a perpetuity. 442 Data management and monitoring This TMG reports to the 480 steering committee, which is part of a wider international advisory group that oversees all activities 481 of the different workstreams of the global health research group. The group will include dependent 482 and independent members who will be invited to attend, at least, annual oversight meetings. 483 484 Dissemination 485 Findings, positive or negative, will be disseminated in international peer-reviewed journals, as well 486 as local, national and international events, workshops and conferences. Whenever possible, girls and 487 their mentors, healthcare providers and community members and stakeholders will be provided with 488 a layperson’s summary of the results, and government and policy makers will also be informed. 489 490 491 Discussion 492 Sierra Leone has one of the highest maternal mortality rates in the world (717 deaths per 100 000 493 livebirths in 2019), and this burden falls primarily on adolescent girls. They are 40-60% more likely to 494 die during childbirth and their infants are also at increased risk of sickness and death [1-2]. More than 465 well as a comprehensive alerting system to identify missing data. Fields including ‘limits’ will be used 466 to avoid entry of erroneous data. The 2YL management team will regularly conduct data monitoring 467 visits to perform validation checks to verify validity and completeness of at least 10% of the source 468 data. The team will also review the audit trail at individual levels if necessary. Training in the trial 469 protocol will be delivered centrally (before trial recruitment) and on site in the field, to ensure local 470 research staff are confident and competent to collect outcome data and ensure its quality, accuracy, 471 and completeness. The final datasets used and/or analysed during the study will be made available 472 from the corresponding author upon reasonable request. 473 465 well as a comprehensive alerting system to identify missing data. Fields including ‘limits’ will be used 466 to avoid entry of erroneous data. The 2YL management team will regularly conduct data monitoring 467 visits to perform validation checks to verify validity and completeness of at least 10% of the source 468 data. The team will also review the audit trail at individual levels if necessary. 474 Trial oversight 474 Trial oversight 475 This study will be supported by a trial management group (TMG) that includes research fellows, 476 research assistants, data collectors, a 2YL programme manager and trial coordinator who will run the 477 implementation and provide organisational support, particularly to the data collectors and teams in 478 the field. The TMG will meet every week, with all co-investigators meeting every two-three months 479 to monitor and review progress, troubleshoot and for strategic planning. This TMG reports to the 480 steering committee, which is part of a wider international advisory group that oversees all activities 481 of the different workstreams of the global health research group. The group will include dependent 482 and independent members who will be invited to attend, at least, annual oversight meetings. 483 484 Dissemination 485 Findings, positive or negative, will be disseminated in international peer-reviewed journals, as well 486 as local, national and international events, workshops and conferences. Whenever possible, girls and 487 their mentors, healthcare providers and community members and stakeholders will be provided with 488 a layperson’s summary of the results, and government and policy makers will also be informed. 489 490 491 Discussion 492 Sierra Leone has one of the highest maternal mortality rates in the world (717 deaths per 100 000 493 livebirths in 2019), and this burden falls primarily on adolescent girls. They are 40-60% more likely to 494 die during childbirth and their infants are also at increased risk of sickness and death [1-2]. More than 495 two thirds of all maternal deaths are caused by haemorrhage, hypertension and sepsis, and about a 496 third are due to unsafe abortions among adolescents [2]. Many of these deaths could be prevented 497 with simple, cost-effective and available interventions, but unfortunately there are often inequities 498 in availability, access and quality of care, and delays in delivery, escalation and referral in Sierra Leone 499 [14]. Adolescent girls are a particularly vulnerable group, often from disadvantaged communities and 500 usually driven by poverty, lack of education and employment opportunities [1,2]. Stigma and 475 This study will be supported by a trial management group (TMG) that includes research fellows, 476 research assistants, data collectors, a 2YL programme manager and trial coordinator who will run the 477 implementation and provide organisational support, particularly to the data collectors and teams in 478 the field. 442 Data management and monitoring Training in the trial 469 protocol will be delivered centrally (before trial recruitment) and on site in the field, to ensure local 470 research staff are confident and competent to collect outcome data and ensure its quality, accuracy, 471 and completeness. The final datasets used and/or analysed during the study will be made available 472 from the corresponding author upon reasonable request. 473 442 Data management and monitoring no 447 internet and very remote PHUs), a paper-based data collection tool will be used and stored 448 anonymously in secure areas of each health facility and of the implementation partners. All data 449 uploaded on the MedSciNet database will be automatically stored and backed up. Data collection 450 and storage will be governed by the UK Data Protection Act 1998. All MedSciNet data is stored on 451 high-capacity servers kept in highly secure monitoring and surveillance systems. 452 13 . CC-BY 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 6, 2023. ; https://doi.org/10.1101/2023.11.05.23298118 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 6, 2023. ; https://doi.org/10.1101/2023.11.05.23298118 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a perpetuity. 465 well as a comprehensive alerting system to identify missing data. Fields including ‘limits’ will be used 466 to avoid entry of erroneous data. The 2YL management team will regularly conduct data monitoring 467 visits to perform validation checks to verify validity and completeness of at least 10% of the source 468 data. The team will also review the audit trail at individual levels if necessary. Training in the trial 469 protocol will be delivered centrally (before trial recruitment) and on site in the field, to ensure local 470 research staff are confident and competent to collect outcome data and ensure its quality, accuracy, 471 and completeness. The final datasets used and/or analysed during the study will be made available 472 from the corresponding author upon reasonable request. 473 474 Trial oversight 475 This study will be supported by a trial management group (TMG) that includes research fellows, 476 research assistants, data collectors, a 2YL programme manager and trial coordinator who will run the 477 implementation and provide organisational support, particularly to the data collectors and teams in 478 the field. The TMG will meet every week, with all co-investigators meeting every two-three months 479 to monitor and review progress, troubleshoot and for strategic planning. 474 Trial oversight The TMG will meet every week, with all co-investigators meeting every two-three months 479 to monitor and review progress, troubleshoot and for strategic planning. This TMG reports to the 480 steering committee, which is part of a wider international advisory group that oversees all activities 481 of the different workstreams of the global health research group. The group will include dependent 482 and independent members who will be invited to attend, at least, annual oversight meetings. 483 491 Discussion 492 Sierra Leone has one of the highest maternal mortality rates in the world (717 deaths per 100 000 493 livebirths in 2019), and this burden falls primarily on adolescent girls. They are 40-60% more likely to 494 die during childbirth and their infants are also at increased risk of sickness and death [1-2]. More than 495 two thirds of all maternal deaths are caused by haemorrhage, hypertension and sepsis, and about a 496 third are due to unsafe abortions among adolescents [2]. Many of these deaths could be prevented 497 with simple, cost-effective and available interventions, but unfortunately there are often inequities 498 in availability, access and quality of care, and delays in delivery, escalation and referral in Sierra Leone 499 [14]. Adolescent girls are a particularly vulnerable group, often from disadvantaged communities and 500 usually driven by poverty, lack of education and employment opportunities [1,2]. Stigma and 14 . CC-BY 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 6, 2023. ; https://doi.org/10.1101/2023.11.05.23298118 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 6, 2023. ; https://doi.org/10.1101/2023.11.05.23298118 doi: medRxiv preprint 501 abandonment, lack of family-based support and delayed care-seeking have been found to be 502 important contributors to maternal adolescent mortality [4]. Social complexities and high rates of 503 child marriage, adolescent pregnancy and gender-based violence (39%, 28%, and ~50% respectively) 504 also prevent girls from realizing their full potential in all aspects of their development [34,35]. 491 Discussion 505 506 The country has a very young population (nearly 65% are under 25 years old) [36], and improving 507 health and wellbeing of these girls (including sexual and reproductive health) remains a top priority, 508 indicated by a number of government policies targeted to this group: the Free Health Care Initiative 509 for pregnant and breastfeeding women (2010), the National Standards for Adolescent and Young 510 People Friendly Health Services (2011), the Reproductive, Maternal, Newborn, Child and Adolescent 511 Health Policy (2017-2021); the National strategy for the reduction of adolescent pregnancy and child 512 marriage 2018-2022 (2018), and the National Policy for Radical Inclusion in Schools (2021). The 513 development of the first multi-agency and cross-ministry National Strategy for the Reduction of 514 Teenage Pregnancy (2013-2015) was impeded by the scarcity of resources and diversion of efforts to 515 the Ebola epidemic which also increased rates of adolescent pregnancy. However, in the aftermath 516 of the epidemic, a revision and update of the national strategy was relaunched in 2018 which 517 included child marriage [37]. 518 519 Much of the attention has been given to primary prevention of adolescent pregnancy and child 520 marriage, but adolescent girls are still getting pregnant and dying during childbirth. It is imperative 521 to also identify programmes or interventions that support them during pregnancy and the parenting 522 period so they can survive and improve their health and development and that of their child. To 523 support and facilitate the integration of pregnant and parenting girls it is crucial to sensitise 524 communities, strengthen existing youth friendly services and work closely with stakeholders 525 (government, gatekeepers, community and family actors). It is now widely recognized that the need 526 to support a holistic agenda where integration and implementation of evidence-based interventions 527 across health, education, and social systems must improve in order to protect, nurture, and support 528 the health and development potential for every child and adolescent [38]. 529 530 These are the philosophical principles by which the 2YL community-based mentoring intervention 531 was initially developed in 2017. Mentored adolescent girls are supported to thrive, not just survive. 532 Youth mentoring programmes have been shown to improve outcomes across academic, behavioral, 533 emotional and social areas of young people’s lives. 491 Discussion 542 In addition, consideration needs to be given to moderating factors (i.e. mentors’, mentees’ and 543 matching characteristics), highlighting the need for more research in this area [41]. Although the 2YL 544 mentoring scheme has been piloted in five sites over four years with promising results, a more robust 545 and formal evaluation is needed to understand the feasibility of 2YL in other communities and how 546 it can address determinants of adolescent maternal mortality and general health and wellbeing, and 547 this is what the 2YL pilot trial is aiming to achieve. 548 549 Since 2021, 2YL has been part of a Global Health Research Group (CRIBS) funded by the UK’s National 550 Institute for Health and Care Research and led by the University of Sierra Leone and King’s College 551 London in collaboration with multiple partners, including the Sierra Leone Ministry of Health and 552 Sanitation, Lifeline Nehemiah Projects (LNP), NGO Welbodi Partnership, the National Emergency 553 Medical Services (NEMS), and the national Midwifery Schools. The group, which will run for 3 years, 554 aims to develop and implement simple, scalable innovations to reduce maternal (including 555 adolescent) and perinatal mortality and build research capacity and expertise in Sierra Leone [42]. 556 The group is uniquely multidisciplinary with expertise in reproductive health, obstetrics and 557 gynecology, nursing, midwifery, public health, gender studies, sociology, implementation science, 558 medical statistics, health policy and economics. 559 560 We believe findings from the 2YL pilot trial will provide more holistic information for communities 561 and local and national decision-makers and refine procedures to inform future scale-up work aiming 562 to reduce mortality among adolescent girls and their babies, and improving their health, educational 563 and socio-economic welfare. The various programmes implemented by the line ministries in Sierra 564 Leone are more institutionally based with little active involvement with communities that would 565 serve to support prevention and sustainability; one of the unique features of the 2YL model is the 566 meaningful community engagement and involvement. There has been national and international 537 orientated programmes are keys for success [39]. Goldner and Ben-Eliyahu [41] tried to unpack the 538 relational processes in community-based youth mentoring which promote high relationship quality 539 and generate the most significant benefits. 491 Discussion They found that long enough, supportive, reliable, 540 trustworthy, and balanced mentoring relationships (clearly articulated goals, structure, and 541 behaviours) serve as building blocks in promoting mentees’ development and minimising adversity. 542 In addition, consideration needs to be given to moderating factors (i.e. mentors’, mentees’ and 543 matching characteristics), highlighting the need for more research in this area [41]. Although the 2YL 544 mentoring scheme has been piloted in five sites over four years with promising results, a more robust 545 and formal evaluation is needed to understand the feasibility of 2YL in other communities and how 546 it can address determinants of adolescent maternal mortality and general health and wellbeing, and 547 this is what the 2YL pilot trial is aiming to achieve. 549 Since 2021, 2YL has been part of a Global Health Research Group (CRIBS) funded by the UK’s National 550 Institute for Health and Care Research and led by the University of Sierra Leone and King’s College 551 London in collaboration with multiple partners, including the Sierra Leone Ministry of Health and 552 Sanitation, Lifeline Nehemiah Projects (LNP), NGO Welbodi Partnership, the National Emergency 553 Medical Services (NEMS), and the national Midwifery Schools. The group, which will run for 3 years, 554 aims to develop and implement simple, scalable innovations to reduce maternal (including 555 adolescent) and perinatal mortality and build research capacity and expertise in Sierra Leone [42]. 556 The group is uniquely multidisciplinary with expertise in reproductive health, obstetrics and 557 gynecology, nursing, midwifery, public health, gender studies, sociology, implementation science, 558 medical statistics, health policy and economics. 559 560 We believe findings from the 2YL pilot trial will provide more holistic information for communities 561 and local and national decision-makers and refine procedures to inform future scale-up work aiming 562 to reduce mortality among adolescent girls and their babies, and improving their health, educational 563 and socio-economic welfare. The various programmes implemented by the line ministries in Sierra 564 Leone are more institutionally based with little active involvement with communities that would 565 serve to support prevention and sustainability; one of the unique features of the 2YL model is the 566 meaningful community engagement and involvement. 491 Discussion At present there is no clear evidence that they 534 can improve physical health, although studies are limited (including for pregnant and parenting 535 adolescents) [39,40]. The evidence suggests that longer mentoring relationships are linked to better 536 d h l f h hi h i i d i i d h d f l 501 abandonment, lack of family-based support and delayed care-seeking have been found to be 502 important contributors to maternal adolescent mortality [4]. Social complexities and high rates of 503 child marriage, adolescent pregnancy and gender-based violence (39%, 28%, and ~50% respectively) 504 also prevent girls from realizing their full potential in all aspects of their development [34,35]. 530 These are the philosophical principles by which the 2YL community-based mentoring intervention 531 was initially developed in 2017. Mentored adolescent girls are supported to thrive, not just survive. 532 Youth mentoring programmes have been shown to improve outcomes across academic, behavioral, 533 emotional and social areas of young people’s lives. At present there is no clear evidence that they 534 can improve physical health, although studies are limited (including for pregnant and parenting 535 adolescents) [39,40]. The evidence suggests that longer mentoring relationships are linked to better 536 outcomes, and the role of the matching process, the training and motivation, and the need for goal- 15 . CC-BY 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 6, 2023. ; https://doi.org/10.1101/2023.11.05.23298118 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 6, 2023. ; https://doi.org/10.1101/2023.11.05.23298118 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a perpetuity. 537 orientated programmes are keys for success [39]. Goldner and Ben-Eliyahu [41] tried to unpack the 538 relational processes in community-based youth mentoring which promote high relationship quality 539 and generate the most significant benefits. They found that long enough, supportive, reliable, 540 trustworthy, and balanced mentoring relationships (clearly articulated goals, structure, and 541 behaviours) serve as building blocks in promoting mentees’ development and minimising adversity. 491 Discussion There has been national and international 567 interest in 2YL which has been featured in the Lancet [10], the 2022 UNFPA State of the World 568 Population Report [43], a programme for the BBC World Service ‘People fixing the World’ [44] and 569 the Evening Standard [45]. We believe 2YL has the potential to save lives and promote health and 570 wellbeing of adolescent girls and babies, and thus become a model of good practice for adolescent 571 pregnancy, to be adopted more widely in Sierra Leone and elsewhere. 572 16 . CC-BY 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 6, 2023. ; https://doi.org/10.1101/2023.11.05.23298118 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a perpetuity. 573 Supplementary information 574 No data are available. 575 576 Trial status 577 The current 2YL protocol is version 2.1, 21 July 2023. Recruitment start time was 4 July 2022; 578 completion is 30 November 2023. 579 580 Abbreviations 581 2YL: 2YoungLives; cRCT: cluster randomised controlled trial; CONSORT: Consolidated Standards of 582 Reporting Trials; EmONC: Emergency Obstetric and Newborn care; ICC: Intra-Cluster Correlation; 583 ISRCTN: International Standard Randomised Controlled Trial Number; KCL: King’s College London; 584 PHU: Peripheral Health Unit; SL: Sierra Leone; SPIRIT: Standard Protocol Items: Recommendations 585 for Interventional Trials; TMG: Trial Management Group; TIDieR: Template for Intervention 586 Description and Replication; WHO: World Health Organization. 587 588 589 Declarations 590 591 Funding 592 This study has undergone full external peer review as part of the funding process. This research is 593 funded by the National Institute for Health and Care Research (NIHR) (ID: NIHR33232) using UK aid 594 from the UK Government to support global health research. JS and CFT are supported by the NIHR 595 Applied Research Collaboration (ARC) South London. JS is a NIHR Senior Investigator and CFT is 596 supported by a NIHR Development and Skills Award (ID: NIHR301603). The views expressed in this 597 publication are those of the author(s) and not necessarily those of the NIHR or the UK Government. 598 (https://fundingawards.nihr.ac.uk/award/NIHR133232) 599 600 Competing interests 601 We declare no competing interests. 602 603 Availability of data and Material: 604 N d t il bl t 573 Supplementary information CFT, LN, MK drafted the initial trial protocol, and PK, AMK, 608 AR, ST, PTS, AHS, JS and PTW reviewed and commented on the initial draft and/or on subsequent 609 revisions. All authors have seen and approved the final version of the manuscript. 610 18 609 revisions. All authors have seen and approved the final version of the manuscript. 610 611 Ethical approval and consent to participate 612 This study has been approved by ethics committee at King’s College London UK (HR/DP-21/22-26320) 613 and the Office of the Sierra Leone Ethics and Scientific Review Committee. Data security will be 614 maintained in accordance with General Data Protection Regulations. Written, informed consent to 615 participate will be obtained from all participants for qualitative interviews, focus group discussions, 616 and photovoice. 617 618 Consent to publication 619 This manuscript does not contain individual personal data from participants. 620 621 Acknowledgements 622 We thank all members of CRIBS Group & collaborators at the University of Sierra Leone, King’s College 623 London; Ministry of Health and Sanitation; Schools of Midwifery; Welbodi Partnership; NEMS; PCMH; 624 UNICEF; WHO SL. Special thank you to CRIBS research assistants and data collectors working for 2YL, 625 as well as all the girls, mentors, relatives, healthcare providers and community stakeholders. 626 627 Name and contact information for sponsor 628 King’s College London (Professor Reza Razavi, reza.razavi@kcl.ac.uk) 629 630 Role of sponsor 631 The study is sponsored by King’s College London (KCL). However, this is an investigator initiated 632 clinical trial, therefore the sponsor played no role in the design of the study and collection, analysis, 633 and interpretation of data and in writing the manuscript. 634 635 636 637 638 639 640 641 642 573 Supplementary information 17 . CC-BY 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 6, 2023. ; https://doi.org/10.1101/2023.11.05.23298118 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 6, 2023. ; https://doi.org/10.1101/2023.11.05.23298118 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a perpetuity. 607 LN, MK, PTW are the co-founders of 2YL. CFT, LN, MK drafted the initial trial protocol, and PK, AMK, 608 AR, ST, PTS, AHS, JS and PTW reviewed and commented on the initial draft and/or on subsequent 609 revisions. All authors have seen and approved the final version of the manuscript. 610 611 Ethical approval and consent to participate 612 This study has been approved by ethics committee at King’s College London UK (HR/DP-21/22-26320) 613 and the Office of the Sierra Leone Ethics and Scientific Review Committee. Data security will be 614 maintained in accordance with General Data Protection Regulations. Written, informed consent to 615 participate will be obtained from all participants for qualitative interviews, focus group discussions, 616 and photovoice. 617 618 Consent to publication 619 This manuscript does not contain individual personal data from participants. 620 621 Acknowledgements 622 We thank all members of CRIBS Group & collaborators at the University of Sierra Leone, King’s College 623 London; Ministry of Health and Sanitation; Schools of Midwifery; Welbodi Partnership; NEMS; PCMH; 624 UNICEF; WHO SL. Special thank you to CRIBS research assistants and data collectors working for 2YL, 625 as well as all the girls, mentors, relatives, healthcare providers and community stakeholders. 626 627 Name and contact information for sponsor 628 King’s College London (Professor Reza Razavi, reza.razavi@kcl.ac.uk) 629 630 Role of sponsor 631 The study is sponsored by King’s College London (KCL). However, this is an investigator initiated 632 clinical trial, therefore the sponsor played no role in the design of the study and collection, analysis, 633 and interpretation of data and in writing the manuscript. 634 635 607 LN, MK, PTW are the co-founders of 2YL. 611 Ethical approval and consent to participate 612 This study has been approved by ethics committee at King’s College London UK (HR/DP-21/22-26320) 613 and the Office of the Sierra Leone Ethics and Scientific Review Committee. Data security will be 614 maintained in accordance with General Data Protection Regulations. Written, informed consent to 615 participate will be obtained from all participants for qualitative interviews, focus group discussions, 616 and photovoice. 18 . CC-BY 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 6, 2023. ; https://doi.org/10.1101/2023.11.05.23298118 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 6, 2023. ; https://doi.org/10.1101/2023.11.05.23298118 doi: medRxiv preprint 643 References 644 1. WHO, UNICEF, UN Population Fund, World Bank Group and the United Nations Population 645 Division. 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CC-BY 4.0 International license It is made available under a perpetuity. 643 References is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 6, 2023. ; https://doi.org/10.1101/2023.11.05.23298118 doi: medRxiv preprint
https://openalex.org/W4212866352	https://jurnal.uns.ac.id/hpe/article/download/54638/34587	Indonesian	null	PERAN KEPERCAYAAN DALAM MEMODERASI PENGARUH SALES PROMOTION, ADVERTISIG DAN RELIGIUSITAS TERHADAP KEPUTUSAN MENBUNG	Jurnal Hukum dan Pembangunan Ekonomi	2,021	cc-by	4,511	IAIN SALATIGA tuttyalawiyah3004@gmail.com1, moclassofyan@gmail.com2 tuttyalawiyah3004@gmail.com1, moclassofyan@gmail.com2 ABSTRACT Penelitian ini bertujuan untuk mengetahui pengaruh sales promotion, advertising dan religiusitas terhadap keputusan menabung di bank syariah dengan kepercayaan sebagai variabel moderating pada Bank syariah Indonesia KC Kudus. Dalam penelitian ini menggunakan metode kuantitatif dengan mengelola data primer dengan pengumpulan data kuesioner yang diberikaan kepada nasabah Bank syariah Indonesia KC Kudus dengan jumlah responden 100 nasabah yang diolah dengan menggunakan data SPSS versi 24 dan dianalisis menggunakan analisis regresi berganda, dengan analisis yang digunakan meliputi uji responden, uji instrument, uji asumsi klasik dan uji moderating. Berdasarkan hasil uji penelitian yang dilakukan yaitu: variabel sales promotion berpengaruh positif dan signifikan terhadap keputusan menabung. variabel advertising memiliki hasil yang negarif tidak signifikan terhadap keputusan menabung, dan variabel religiusitas memiliki hasil yang positif dan signifikan terhadap keputusan menabung. dan terdapat variabel moderating untuk memperkuat atau memperlemah variabel yang di uji. Kepercayaan dapat memperkuat adanya pengaruh sales promotion terhadap keputusan menabung, kepercayaan memperlemah hubungan advertising terhadap keputusan menabung, dan kepercayaan dapat memperkuat adanya hubungan religiusitas terhadap keputusan menabung. y g g p p g Keyword: Kepercayaan Sales Promotion, Advertising dan Religiusitas, Keputusan Menabung y g g p p g Keyword: Kepercayaan Sales Promotion, Advertising dan Religiusitas, Keputusan Menabung © Author(s). This work is licensed under a Creative Commons Attribution 4.0 International License . Published by Postgraduate Program, Master of Laws, Faculty of Law, Universitas Sebelas Maret, Indonesia Tutty Alawiyah1, Mochlasin2 Tutty Alawiyah1, Mochlasin2 Jurnal Hukum dan Pembangunan Ekonomi, Volume 9, Nomor 2, 2021 257 ISSN (Print) 2338-1051, ISSN (Online) 2777-0818 Jurnal Hukum dan Pembangunan Ekonomi, Volume 9, Nomor 2, 2021 257 ISSN (Print) 2338-1051, ISSN (Online) 2777-0818 Jurnal Hukum dan Pembangunan Ekonomi, Volume 9, Nomor 2, 2021 257 ISSN (Print) 2338-1051, ISSN (Online) 2777-0818 1 Asmarantaka, R. W., Atmakusuma, J., Muflikh, Y. N., & Rosiana, N. (2018). Konsep Pemasaran Agribisnis : Pendekatan Ekonomi Dan Manajemen. Jurnal Agribisnis Indonesia, 5(2), 151. https://doi.org/10.29244/jai.2017.5.2.151-172 2 Njoto, D. P., & Sienatra, K. B. (2018). Pengaruh Promosi Terhadap Keputusan Pembelian Konsumen Wenak Tok. Pengaruh Harga Diskon Dan Persepsi Produk Terhadap Nilai Belanja Serta Perilaku Pembelian Konsumen, 7(9), 27–44. 3 Firmansyah, A. (2019). No Title. In Pemasaran (Konsep dan Dasar) (pp. 11–13). Qiara Media. INTRODUCTION Keberadan bank di dunia perekonomian di era sekarang merupakan kebutuhan yang memilki hubungan antara perekonomian dan perbankan yang keduanya sulit untuk dipisahkan. Peran bank disini memberikan modal atau sebagai jasa penitipan bagi pelaku bisnis untuk meningkatkan suatu usaha yang dijalankan. Bank disini digunakan untuk memenuhi kebutuhan dalam kegiatan perekonomianya yang bersifat konsumtif, produktif, properti, dengan berbagai layanan seperti asuransi, investasi, gadai, tabungan haji, tabungan 258 Jurnal Hukum dan Pembangunan Ekonomi, Volume 9, Nomor 2, 2021 ISSN (Print) 2338-1051, ISSN (Online) 2777-0818 258 Jurnal Hukum dan Pembangunan Ekonomi, Volume 9, Nomor 2, 2021 ISSN (Print) 2338-1051, ISSN (Online) 2777-0818 pensiun dan lain sebagainya sebagai media untuk mempermudahkan kegiatan perekonomia atau lainya. Bank sebagai lembaga yang bersifat kepercayaan yang tidak hanya untuk individu dan masyarakat, tetapi juga berperan dalam pertumbuhan perekonomian suatu negara, selain itu bank juga membantu dalam kegiatan transaksi, produksi, serta komunikasi yang fungsinya sebagai lembaga yang melakukan kegiatan pembiayaan.1 pensiun dan lain sebagainya sebagai media untuk mempermudahkan kegiatan perekonomia atau lainya. Bank sebagai lembaga yang bersifat kepercayaan yang tidak hanya untuk individu dan masyarakat, tetapi juga berperan dalam pertumbuhan perekonomian suatu negara, selain itu bank juga membantu dalam kegiatan transaksi, produksi, serta komunikasi yang fungsinya sebagai lembaga yang melakukan kegiatan pembiayaan.1 Oleh karena itu buat pengupayaan kenaikan mutu aktivitas operasional- nya, perbankan syariah berupaya semaksimal bisa jadi buat membagikan jasa yang diperlukan nasabah secara luas serta bisa merata, bank syariah wajib melihat pangsa pasar yang dibutuhkan oleh masyarakat dengan memperhatikan apa yang dibutukan nasabah yang akan mencerminkan mengapa seorang itu harus melakukan pemeblian jasa dan bagaimana seorang itu dapat memilih dan menabung maupun yang menjaminkan sehingga dapat meningkatkan aktifitas dalam perbankan Syariah.2 Dilihat dari perkembanganya bank Syariah di kota Kudus berkembang begitu pesat dengan penduduk yang mayoritas muslim, dan kesadaran tentang keagamaan menjadikan bank Syariah di kota banyak diminati oleh masyarakat kota Kudus fenomena ini sangat menarik untuk saya lakukan dengan kajian yang lebih jauh tentang problematika yang terjadi, dan peneliti nengadakan tema “ Pengaruh sales promotion, advertising dan religiutas terhadap keputusan menabung di bank syariah melalui kepercayaan sebagai variabel moderating ( studi kasus terhadap nasabah Bank Syariah KC Kudus). Perilaku Konsumen Sikap konsumen ialah suatu aksi langsung, turut serta memperoleh, komsumsi serta menghabiskan produk ataupun jasa. Sikap konsumen dapat dikatakan proses pembelian, pada saat mengevaluasi, pencarian produk serta jasa dan sikap konsumen merupakan sesuatu yang hendak mendasari konsumen membuat keputusan yang ada kaitannya dengan prosses mengkonsumsi suatu benda ataupun jasa. Sikap konsumen ialah perihal yang sangat mendasar untuk memberikan keputusan saat membeli benda ataupun jasa. Pastinya selaku konsumen, akan memikirkan terlebih dulu apa yang wajib disantap mulai dari harga, model, wujud, kemasan serta mutu dan guna dari khasiat benda tersebut.3 © Author(s). This work is licensed under a Creative Commons Attribution 4.0 International License. Published by Postgraduate Program, Master of Laws, Faculty of Law, Universitas Sebelas Maret, Indonesia © Author(s). This work is licensed under a Creative Commons Attribution 4.0 International License. Published by Postgraduate Program Master of Laws Faculty of Law Universitas Sebelas Maret Indonesia ( ) Published by Postgraduate Program, Master of Laws, Faculty of Law, Universitas Sebelas Maret, Indonesia 4 Suprihati, & Utami, W. B. (2015). Analisis Faktor - Faktor Yang Mempengaruhi Perilaku Di Kelurahan Gonilan Kabupaten Sukoharjo. Jurnal Paradigma, 13(01), 108. 5 Setyorini, A. P., & Ratno, F. A. (2020). Pengaruh Promotional Mix Terhadap Keputusan Nasabah Menabung Di Bank Syariah. Jurnal Studi Manajemen Dan Bisnis, 7(2), 83–92. https://doi.org/10.21107/jsmb.v7i2.9045 Keputusan Pengambilan keputusan merupakan sesuatu proses buat memperhitungkan serta memilah dari bermacam alternatif yang cocok dengan kepentingan tertentu dengan menghasilkan sesuatu opsi mana yang sangat dapat digunakan cocok dengan kebutuhanya. Menarangkan keputusan pembelian merupakan sesuatu proses intograsi yang digunakan buat mencampurkan pengetahuan buat mengevaluasi yang lebih dari satu buat memilah satu. Sebaliknya keputusan konsumen merupakan pendekatan untuk menuntaskan permasalahan buat membeli benda ataupun jasa dalam penuhi keinginan serta kebutuhanya.5 © Author(s). This work is licensed under a Creative Commons Attribution 4.0 International License . Published by Postgraduate Program, Master of Laws, Faculty of Law, Universitas Sebelas Maret, Indonesia Sales Promotin Sales promotion adalah program promosi yang paritel dalam mendorong terjadinya penjualan untuk meningkatkan penjualan atau dalam rangka untuk mempertahankan minat pelanggan untuk tetap belanja di tempatnya, sales promotion terdiri dari dua dimensi yang berkaitan dengan uang dan tidak berkaitan dengan uang. Yang berkaitan dengan uang promosi penjuaalan ini contohnya potongan harga atau discount. Sedangkan yang tidak ada keterkaitanya dengan uang yaitu promosi yang tidak memberikan intensif secara langsung dan lebih besar hubungan langsung dengan konsumen. Tujuan sales promotion ini adalah untuk menarik para mpembeli dan masyarakat yang belum mengkonumsi dengan meningkatkan volume penjualan dalam rangka memperluas pansa pasar. Konsep Pemasaran Pemasaran ialah aktivitas yang sangat berarti pada dunia usaha yang terus menjadi tumbuh dimasa globalisasi ini. Di dalam kegiatan pemasaran terdapat aspek yang akan mendukung keberhasilan industri dalam mengalami persaingan yang terus menjadi ketat ini, oleh sebab itu industri wajib merancang serta melakukan kegaitan pemasaran dengan sebaikbaiknya. Dalam perihal ini pemasaran mencakup aktivitas yang sangat luas meliputi seluruh kegiatan industri dalam penuhi kebutuhan serta kemauan warga lewat sesuatu proses penciptaan serta penawaran yang berbentuk benda maupun jasa. Jurnal Hukum dan Pembangunan Ekonomi, Volume 9, Nomor 2, 2021 259 ISSN (Print) 2338-1051, ISSN (Online) 2777-0818 259 Pemasaran itu suatu proses sosial yang di dalamnya orang serta kelompok hendak memperoleh apa yang mereka butuhkan, serta apa yang diinginkan dengan menghasilkan, menawarkan secara leluasa untuk mempertukarkan produk yang bernilai dengan pihak yang lain. Pemasaran tidak cuma dicoba dengan komunikasi antara mulut dengan mulut saja, namun pula dapat dengan wujud foto serta iklan selaku media pemasarannya.4 260 Jurnal Hukum dan Pembangunan Ekonomi, Volume 9, Nomor 2, 2021 ISSN (Print) 2338-1051, ISSN (Online) 2777-0818 260 Jurnal Hukum dan Pembangunan Ekonomi, Volume 9, Nomor 2, 2021 ISSN (Print) 2338-1051, ISSN (Online) 2777-0818 Religiusitas g Religiusitas adalah sumber dari segala sesuatu yang menjadi pedoman atau tolak ukur individu dalam mencari kebenaran untuk melakukan suatu ibadah bagi seorang individu, religiusitas cenderung kepada besarnya sikap seseorang kepada kepatuhan dan pengabdianya terhadap agama yang di anutnya. © Author(s). This work is licensed under a Creative Commons Attribution 4.0 International License. Published by Postgraduate Program, Master of Laws, Faculty of Law, Universitas Sebelas Maret, Indonesia © Author(s). This work is licensed under a Creative Commons Attribution 4.0 International License. Published by Postgraduate Program, Master of Laws, Faculty of Law, Universitas Sebelas Maret, Indonesia Advertising Merupakan fasilitas promosi yang digunakan oleh sesuatu lembaga buat menginformasikan seluruh produk ataupun jasa yang digunakan oleh bank. Dalam periklanan ini membagikan data yang bisa membagikan khasiat pada produk, harga dan keunggulankeunggulan produk yang dibanding dengan produk yang sejenis yang ditawarkan oleh pesaing. Dalam tujuan promosi memakai iklan ini tujuan buat menarik serta bisa pengaruhi calon nasabah serta nasabah lamanya. Iklan merupakan wujud dari presentasi non- pribadi serta sesuatu promosi dari gagasan, benda ataupun jasa oleh sesuatu sponsos yang wajib dibayar. Advertising merupakan wujud dari perlengkapan promosi yang umumnya digunakan buat memusatkan komunikasi secara persuasif pada pembeli dengan memilki sasaran dari masyarakat dimana bentuknya dalam menyajikan iklanya bersifat sangat non-personal. Kepercayaan Keyakinan merupakan kemauan buat tergantung kepada orang lain dalam aktivitas kerjasama yang bisa diyakini, kepercayaan dalam ikatan kerjasama memilki makna kepercayaan didalam industri yang bearti pihak patner dipercayai hendak melaksanakan aksi yang nantinya hendak membawa kepada keuntungan yang tertentu serta tidak hendak melaksanakan perbuatan yang nantinya merugikan industri. Mendefinisikan kalau keyakinan ialah harapan yang bisa dipegang oleh konsumen kepada penyedia jasanya yang bisa dipercaya serta nantinya bisa diandalkan untuk bisa membagikan cocok apa yang sudah dijanjikan. Aspek yang bisa diambil dari definisi merupakan keyakinan ialah sesuatu kepercayaan serta harapan terhadap atensi yang dipercaya selaku akibat dari keahlianya serta dari mutu lembaga tersebut. Keyakinan itu sendiri hendak mencuat selaku hasil dari kehandalan serta mutu dari lembaga yang hendak ditunjukkan lewat bermacam perilaku yang semacam konsitensi, komponen, adil serta bisa bertanggung jawab. Kerangka Penelitian Kerangka Penelitian Kerangka Penelitian H1 H2 H3 H4 H5 H6 Advertising X ( 2) Religiusitas ( X 3) Kepercayaan (Z) Keputusan menabung ( Y ) Sales Promotion 1) X ( Sales Promotion 1) X ( H1 Keputusan menabung ( Y ) Advertising X ( 2) Religiusitas ( X 3) Kepercayaan (Z) Kerangka penelitian diatas menggambarkan bagaimana hubungan variabel bebas ke variabel terkait, melalui variabel pemediasi. Dalam hal ini sales promotion, advertising, religiusitas (X) sebagai variabel independent mempengaruhi keputusan (Y) melalui kepercayaan sebagai varibel mediasi (Z). Jadi hipotesis dalam penelitian ini adalah sebagai berikut: H1 : Sales promotion berpegaruh positif signifikan terhadap keputusan menabung H2 : Advertising berpengaruh negative signifikan terhadap keputusan menabung H3 : Religiusitas berpengaruh positif signifikan terhadap keputusan menabung Jurnal Hukum dan Pembangunan Ekonomi, Volume 9, Nomor 2, 2021 261 ISSN (Print) 2338-1051, ISSN (Online) 2777-0818 261 H4 : Kepercayaan dapat meningkatkan pengaruh sales promotion terhadap keputusan menabung. H4 : Kepercayaan dapat meningkatkan pengaruh sales promotion terhadap keputusan menabung. g H5 : Kepercayaan dapat meningkatkan pengaruh advertising terhadap keputusan menabung g H5 : Kepercayaan dapat meningkatkan pengaruh advertising terhadap keputusan menabung H6 : Kepercayaan dapat meningkatkan pengaruh religiusitas terhadap keputusan menabung H6 : Kepercayaan dapat meningkatkan pengaruh religiusitas terhadap keputusan menabung METHOD Populasi yang digunakan didalam penelitian ini adalah nasabah BSI Cabang Kudus. Sampel diambil berdasarkan nasabah yang sedang melakukan transaksi. Dalam pengumulan data, peneliti menggunakan probability sampling dengan teknik random sampling, yaitu teknik pengambilan sampel yang memberikan peluang yang sama bagi setiap anggotanya, populasi untuk dijadikan sampel. Penelitian ini menggunakan analisis IMB SPS Statistic 24. Analisis ini dilakukan untuk menguji bagaimana pengaruh variabel independent terhadap variabel dependen melalui variabel pemediasi. © Author(s). This work is licensed under a Creative Commons Attribution 4.0 International License. Published by Postgraduate Program, Master of Laws, Faculty of Law, Universitas Sebelas Maret, Indonesia © Author(s). This work is licensed under a Creative Commons Attribution 4.0 International License. Published by Postgraduate Program, Master of Laws, Faculty of Law, Universitas Sebelas Maret, Indonesia RESULTS & DISCUSSION Coefficientsa Model Unstandardized Coefficients Standardized Coefficients t Sig. B Std. Error Beta 1 (Constant) 29.929 6.134 4.879 .000 Sales Promotion .209 .069 .300 3.023 .003 Advertising .328 .091 .031 3.309 .758 Religiusitas .308 .070 .011 1.114 .010 a. Dependent Variable: Kepercayaan Uji T T Coefficientsa Model Unstandardized Coefficients Standardized Coefficients t Sig. B Std. Error Beta 1 (Constant) 29.929 6.134 4.879 .000 Sales Promotion .209 .069 .300 3.023 .003 Advertising .328 .091 .031 3.309 .758 Religiusitas .308 .070 .011 1.114 .010 a. Dependent Variable: Kepercayaan © Author(s). This work is licensed under a Creative Commons Attribution 4.0 International License . Published by Postgraduate Program, Master of Laws, Faculty of Law, Universitas Sebelas Maret, Indonesia 262 Jurnal Hukum dan Pembangunan Ekonomi, Volume 9, Nomor 2, 2021 ISSN (Print) 2338-1051, ISSN (Online) 2777-0818 Berdasarkan hasil uji persial pada table diatas dapat ditarik kesimpulan bahawa: a. Pengaruh sales promotion terhadap kepercayaan Berdasarkan hasil uji di atas bahwa pengaruh sales promotion terhadap kepercayaan memiliki pengaruh yang positif signifikan dengan nilai T hitungnya 3.023 dan nilai sig 0,03 < 0,05. b. Pengruh advertising terhadap kepercayaan Berdasarkan hasil uji di atas bahwa pengaruh advertising terhadap kepercayaan memiliki pengaruh yang positif tidak signifikan dengan nilai T hitungnya 3.309 dan nilai sig 0,758 > 0,05. c. Pengaruh religiusitas terhadap kepercayaan Berdasarkan hasil uji di atas bahwa pengaruh religiusitas terhadap kepercayaan memiliki pengaruh yang positif signifikan dengan nilai T hitung 1.114 dan nilai signifikan 0,10 < 0,05. Coefficientsa Model Unstandardized Coefficients Standardized Coefficients T Sig. B Std. Error Beta 1 (Constant) 22.793 5.696 4.001 .000 Sales Promotion .136 .276 .175 2.789 .037 Advertising -.014 .059 -.022 -.231 .818 Religiusitas 225 .860 .043 2.422 .044 Kepercayaan .280 .085 .330 3.303 .001 a. Dependent Variable: Keputusan Coefficientsa Model Unstandardized Coefficients Standardized Coefficients T Sig. B Std. Error Beta 1 (Constant) 22.793 5.696 4.001 .000 Sales Promotion .136 .276 .175 2.789 .037 Advertising -.014 .059 -.022 -.231 .818 Religiusitas 225 .860 .043 2.422 .044 Kepercayaan .280 .085 .330 3.303 .001 a. Dependent Variable: Keputusan Berdasarkan hasil uji persial pada table diatas dapat ditarik kesimpulan bahawa: a. Pengaruh Sales Promotion terhadap keputusan menabung Yang dilihat dari uji persial di atas memiliki pengaruh yang positif signifikan terhadap keputusan menabung dengan nilai T hitung 2.789 dn nilai sig 0,37 < 0,05. b. RESULTS & DISCUSSION Pengaruh advertising terhadap keputusan menabung yang dilihat dari hasil uji di atas berpengaruh negatif tidak signifikan terhadap keputusan menabung dengan T hitung -231 dan nilai sig 0,818 > dari 0,05. b. Pengaruh advertising terhadap keputusan menabung yang dilihat dari hasil uji di atas berpengaruh negatif tidak signifikan terhadap keputusan menabung dengan T hitung -231 dan nilai sig 0,818 > dari 0,05. Jurnal Hukum dan Pembangunan Ekonomi, Volume 9, Nomor 2, 2021 263 ISSN (Print) 2338-1051, ISSN (Online) 2777-0818 263 c. Pengaruh religiusitas terhadap keputusan menabung dapat dilihat dari table uji diatas, bahwa pengaruh religiusitas terhadap keputusan menabung adalah positif signifikan dengan nilai T hitung 2.422 dan nilai sig 0,44 < 0,05. c. Pengaruh religiusitas terhadap keputusan menabung dapat dilihat dari table uji diatas, bahwa pengaruh religiusitas terhadap keputusan menabung adalah positif signifikan dengan nilai T hitung 2.422 dan nilai sig 0,44 < 0,05. d. Pengaruh kepercayaan terhadap keputusan menabung dapat dilihat dari table uji diatas bahwa kepercayaan (z) memiliki pengaruh positif signifikan terhadap keputusan menabung dengan nilai T hitung-nya 3.303 dan nilai sig 0,001 < 0,05. d. Pengaruh kepercayaan terhadap keputusan menabung dapat dilihat dari table uji diatas bahwa kepercayaan (z) memiliki pengaruh positif signifikan terhadap keputusan menabung dengan nilai T hitung-nya 3.303 dan nilai sig 0,001 < 0,05. a. Sales Promotion Model Summary Model R R Square Adjusted R Square Std. Error of the Estimate 1 .362a .131 .104 4.06474 a. Predictors: (Constant), Sales promotion x Kepercayaan, Sales Promotion, Kepercayaan Dari tabel-tabel diatas ada dua uji dalam uji moderating yang pertama uji pengaruh sales promotion (x1) terhadap keputusan menabung (y) dan varibel moderasi kepercayaan (z). Dengan hasil dari tabel pertama nilai dari Rsquare regresi sebesar 0,030 sehingga dapat dikatakan bahwa variabel sales promotion terhadap keputusan menabung sebesar 3%. Sedangkan uji regresi yang kedua setelah adanya variabel moderasi (kepercayaan) pada persamaan regresi nilai yang dihasilkan meningkat menjadi 0,131 atau 13,1%. Dengan demikian maka dapat disimpulkan bahwa hasil hipotesis dapat diterima, dan dapat dikatakan bahwa keberdaan kepercayaan dapat memperkuat dan meningkatkan pengaruh sales promotion terhadap keputusan menabung. b. Advertising Model R R Square Adjusted R Square Std. Error of the Estimate 1 .319a .102 .074 4.13327 a. Predictors: (Constant), Advertising x Kepercayaan, Kepercayaan, Advertising b. Advertising Model R R Square Adjusted R Square Std. Error of the Estimate 1 .319a .102 .074 4.13327 a. Predictors: (Constant), Advertising x Kepercayaan, Kepercayaan, Advertising Uji Moderating a. Sales Promotion © Author(s). This work is licensed under a Creative Commons Attribution 4.0 International License. Published by Postgraduate Program, Master of Laws, Faculty of Law, Universitas Sebelas Maret, Indonesia c. Religiusitas Dari tabel diatas pada persamaan regresi pertama atau tabel pertama nilai dari Rsquare sebesar 0,041 sehingga dapat dikatakan bahwa variabel religiusitas berpengaruh terhadap variabel keputusan menabung sebesar 4,1%. Dan ditabel kedua setelah adanya variabel kepercayaan sebagai variabel moderasi pada persamaan regresi nilai Rsquare meningkat menjadi 0,106 atau 10,6%. Maka dengan demikian bahwa hipotesis dapat diterima dengan adanya variabel kepercayaan sebagai variabel moderasi dengan meningkatnya nilai Rsquare tersebut dapat memperkuat hubungan variabel religiusitas terhadap keputusan menabung. b. Advertising © Author(s). This work is licensed under a Creative Commons Attribution 4.0 International License . Published by Postgraduate Program, Master of Laws, Faculty of Law, Universitas Sebelas Maret, Indonesia 264 Jurnal Hukum dan Pembangunan Ekonomi, Volume 9, Nomor 2, 2021 ISSN (Print) 2338-1051, ISSN (Online) 2777-0818 Dari tabel-tabel diatas ada dua uji dalam uji moderating yang pertama uji pengaruh advertising (x2) terhadap keputusan menabung(y) dan varibel moderasi kepercayaan (z). Nilai Rsquare pada persamaan tabel pertama sebesar 0,010 sehingga dapat dikatakan bahwa variabel dari advertising berpengaruh terhadap variabel keputusan menabung, dengan nilai sebesar 1%. Sedangkan dari hasil uji kedua menggunakan variabel moderasi pada persamaan regresi yang kedua dengan nilai Rsquare meningkat menjadi 0,102 atau 10,2%. Dengan demikian itu dapat disimpulkan bahwa hipotesis dapat diterima dengan dikatakanya bahwa keberadaan variabel kepercayaan sebagai variabel moderasi dapat memperkuat dengan meningkatnya Rsquare sebesar 10,2%. c. Religiusitas Model Summary Model R R Square Adjusted R Square Std. Error of the Estimate 1 .325a .106 .078 4.12458 a. Predictors: (Constant), Religiusitas x Kepercayaan, Kepercayaan, Religiusitas c. Religiusitas Pengaruh sales promotion terhadap keputusan menabung dengan kepercayaan sebagai variabel moderasi. g Meningkatnya nilai R2 dari nilai 0,030 menjadi 0,131 mengidtifikasikan bahwa variabel kepercyaan dapat memperkuat minat pegaruh sales promotion terhadap keputusan menabung. Hasil ini sesuai dengan penelitin yang dilakukan oleh Wijayani (2017), menyatakan dalam penelitianya semakin baik kegiatan promosi dilakukan, maka masyarakat semakin mengerti tentang bank syariah beserta apa saja isi dari bank syariah tersebut, promosi dibutuhkan untuk memberikan informasi secara actual untuk mendapatkan kepercayaan dari nasabah, semakin tinggi tingkat kepercayaan semakin tinggi pula minat untuk memutuskan menabung di bank syariah. Hasil tersebut di dukung penelitian dari Halik (2016) dalam penelitianya bauran pemasaran jasa dapat digunakan sebagai kegiatan yang berkaitan dengan menumbuhkan kepercayaan untuk menciptakan sebuah komitmen nasabah melalui kegiatan dari bauran pemasaran. Pengaruh religiusitas terhadap keputusan menabung. Pengujian yang dilakukan berkaitan dengan penelitian pengaruh religiusitas terhadap keputusan menabung memberikan hasil yang positif signifikan terhadap keputusan menabung dengan nilai sig 0,044 < 0,05 dengan nilai koefisien 0,225 sehingga terdapat pengaruh yang positif dan signifikan antara religiusitas dengan keputusan menabung. Bahwa religiusitas sebagai acuan utuk berpedoman hidup dalam melakukan kegiatan sehari-hari begitu pula dalam melakukan transaksi sebagai umat muslim hendaknya menjahui riba dengan menggunka jasa syariah. Dengan didukung penelitian yang dilakukan oleh Satrio dan Siswanto (2016) dan Oktaviani (2018) Dengan adanya individu yang yang memiliki sifat religiusitas akan memilih bank syariah untuk kegiatan ekonominya. Hal serupa juga di dukung oleh penelitian dari Ningsih (2017) dan Riskyono (2017) yang menyatakan bahwa religiusitas berpengaruh positif terhadap minat menabung. © Author(s). This work is licensed under a Creative Commons Attribution 4.0 International License . Published by Postgraduate Program, Master of Laws, Faculty of Law, Universitas Sebelas Maret, Indonesia Pengaruh advertising terhadap keputusan menabung Pengujian yang dilakukan berkaitan dengan penelitian pengaruh advertising terhadap keputusan menabung memberikan hasil yang negatif tidak signifikan dengan nilai sig 0,818 > 0,05 dengan koefisienya -0,136 sehingga tidak dapat berpengaruh yang positif dan menurunkan antara variabel advertising dengan variabel keputusan menabung. Darna dan Dita (2013) dalam penelitiannya mengemukakan bahwa sebagai nasabah tidak hanya melihat adanya iklan bank syariah melalui media masa saat nasabah tersebut memutuskan mejadi nasabah di bank syariah ada beberapa faktor untuk mempengaruhi keputusan untuk menabung di bank syriah yaitu dipengaruhi oleh lingkunganya, strategi pemasaranya dan pengetahuanya. Didukung penelitian dari Riani dkk (2017) dan Arman (2015) yang menyatakan bahwa advertising berpengaruh positif dan tidak signifikan terhadap keputusan. Pengaruh sales promotion terhadap keputusan menabung Pengujian yang dilakukan berkaitan dengan penelitian pengaruh sales promotion terhadap keputusan menabung memberikan hasil yang signifikan dengan nilai signifikanya sebesar 0,037 < 0,05 dengan koefisienya 0,136 sehingga terdapat pengaruh yang positif signifikan terhadap keputusan menabung. Sales promotion yang dilakukan dengan dengan memberikan hadiah, souvenir dan kebebasan biaya admin yang dilakukan oleh bank syariah dapat menyatakan bahwa proses pemilihan untuk menindak lanjuti nasabah dalam memilih lembaga keuagan oleh karena itu dibutuhkan informasi yang lengkap, terpercaya dan actual untuk pengambilan keputusan. Berdasarkan hal ini nasabah membutuhkan suaru informasi yang lengkap dari promosi penjualan. Didukung oleh penelitian oleh Girang (2018), Diana dkk (2018) dan Dea, krisna (2018) dan Kamsir (2004) yang menyatakan promosi penjualan berpengaruh positif signifikan terhadap keputusan. Jurnal Hukum dan Pembangunan Ekonomi, Volume 9, Nomor 2, 2021 265 ISSN (Print) 2338-1051, ISSN (Online) 2777-0818 265 Pengaruh religiusitas terhadap keputusan menabung dengan kepercayaan sebagai variabel moderasii Meningkatnya niali R2 dari 0,041 menjadi 0,106 mengidentifikasikan bahwa kepercayaan memperkuat pengaruh religiusitas terhadap keputusan menabung. hasil ini sama dengan penelitian Selanjutnya penelitian Halik (2006) hasil penelitianya menunjukkan nilai religiusitas pada kepercayaan berpengaruh positif dan signifikan, religiusitas dapat digunakan dasar untuk membuat kebijakan di bank syariah, kepercayaan layak sebagai variabel moderating. Pengaruh advertising terhadap keputusan menabung dengan kepercayaan sebagai variabel moderasi g Meningkatnya nilai R2 dari 0, 010 menjadi 0,131 mmenginditifikasikan bahwa variabel kepercayaan memperkuat pengaruh advertising terhadap keputusan menabung. semakin tinggi tingkat kepercayaan nasabah yag didapatkan dari iklan yang mereka lihat akan 266 Jurnal Hukum dan Pembangunan Ekonomi, Volume 9, Nomor 2, 2021 ISSN (Print) 2338-1051, ISSN (Online) 2777-0818 memperkuat keinginan mereka untuk menabung di bank syariah. Hasil ini sesuai dengan penelitian yang dilakukan oleh Rohana (2019) menunjukkan bahwa kepa dapat memediasi dengan baik advertising terhadap keputusan. Apabila advertising disampaikan dengan menarik maka akan semakin berpengaruh terhadap minat seseorang dalam melakukan keputusan menabung di bank syariah. CONCLUSION Berdasarkan hasil uji analisis data dan penjelasan yang telah dijabarkan pada uraian pengaruh sales promotion, advertising dan religiusitas terhadap keputusan menabung dengan kepercayaan sebagai variabel moderasi pada PT Bank Syariah Indonesia KC Kudus Ahmad Yani 1 maka dapat disimpulkan sebagai berikut: Yani 1, maka dapat disimpulkan sebagai berikut: 1. Berdasarkan dari hasil uji T (Persial) dapat diambil kesimpulkan sales promotion secara persial memiliki pengaruh yang positif signifikan terhadap keputusan menabung. 2. Berdasarkan dari hasil uji T (Persial) dapat diambil kesimpulan bahwa advertising secara persial memiliki hasil yang negatif tidak signifkan terhadap keputusan menabung. g 3. Berdasrkan hasil dari hasil uji T (Persial) dapat diambil kesimpulan bahwa religiusitas secara persial memiliki hasil yang positif signifikan terhadap keputusan menabung. 4. Berdasarkan dari hasil uji MRA kepercayaan dapat memperkuat hubunga pengaruh sales promotion terhadap keputusan menabung. 5. Berdasarkan hasil dari uji MRA kepercayaan dapat memperkuat hubungan pengaruh advertising terhadap keputusan menabung. 6. Berdasarkan hasil dari uji MRA kepercayaan dapat memperkuat hubungan pengaruh religiusitas terhadap keputusan menabung. © Author(s). This work is licensed under a Creative Commons Attribution 4.0 International License. Published by Postgraduate Program, Master of Laws, Faculty of Law, Universitas Sebelas Maret, Indonesia Jurnal Hukum dan Pembangunan Ekonomi, Volume 9, Nomor 2, 2021 267 ISSN (Print) 2338-1051, ISSN (Online) 2777-0818 267 © Author(s). This work is licensed under a Creative Commons Attribution 4.0 International License . Published by Postgraduate Program, Master of Laws, Faculty of Law, Universitas Sebelas Maret, Indonesia REFERENCES Alma, B. (2009). Manajemen pemasaran dan pemasaran jasa edisi Revisi. Cetakan Kelima. CV. 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https://openalex.org/W2510405261	https://europepmc.org/articles/pmc5006766?pdf=render	English	null	Complete supine PCNL: ultrasound vs. fluoroscopic guided: a randomized clinical trial	International Braz J Urol	2,016	cc-by	3,485	Keywords: Materials and Methods: In this randomized clinical trial study from January 2009 to September 2010, 26 of 51 patients with renal stones underwent csPCNL with ultraso- nographic guidance in all steps of the procedure (group A), and the other 25 patients underwent standard fluoroscopically guided csPCNL (group B). All of the patients un- derwent PCNL in the complete supine position. Statistical analysis was performed with SPSS16 software. Results: Mean BMI was 28.14 in group A and 26.31 in group B (p=0.30). The mean stone burden was 26.48 and 30.44 in groups A and B, respectively (p=0.20). The stone free rate was 88.5% in group A and 75.5% in group B, that was no significant (p=0.16). Overall 2 patients (7.7%) in group A and 6 patients (24%) in group B had complications (p=0.11). Mean operative time in group A was 88.46 minutes, and in group B it was 79.58 minutes (p=0.39). Mean hospital stay was 69.70 and 61.79 hours in group A and B, respectively (p=0.22). There was no visceral injury in groups. Accepted after revision: October 12, 2015 Conclusions: This randomized study showed that totally ultrasonic had the same outcomes of fluoroscopically csPCNL. Ultrasonography can be an alternative rather than fluorosco- py in PCNL. We believe that more randomized studies are needed to allow endourologists to use sonography rather than fluoroscopy in order to avoid exposition to radiation. doi: 10.1590/S1677-5538.IBJU.2014.0291 Vol. 42 (4): 710-716, July - August, 2016 doi: 10.1590/S1677-5538.IBJU.2014.0291 Vol. 42 (4): 710-716, July - August, 2016 ORIGINAL ARTICLE Siavash Falahatkar 1, Aliakbar Allahkhah 1, Majid Kazemzadeh 1, Ahmad Enshaei 1, Maryam Shakiba 1, Fahimeh Moghaddas 1 Siavash Falahatkar 1, Aliakbar Allahkhah 1, Majid Kazemzadeh 1, Ahmad Enshaei 1, Maryam Shakiba 1, Fahimeh Moghaddas 1 1 Urology Research Center, Guilan University of Medical Sciences, Guilan, Iran ABSTRACT ___________ Keywords: Nephrostomy, Percutaneous; Ultrasonography; Fluoroscopy; Supine Position Int Braz J Urol. 2016; 42: 710-6 _____________________ Submitted for publication: June 15, 2014 _____________________ Accepted after revision: October 12, 2015 Introduction and Hypothesis: To compare complications and outcomes of complete supine percutaneous nephrolithotomy (csPCNL) with ultrasound guided and fluorosco- pically guided procedure. ibju \| ultrasound vs. fluoroscopic guided PCNL the Amplatz sheath were not exactly visible by ultrasonography. ultrasound-guided csPCNL procedure with the urological community. In group B, we performed all the above steps of csPCNL with the guidance of fluorosco- py. Our technique was a one-shot dilatation in both groups. INTRODUCTION to some level of radiation by fluoroscopy during PCNL. The side effects of extensive radiation are well known. Thus, the ultrasound-guided PCNL can be an alternative method to decrease the ra- diation exposure hazard to the surgeon (4-6). Percutaneous nephrolithotomy (PCNL) is a common method for treatment of kidney stones (1, 2). All of the steps in PCNL should be perfor- med with proper image guidance. The imageless PCNL should never be applied because it is dange- rous to vital structures (3).l The purpose of present study is to com- pare complications and outcomes in patients who underwent complete supine percutaneous nephro- lithotomy (csPCNL) with these two methods and to share the experience of the authors with totally The popular imaging of PCNL is fluorosco- py, so the patient and surgical team are exposed 710 MATERIAL AND METHODS In this randomized clinical trial stu- dy from January 2009 to September 2010, 51 patients with renal stones were selected for csPCNL. All participants were informed about the surgical method and consent. We used to- tally ultrasonographic guidance in all steps of the procedure during csPCNL in 26 of our pa- tients (group A), whereas the other 25 patients underwent standard fluoroscopically guided csPCNL (group B). All patients in both groups performed PCNL in the complete supine position without any towel under the patient’s flank and with no change in leg position. For all patients, routine blood and urine tests, coagulation pro- file and imaging series, including intravenous urogram and ultrasonography, were carried out and medical conditions were studied. In this study, the items including side of renal unit, stone burden, stone-free rate, compli- cations (extravasation, colon injury, fever, etc.), and the history of previous open renal surgery or previous ESWL, mean hospital stay, mean operati- ve time, body mass index (BMI), serum creatinine before the operation, and hemoglobin before and after the csPCNL were studied. In group A, after removal of the stone(s), ultrasonography was used to detect any residual stones, hematoma, or extravasation of urine out- side of the kidney.l In the fluoroscopic group, residual stones and extravasation were checked by fluoroscopy. We performed tubeless PCNL except in patients with severe extravasation, ureteral obstruction, severe hemorrhage, or large residual stone. Inclusion criteria were patients with sin- gle large pelvic stone, lower caliceal stone, sto- nes in the pelvis and lower calyx, middle cali- ceal stones, and non-opaque stones (staghorn stones) with hydronephrosis. Statistical analysis was performed with SPSS16 software. A P value of less than 0.05 was considered statistically significant. This study was approved by ethical review committee of Guilan University of Medical Scien- ce and the trial registered at http://www.irct.ir (IRCT138805251853N3). Exclusion criteria’s in this study were multiple stones in multiple calyxes, staghorn stones (except non-opaque stones), urinary tract anomalies, single kidney and morbid obesity and non-opaque stones (staghorn stones) wi- thout hydronephrosis. RESULTS Total number of patients in both groups was 51 (26 patients in group A and 25 patients in group B). Demographic data and stone characteris- tics of two groups are shown in Table-1. In group A, mean age was 48.41 years and in group B it was 51.17 years (p=0.46). Mean BMI was 28.14 in group A and 26.31 in group B (p=0.30). The mean hemoglobin level before operation was 12.81 and 13.38 in groups A and B, respectively (p=0.23). The mean stone burden was 26.48 and 30.44 in groups A and B, respectively (p=0.20). The sto- ne burden was detected on the basis of maximum diameter of stones on the KUB or ultrasonography. 4 patients (15.4%) in group A and 7 patients (28%) in group B had coexisting disease (p=0.44). All of All of the patients underwent general anesthesia, and a 5F ureteral catheter was pla- ced transurethrally for injection of saline or contrast media. Injection of saline obtained mild dilatation of collecting system and this was use- ful especially for the totally ultrasound-guided PCNL group. In group A, ultrasonography was used to observe the location of the kidney, needle entrance point, urinary tract dilatation and to check for residual stone at the end of csPCNL. Because the Rouch guidewire is more rigid, and in order to not miss the access, we used this type of guidewire, although the guidewire was clearly visible but the Amplatz dilatators and 711 ibju \| ultrasound vs. fluoroscopic guided PCNL Table 1 - This table showed the demographic data of two groups according to method of study. Ultrasonographic Group Fluoroscopic Group P_Value Total N 26 25 - Sex Male (%) 17 (65.4) 15 (60) 0.69 Female (%) 9 (34.6) 10 (40) Age (Year) 48.41 51.17 Mean (SD) (13.22) (11.82) 0.46 BMI (Kg/m2) 28.17 26.31 Mean (SD) (4.17) (5.88) 0.30 Serum Cr. RESULTS before the Operation, 1.45 1.16 Mean (SD) (1.60) (0.28) 0.38 Hb before the Operation, 12.81 13.38 Mean (SD) (1.78) (1.56) 0.23 Stone Size (mm), 26.48 30.44 Mean (SD) (10.90) (11) 0.20 Number of Stones, 1.42 1.58 Mean (SD) (0.50) (0.50) 0.26 Side,n (%) Right 15 (57.7) 17 (68) 0.51 Left 10 (38.5) 8 (32) Co-existing Disease,n (%) Yes 4 (15.4) 7 (28) 0.44 No 22 (84.6) 19 (76) Previous open or percutaneous surgery, n (%) Yes 6 (23.1) 7 (28) 0.68 No 20 (76.9) 18 (72) Previous ESWL, n (%) Yes 11 (42.3) 13 (52) 0.48 No 15 (57.7) 12 (48) Table 1 - This table showed the demographic data of two groups according to method of study. the patients underwent general anesthesia and the access was sub costal in all patients. Intra and postoperative parameters of the two groups are shown in Table-2. The stone free rate was 88.5% in group A and 75.5% in group B, that was no significant (p=0.16). Overall, 2 patients (7.7%) in group A and 6 patients (24%) in group B had com- plications (p=0.11). In group A, 1 patient (3.8%) had fever, and in group B, 4 patients (16%) needed transfusion and 2 patients (8%) had fever (Grade I and II of the Clavien Classification of Surgical Complications). Mean operation time in group A was 88.46 minutes, and in group B, it was 79.58 minutes (p=0.39). Mean hospital stay was 69.70 and 61.79 hours in groups A and B, respectively (p=0.22). There was no complications compatible with Grade III to V of the Clavien Classification of Surgical Complications in both groups. transfusion and 2 patients (8%) had fever (Grade I and II of the Clavien Classification of Surgical Complications). Mean operation time in group A was 88.46 minutes, and in group B, it was 79.58 minutes (p=0.39). Mean hospital stay was 69.70 and 61.79 hours in groups A and B, respectively (p=0.22). There was no complications compatible with Grade III to V of the Clavien Classification of Surgical Complications in both groups. 712 ibju \| ultrasound vs. fluoroscopic guided PCNL DISCUSSION Th f d l d it it py, ultrasonography, or computed tomography (CT) guidance (7-9). T d th i k f di ti Table 2 - This table showed the comparison of results after the procedure between two groups. RESULTS Ultrasonographic Group Fluoroscopic Group P_Value Total N 26 25 - Stone free rate (%) Stone free 20 (77) 17 (71) Residual stone<5mm 3 (11.5) 1 (4.5) 0.16 Residual stone>5mm 3 (11.5) 6 (27.3) Complications Yes 2 (7.7) 6 (24) 0.1 No 24 (92.3) 19 (76) Nephrostomy tube Yes 2 (8.7) 1 (4.3) 0.55 No 21 (91.3) 22 (95.7) Duration of access to target calyx (sec) 14.36 14.78 0.08 Mean (SD) (14.84) (25.54) Duration of entrance to target calyx (sec) 84.87 41.22 0.07 Mean (SD) (112.83) (48.51) Duration of 9Fr dilator dilatation (sec) 22.48 23.39 0.78 Mean (SD) (26.7) (37.7) Duration of Amplatz dilator dilatation (sec) 32.72 15.57 0.77 Mean (SD) (82.45) (15.94) Duration of Amplatz sheath insertion (sec) 17.46 12.41 0.28 Mean (SD) (26.72) (15.67) Hb Drop after the operation 1.11 1.14 Mean (SD) (1.35) (1.52) 0.93 Operating time, min 88.46 79.58 Mean (SD) (39.49) (32.6) 0.39 Hospital stay, hour 69.70 61.79 Mean (SD) (18.87) (25.22) 0.22 Extravasation (%) 0 0 - Pseudo aneurism (%) 0 0 - Fever, n (%) 1(3.8) 2(8) - Colon injury (%) 0 0 - Table 2 - This table showed the comparison of results after the procedure between two groups. ibju \| ultrasound vs. fluoroscopic guided PCNL with ultrasound-guided percutaneous nephro- lithotomy were 45.7 - 69.6% and 82.6 - 96.5%, respectively (21, 26). In our study, similar to the others, the stone-free rate was 88.46% and 72%, without any significant statistical difference in groups A and B, respectively (p=0.16). Some studies reported that PCNL under ultrasonography guidance in the flank or prone position has high success rates and limited com- plications and can be a safe and effective al- ternative to fluoroscopy in experienced hands (11-18). The mean operative time was 120±68 mi- nutes (range 45-350) in one study. The real-time ultrasound can be used to guide the percutaneous puncture (26). In another study mean (range) of operative time was 111 (70-180) minutes. They emphasized that ultrasonographic - guided PCNL is feasible but fluoroscopy must be present in the operating room (27). Mean operative time was re- ported as 88.92 and 79.28 minutes in sonographic and fluoroscopic groups, respectively (20). In the current study mean operative time was similar to other studies without any significant statistical di- fference (p-value=0.39). Ultrasound-guided PCNL without fluoros- copy has some advantages and disadvantages. Advantages: Avoidance of X-ray exposure, no necessity of lead shield, all organs on the way of access are visible, search for residual stones at the end of the procedure especially for non-opaque stones. Disadvantages: Endourologists are unfa- miliar with ultrasonography, and poor echogeni- city of the Amplatz dilatator and Amplatz sheath (11, 12, 19, 20). Nowadays, PCNL is considered a generally safe management option with a low incidence of complications and is the method of choice for tre- atment of renal stones (11, 21, 22). Hospital stay was 3.6 days (range 2-8 days) in one study and other studies reported 2.7 to 4.1 days (5, 12, 20 24, 27). In our study, hospital stay was similar to other studies without any signifi- cant difference (p=0.22). PCNL is done in the prone, flank, semi-su- pine, and csPCNL positions. We performed csPCNL in our patients due to better control of the airway, better tolerance for patients especially with car- diopulmonary disease, easier to perform ureteros- copy or TUL, better drainage and evacuation of stones by the Amplatz sheath, possibility to chan- ge regional anesthesia to general anesthesia, and probably less risk of colon injury. These are some advantages of csPCNL (13). Although we had seen more complications in fluoroscopic group, they were not significant. DISCUSSION py, ultrasonography, or computed tomography (CT) guidance (7-9). The scope of endourology despite its short age has been widened. The first step in percutaneous procedures is to access to the col- lecting system, usually performed by fluorosco- To reduce the risk of radiation exposure, using ultrasonography for PCNL can be an al- ternative imaging method to fluoroscopy as the first and standard imaging technique (10, 11). 713 ibju \| ultrasound vs. fluoroscopic guided PCNL 9. Lee WJ. Advances in percutaneous nephrostomy. Yonsei Med J. 1990;31:285-300. study. We achieved access in all patients and we believe that ultrasound-guided csPCNL in obese patients is more difficult but it is safe and feasible. Sometimes it was imperative to draw up the fatty abdomen with a strip band for preventing any en- cumbrance during the procedure. 10. Grasso M: Techniques for percutaneous renal access. Textbook of Endourology. Philadelphia, WB Saunders. 1997; pp.101-2. 11. Basiri A, Ziaee AM, Kianian HR, Mehrabi S, Karami H, Moghaddam SM. Ultrasonographic versus fluoroscopic access for percutaneous nephrolithotomy: a randomized clinical trial. J Endourol. 2008;22:281-4. PCNL is feasible and safe in the supine po- sition (13, 29-32). 12. Hosseini MM, Hassanpour A, Farzan R, Yousefi A, Afrasiabi MA. Ultrasonography-guided percutaneous nephrolithotomy. J Endourol. 2009;23:603-7. ibju \| ultrasound vs. fluoroscopic guided PCNL In this study we found no extravasation in both groups. This result was similar to others (20, 21). Some studies reported 4-9% with pos- toperative fever (12, 18). Other studies reported 26.3-27.6% postoperative fever and the patients responded to antibiotics (21, 27). In this study fe- ver had no effect on the results of our study. All of the patients with fever were cured with appropria- te antipyretics and antibiotics. Septic shock was not a major complication in our patients. Because of levitation of the colon in the abdominal cavity in the supine position it is less affected to injury when puncture site is in the pos- terior axillary line (23). The access to the kidney is important in PCNL and usually is performed by fluoroscopy, ultrasonography, or computed tomography gui- dance with rates of success of 86.7-100% (9, 11, 20, 24-26). The success rate in achieving access in our study was 100% in both groups. The gaining access to the collecting system under ultrasono- graphic guidance was similar to the fluoroscopic- -guided access. In other studies, like ours, no severe com- plications such as colon damage, pneumothorax or hydrothorax or any adjacent injuries occurred (24, 26). We had no patient with injury to adjacent organs during csPCNL till now. Totally ultrasound-guided PCNL is feasible and safe in patients with a history of renal surgery (28). Some studies showed that the stone-free rate in percutaneous nephrolithotomy with ultra- sonography guidance varied from 66.6 to 94.7% (5, 7, 12, 18, 20, 27). Other studies showed that primary stone-free rate and total stone-free rate In current study mean BMI was similar to the other studies without any significant statistical difference between the two groups (p-value=0.3); therefore, BMI had no effect on the results of our 714 ibju \| ultrasound vs. fluoroscopic guided PCNL CONCLUSIONS This randomized study showed that totally ultrasonic csPCNL had the same outcomes of fluo- roscopically-guided csPCNL. We believe that more randomized studies are needed to allow endouro- logists to use sonography rather than fluoroscopy to avoid exposing the radiation. 13. Falahatkar S, Moghaddam AA, Salehi M, Nikpour S, Esmaili F, Khaki N. Complete supine percutaneous nephrolithotripsy comparison with the prone standard technique. J Endourol. 2008;22:2513-7. 14. Fu YM, Chen QY, Zhao ZS, Ren MH, Ma L, Duan YS, Ultrasound-guided minimally invasive percutaneous nephrolithotomy in flank position for management of complex renal calculi. Urology. 2011;77:40-4. CONFLICT OF INTEREST 15. Alan C, Koçoğlu H, Ateş F, Ersay AR. Ultrasound-guided X-ray free percutaneous nephrolithotomy for treatment of simple stones in the flank position. Urol Res. 2011;39:205-12. None declared. 16. Agarwal M, Agrawal MS, Jaiswal A, Kumar D, Yadav H, Lavania P. Safety and efficacy of ultrasonography as an adjunct to fluoroscopy for renal access in percutaneous nephrolithotomy (PCNL). BJU Int. 2011;108:1346-9. _______________________ Correspondence address: Aliakbar Allahkhah, MD Urology Research Center, Guilan University of Medical Sciences Urology Research Center, Razi Hospital Sardare Jangal Street, Rasht, Iran Fax: + 98 131 552-5259 E-mail: mallahkhah@yahoo.com REFERENCES 17. Yan S, Xiang F, Yongsheng S. Percutaneous nephrolithotomy guided solely by ultrasonography: a 5-year study of >700 cases. BJU Int. 2013;112:965-71. 1. Fernström I, Johansson B. Percutaneous pyelolithotomy. A new extraction technique. Scand J Urol Nephrol. 1976;10:257-9. 1. Fernström I, Johansson B. Percutaneous pyelolithotomy. A new extraction technique. Scand J Urol Nephrol. 1976;10:257-9. 18. Basiri A, Ziaee SA, Nasseh H, Kamranmanesh M, Masoudy P, Heidary F, et al. Totally ultrasonography-guided percutaneous nephrolithotomy in the flank position. J Endourol. 2008;22:1453-7. 2. Steele D, Marshall V. Percutaneous nephrolithotomy in the supine position: a neglected approach? J Endourol. 2007;21:1433-7. 19. Falahatkar S, Allahkhah A: Recent developments in percutaneous nephrolithotomy: benefits of the complete supine position. Urotoday Int J. 2010;3(2). 3. Kalogeropoulou C, Kallidonis P, Liatsikos EN. Imaging in percutaneous nephrolithotomy. J Endourol. 2009;23:1571- 7. 20. Osman M, Wendt-Nordahl G, Heger K, Michel MS, Alken P, Knoll T. Percutaneous nephrolithotomy with ultrasonography-guided renal access: experience from over 300 cases. BJU Int. 2005;96:875-8. 4. Rao PN, Faulkner K, Sweeney JK, Asbury DL, Sambrook P, Blacklock NJ. Radiation dose to patient and staff during percutaneous nephrostolithotomy. Br J Urol. 1987;59:508- 12. 21. Saxby MF, Sorahan T, Slaney P, Coppinger SW. A case-control study of percutaneous nephrolithotomy versus extracorporeal shock wave lithotripsy. Br J Urol. 1997;79:317-23. 5. Karami H, Arbab AH, Rezaei A, Mohammadhoseini M, Rezaei I. Percutaneous nephrolithotomy with ultrasonography- guided renal access in the lateral decubitus flank position. J Endourol. 2009;23:33-5. 22. Steele D, Marshall V. Percutaneous nephrolithotomy in the supine position: a neglected approach? J Endourol. 2007;21:1433-7. 6. Majidpour HS. Risk of radiation exposure during PCNL. Urol J. 2010;7:87-9. 7. Etemadian M, Amjadi M, Simforoosh N. Transcutaneous ultrasound guided nephrolithotomy: the first report from Iran. Urol J. 2004;1:82-4. 23. Falahatkar S, Neiroomand H, Enshaei A, Kazemzadeh M, Allahkhah A, Jalili MF. Totally ultrasound versus fluoroscopically guided complete supine percutaneous nephrolithotripsy: a first report. J Endourol. 2010;24:1421-6. 8. Inglis JA, Tolley DA, Law J. Radiation safety during percutaneous nephrolithotomy. Br J Urol. 1989;63:591-3. 715 ibju \| ultrasound vs. fluoroscopic guided PCNL ibju \| ultrasound vs. fluoroscopic guided PCNL 30. Rana AM, Bhojwani JP, Junejo NN, Das Bhagia S. Tubeless PCNL with patient in supine position: procedure for all seasons?--with comprehensive technique. Urology. 2008;71:581-5. 24. Karami H, Rezaei A, Mohammadhosseini M, Javanmard B, Mazloomfard M, Lotfi B. Ultrasonography-guided percutaneous nephrolithotomy in the flank position versus fluoroscopy- guided percutaneous nephrolithotomy in the prone position: a comparative study. J Endourol. 2010;24:1357-61. 31. Valdivia Uría JG, Valle Gerhold J, López López JA, Villarroya Rodriguez S, Ambroj Navarro C, Ramirez Fabián M, et al. Technique and complications of percutaneous nephroscopy: experience with 557 patients in the supine position. J Urol. 1998;160:1975-8. 25. Montanari E, Serrago M, Esposito N, Rocco B, Kartalas-Goumas I, Del Nero A, et al. Ultrasound-fluoroscopy guided access to the intrarenal excretory system. Ann Urol (Paris). 1999;33:168-81. 32. Ng MT, Sun WH, Cheng CW, Chan ES. Supine position is safe and effective for percutaneous nephrolithotomy. J Endourol. 2004;18:469-74. 26. Zhou X, Gao X, Wen J, Xiao C. Clinical value of minimally invasive percutaneous nephrolithotomy in the supine position under the guidance of real-time ultrasound: report of 92 cases. Urol Res. 2008;36:111-4. 27. Basiri A, Mohammadi Sichani M, Hosseini SR, Moradi Vadjargah A, Shakhssalim N, Kashi AH, et al. X-ray-free percutaneous nephrolithotomy in supine position with ultrasound guidance. World J Urol. 2010;28:239-44. 28. Falahatkar S, Panahandeh Z, Ashoori E, Akbarpour M, Khaki N. What is the difference between percutaneous nephrolithotomy in patients with and without previous open renal surgery? J Endourol. 2009;23:1107-10. 29. Shoma AM, Eraky I, El-Kenawy MR, El-Kappany HA. Percutaneous nephrolithotomy in the supine position: technical aspects and functional outcome compared with the prone technique. Urology. 2002;60:388-92. 716
W2056394032.txt	https://zenodo.org/records/2184218/files/article.pdf	de		Einen elektrisch geheizten Trockenschrank	Analytical and bioanalytical chemistry/Analytical & bioanalytical chemistry	1,901	public-domain	415	Operationen, Apparate und Reagentien. 113 Ueber die Einwirkung troekener Bromwasserstoffs~ure a u f Glas beriehtet B e r t h e 1o t 1), dass, allerdings erst nach sehr langer Zeit~ unter dem Einfluss des Sonnenlichtes eine Umsetzung mit den Silicaten ( u n d Sulfaten?) des Glases eintritt, in Folge deren sich eine geringe Menge fltissigen Wassers bildet. Zur Unterscheidung des Ozons von salpetriger S~ure and Wasserstoffsuperoxyd benutzen E. E r l w e i n und T h. W e y l 2) das Methaphenylendiamin~ welches sich mit Ozon sowohl in saurer als aueh ill alkalischer LSsung r o t h f~trbt, w~thrend es in alkalischer LSsung dureh salpetrige S~ure, Wasserstoffsuperoxyd und atmosph~irische Luft nicht gef~rbt wird. S t a t t . des Metaphenylendiamins kann man aueh die 0rtho- oder Paraverbindung anwenden~ doch ist erstere bequemer, weil sie als Reagens t~blieh ist. Zur Ausftthrung der Reaction werden 25 cc einer LSsung yon 0 , 1 - - 0 , 2 g des salzsauren Salzes in 9 0 c c Wasser und l O c c Natronlauge yon 5°/o benutzt. Die LSsung muss jedesmal frisch bereitet werden~ da sie sich bei l ~ n g e r e m Stehen an der Luft f~rbt. Die dureh Einwirkung des 0zons erhaltene rothe LSsung wird mit eoncentrirter Schwefels~ure intensiv dunkelroth. Durch Zink und Salzoder S'ehwefels~ure oder durch Kochen mit Zinkstaub und Alkali tritt Entf~rbung ein. Beim Schiittelu mit I,uf~ wird aber clann die F~trbung wieder hervorgerufen. l~inen elektrisoh geheizten Troekenschrank empfehlen W a r m b r u n n ~ Q u i l i t z u. Co. ~) Die speeielle Einrichtung der Heizvorrichtung ist aus der VerOffentlichung nieht zu ersehen. Bei einer Spannung yon 110 V. und einer Stromst~irke yon 4 A. 15sst sich eine Temperatur yon 180 o erreiehen. F i l t r i r t r i c h t e r mit L~tngsrippen und dazwischen liegemen schr~gel~ Rippen zur Vermeidung diehten Anlegens des Filtrirpapiers haber~ J . M . E . R i e d e l und O . F . J . G r a h l 4 ) angegeben und unter Patentschutz gestellt. Die Trichter haben unten an der Stelle des Uebergangs yore Conus in den Stiel eine kleine kugelartige Erweiterung. 1) Annales do Chimie et do Physique [7. S~r.] '21, 266. 2) Ber. d deutsch, chem. Gesellsch. zu Berlin ~1, 3158. 3) Chemiker-Zeitung ~ , 905. ~) Zeitschrift f. angew. Chemie 1900. S. 447.
https://openalex.org/W4319957201	https://wes.copernicus.org/preprints/wes-2022-109/wes-2022-109.pdf	English	null	Reply on RC1	null	2,023	cc-by	19,087	Damping analysis of Floating Offshore Wind Turbine (FOWT): a new control strategy reducing the platform vibrations Matteo Capaldo1 and Paul Mella2 1TotalEnergies OneTech, Palaiseau, France 1,2Ecole Polytechnique, Institut Polytechnique de Paris, Palaiseau, France Correspondence: Matteo Capaldo (matteo.capaldo@totalenergies.com) Abstract. In this paper, the coupled dynamics of the floating platform and the wind turbine rotor is analysed. In particular, the damping is explicitly derived from the coupled equations of rotor and floating platform. The analysis of the damping leads to the study of the instability phenomena obtaining the explicit conditions that lead to the Non Minimum Phase Zero (NMPZ). Two NMPZs are analysed, one related to the rotor dynamics and the other one to the platform pitch dynamics. The latter is a novelty and an explicit condition is introduced in this work for its verification. In the second part of the paper, from the 5 analysis of the damping of the floating platform, a new strategy for the control of Floating Offshore Wind Turbines (FOWTs) is proposed. This strategy allows one to impose to the controller an explicit level of damping in the platform pitch motion without changing the period of platform pitching. Finally the new strategy is compared to the one without compensation by performing aero-hydro-servo-elastic numerical simulations of a reference FOWT. Generated power, movements, blade pitch 5 and tower base fatigue are compared showing that the new control strategy can reduce fatigue in the structure without affecting 10 the power production. 10 https://doi.org/10.5194/wes-2022-109 Preprint. Discussion started: 21 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/wes-2022-109 Preprint. Discussion started: 21 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. 1 Introduction Wind energy is an important source of renewable energy and it has a very high potential in both onshore and offshore. In terms of installed capacity, onshore wind is still the largest contribution. However, in the next years, the new annual offshore installed capacity is estimated to exceed 30 GW by 2030, in order to stay on-track for a netzero/1.5◦C pathway (Lee et al., 2022). 15 In offshore, there is a growing interest in floating foundations. In fact, FOWTs would allow access to good wind resource locations that are not suitable for fixed-bottom foundations. Wind energy is an important source of renewable energy and it has a very high potential in both onshore and offshore. In terms of installed capacity, onshore wind is still the largest contribution. However, in the next years, the new annual offshore installed capacity is estimated to exceed 30 GW by 2030, in order to stay on-track for a netzero/1.5◦C pathway (Lee et al., 2022). 15 In offshore, there is a growing interest in floating foundations. In fact, FOWTs would allow access to good wind resource locations that are not suitable for fixed-bottom foundations. 15 capacity is estimated to exceed 30 GW by 2030, in order to stay on-track for a netzero/1.5◦C pathway (Lee et al., 2022). 15 In offshore, there is a growing interest in floating foundations. In fact, FOWTs would allow access to good wind resource locations that are not suitable for fixed-bottom foundations. In that context, the levelized cost of energy (LCOE) of offshore wind farms needs to be decreased to be competitive with respect to onshore wind. This is especially true for the FOWTs. One effective way to achieve this objective is to investigate In that context, the levelized cost of energy (LCOE) of offshore wind farms needs to be decreased to be competitive with respect to onshore wind. This is especially true for the FOWTs. One effective way to achieve this objective is to investigate respect to onshore wind. This is especially true for the FOWTs. One effective way to achieve this objective is to investigate different strategies for the control of the FOWTs in order to improve it with respect to the LCOE. As explained in (Bianchi et 20 al., 2007), the minimisation of the LCOE involves a series of partial objectives, energy capture, mechanical loads and power quality. 1 Introduction Disturbance accommodating controller (DAC) is evaluated in (Menezes et al., 2018) and it is coupled with individual pitch control (IPC) in (Namik et al., 2011). A nonlinear pitch and torque controller using wind preview the pitch controller. In (Lackner et al., 2009) and (Lenfest et al., 2020), the platform pitch velocity is used to adjust the rated 40 speed set-point to reduce platform motions. However, the platform pitch damping analysis is not investigated and the link with the compensation parameter is not given. Higher order controllers, such as a linear quadratic regulator (LQR) are applied and evaluated in (Ma et al., 2018). Disturbance accommodating controller (DAC) is evaluated in (Menezes et al., 2018) and it is coupled with individual pitch control (IPC) in (Namik et al., 2011). A nonlinear pitch and torque controller using wind preview the pitch controller. In (Lackner et al., 2009) and (Lenfest et al., 2020), the platform pitch velocity is used to adjust the rated 40 speed set-point to reduce platform motions. However, the platform pitch damping analysis is not investigated and the link with the compensation parameter is not given. Higher order controllers, such as a linear quadratic regulator (LQR) are applied and evaluated in (Ma et al., 2018). Disturbance accommodating controller (DAC) is evaluated in (Menezes et al., 2018) and it is coupled with individual pitch control (IPC) in (Namik et al., 2011). A nonlinear pitch and torque controller using wind preview is designed in (Sarkar et al., 2020) and (Schlipf et al., 2013), giving promising results. 45 The control strategy proposed in this work shares some similarities to the one introduced in (Lackner et al., 2009) and (Lenfest et al., 2020). As introduced in this paper, in fact, the rated generator speed is no longer a constant value but a function of the platform pitch velocity and the blade pitch is used to damp the floating platform pitch. However, differently from (Lackner et al., 2009), the ratio between proportional and integral gains of the correction can be considered different for the is designed in (Sarkar et al., 2020) and (Schlipf et al., 2013), giving promising results. 45 The control strategy proposed in this work shares some similarities to the one introduced in (Lackner et al., 2009) and (Lenfest et al., 2020). 1 Introduction For a FOWT, when the platform has 30 a forward motion, the rotor experiences an increasing wind speed. Consequently, the blade pitch control increases the angle of tt k t f th d h d th d i t d l t th t d H it l d th t The nature and the set of control parameters leading to the instability can variate from one platform design to another one, e.g., barge, spare, semi-sub or tension-leg platform. However, for each of the platforms, there are sets of design and control parameters leading to oscillating stability or instability (Fleming et al., 2014). The issue come from the fact that in above rated wind speed, the blade pitch regulates the speed by increasing the angle of attack to feather. For a FOWT, when the platform has 30 a forward motion, the rotor experiences an increasing wind speed. Consequently, the blade pitch control increases the angle of attack to feather and, hence, reduces the aerodynamic torque and regulates the rotor speed. However, it also reduces the rotor thrust that induces a further forward motion. So the blade pitch control amplifies the original forward motion of the platform because the floating platform surge and pitch natural frequencies are in the bandwidth of the blade pitch control. wind speed, the blade pitch regulates the speed by increasing the angle of attack to feather. For a FOWT, when the platform has 30 a forward motion, the rotor experiences an increasing wind speed. Consequently, the blade pitch control increases the angle of attack to feather and, hence, reduces the aerodynamic torque and regulates the rotor speed. However, it also reduces the rotor thrust that induces a further forward motion. So the blade pitch control amplifies the original forward motion of the platform because the floating platform surge and pitch natural frequencies are in the bandwidth of the blade pitch control. wind speed, the blade pitch regulates the speed by increasing the angle of attack to feather. For a FOWT, when the platform has 30 a forward motion, the rotor experiences an increasing wind speed. Consequently, the blade pitch control increases the angle of attack to feather and, hence, reduces the aerodynamic torque and regulates the rotor speed. However, it also reduces the rotor thrust that induces a further forward motion. 1 Introduction So the blade pitch control amplifies the original forward motion of the platform because the floating platform surge and pitch natural frequencies are in the bandwidth of the blade pitch control. Solutions exist to avoid this phenomenon. The first one and the most common in literature is to reduce the blade pitch control 35 proportional and integral gains in order to reduce its bandwidth and exclude the platform pitch and surge natural frequencies (Jonkman et al., 2008; Larsen et al., 2007). However, this solution do not completely solve the problem and, moreover, the price to pay is to have a less reactive blade pitch control that allows important over-speeds of the rotor. Alternative methods use additional sensing, such as nacelle fore-aft acceleration measurements or platform gyroscopes, to improve the performance of Solutions exist to avoid this phenomenon. The first one and the most common in literature is to reduce the blade pitch control 35 proportional and integral gains in order to reduce its bandwidth and exclude the platform pitch and surge natural frequencies (Jonkman et al., 2008; Larsen et al., 2007). However, this solution do not completely solve the problem and, moreover, the price to pay is to have a less reactive blade pitch control that allows important over-speeds of the rotor. Alternative methods use additional sensing, such as nacelle fore-aft acceleration measurements or platform gyroscopes, to improve the performance of Solutions exist to avoid this phenomenon. The first one and the most common in literature is to reduce the blade pitch control 35 proportional and integral gains in order to reduce its bandwidth and exclude the platform pitch and surge natural frequencies (Jonkman et al., 2008; Larsen et al., 2007). However, this solution do not completely solve the problem and, moreover, the price to pay is to have a less reactive blade pitch control that allows important over-speeds of the rotor. Alternative methods use additional sensing, such as nacelle fore-aft acceleration measurements or platform gyroscopes, to improve the performance of the pitch controller. In (Lackner et al., 2009) and (Lenfest et al., 2020), the platform pitch velocity is used to adjust the rated 40 speed set-point to reduce platform motions. However, the platform pitch damping analysis is not investigated and the link with the compensation parameter is not given. Higher order controllers, such as a linear quadratic regulator (LQR) are applied and evaluated in (Ma et al., 2018). 1 Introduction As introduced in this paper, in fact, the rated generator speed is no longer a constant value but a function of the platform pitch velocity and the blade pitch is used to damp the floating platform pitch. However, differently from (Lackner et al., 2009), the ratio between proportional and integral gains of the correction can be considered different for the is designed in (Sarkar et al., 2020) and (Schlipf et al., 2013), giving promising results. 45 The control strategy proposed in this work shares some similarities to the one introduced in (Lackner et al., 2009) and (Lenfest et al., 2020). As introduced in this paper, in fact, the rated generator speed is no longer a constant value but a function of the platform pitch velocity and the blade pitch is used to damp the floating platform pitch. However, differently from (Lackner et al., 2009), the ratio between proportional and integral gains of the correction can be considered different for the platform pitch motion and the rotor speed. This choice, in fact, can be not optimal for some cases. Differently from (Lenfest 50 et al., 2020), a mathematical frame is given to define the compensation to the platform pitch and the explicit analytical form of it is given. Moreover, a second compensation is considered in the control strategy proposed in this work. It is to solve the Non Minimum Phase Zero (NMPZ) effects caused by the blade pitch on the rotor rotational dynamics. It is already introduced in (Stockhouse et al., 2021) and it is, in this work, analytically developed. The study of the NMPZs brings to a new NMPZ platform pitch motion and the rotor speed. This choice, in fact, can be not optimal for some cases. Differently from (Lenfest 50 et al., 2020), a mathematical frame is given to define the compensation to the platform pitch and the explicit analytical form of it is given. Moreover, a second compensation is considered in the control strategy proposed in this work. It is to solve the Non Minimum Phase Zero (NMPZ) effects caused by the blade pitch on the rotor rotational dynamics. It is already introduced in (Stockhouse et al., 2021) and it is, in this work, analytically developed. The study of the NMPZs brings to a new NMPZ phenomenon, described for the first time in this work. This is the NMPZ caused by the blade pitch on the platform dynamics. 1 Introduction These objectives are actually closely related and sometimes conflicting and they should not be pursued separately. Hence, the question is to find a well balanced compromise among them. Considering FOWTs, this optimization problem increases in complexity since the movements of the floating platform interact with the feedback control loop. Moreover, the different strategies for the control of the FOWTs in order to improve it with respect to the LCOE. As explained in (Bianchi et 20 al., 2007), the minimisation of the LCOE involves a series of partial objectives, energy capture, mechanical loads and power quality. These objectives are actually closely related and sometimes conflicting and they should not be pursued separately. Hence, the question is to find a well balanced compromise among them. Considering FOWTs, this optimization problem increases in complexity since the movements of the floating platform interact with the feedback control loop. Moreover, the 1 1 https://doi.org/10.5194/wes-2022-109 Preprint. Discussion started: 21 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. coupling between the platform movements, the rotor dynamics and the blade pitch control can lead to oscillating (or not 25 damped) stability or even to unstable conditions (Larsen et al., 2007). coupling between the platform movements, the rotor dynamics and the blade pitch control can lead to oscillating (or not 25 damped) stability or even to unstable conditions (Larsen et al., 2007). p g p , y p g ( 5 damped) stability or even to unstable conditions (Larsen et al., 2007). The nature and the set of control parameters leading to the instability can variate from one platform design to another one, e.g., barge, spare, semi-sub or tension-leg platform. However, for each of the platforms, there are sets of design and control parameters leading to oscillating stability or instability (Fleming et al., 2014). The issue come from the fact that in above rated The nature and the set of control parameters leading to the instability can variate from one platform design to another one, e.g., barge, spare, semi-sub or tension-leg platform. However, for each of the platforms, there are sets of design and control parameters leading to oscillating stability or instability (Fleming et al., 2014). The issue come from the fact that in above rated wind speed, the blade pitch regulates the speed by increasing the angle of attack to feather. 1 Introduction In Section 2, the equations of the FOWT system are described with the considered degrees of freedom and their coupling terms Section 2 1 presents the control model considered in this work The closed loop The chosen strategy is, then, compared to the one which does not consider a platform pitch compensation. The latter neglects 65 the following phenomenon: the blade feather reduces the aerodynamic thrust and, thus, a part of the opposing force on the platform is neglected. The chosen strategy is, then, compared to the one which does not consider a platform pitch compensation. The latter neglects 65 the following phenomenon: the blade feather reduces the aerodynamic thrust and, thus, a part of the opposing force on the platform is neglected. The document is organized as follows. In Section 2, the equations of the FOWT system are described with the considered degrees of freedom and their coupling terms. Section 2.1 presents the control model considered in this work. The closed loop The document is organized as follows. In Section 2, the equations of the FOWT system are described with the considered degrees of freedom and their coupling terms. Section 2.1 presents the control model considered in this work. The closed loop feedback system is then analysed in Section 2.3, leading to the definition of the conditions for the NMPZs. For this controller, a 70 new control strategy dedicated to FOWTs, named ζplt-fixed is presented and analytically derived in Section 2.4 and 2.5. Some numerical tests are presented and commented in Section 3. feedback system is then analysed in Section 2.3, leading to the definition of the conditions for the NMPZs. For this controller, a 70 new control strategy dedicated to FOWTs, named ζplt-fixed is presented and analytically derived in Section 2.4 and 2.5. Some numerical tests are presented and commented in Section 3. feedback system is then analysed in Section 2.3, leading to the definition of the conditions for the NMPZs. For this controller, a 70 new control strategy dedicated to FOWTs, named ζplt-fixed is presented and analytically derived in Section 2.4 and 2.5. Some numerical tests are presented and commented in Section 3. 1 Introduction 55 This new phenomenon is analytically developed leading to the explicit condition to verify it. However, the compensations proposed by this model can’t correct it. phenomenon, described for the first time in this work. This is the NMPZ caused by the blade pitch on the platform dynamics. 55 This new phenomenon is analytically developed leading to the explicit condition to verify it. However, the compensations proposed by this model can’t correct it. The novelty of this work is related to the FOWT damping analysis, i.e., the damping obtained by coupling the rotor and the platform pitch dynamics. This damping is explicitly derived from the coupled equations of rotor and floating platform. This 2 2 https://doi.org/10.5194/wes-2022-109 Preprint. Discussion started: 21 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. analysis leads to the study of the instability phenomena underlining the conditions leading to the NMPZ. One new NMPZ, 60 never discussed in literature, is discovered and analysed in this work. The domain of the instability of the platform is explicitly derived from the coupled system of equations. The control strategy proposed relies on this analysis and it allows to impose an explicit level of damping in the platform pitch motion to the controller without changing the period of platform pitching. This explicit form of the damping in the platform pitch dynamics is a novelty of this work. analysis leads to the study of the instability phenomena underlining the conditions leading to the NMPZ. One new NMPZ, 60 never discussed in literature, is discovered and analysed in this work. The domain of the instability of the platform is explicitly derived from the coupled system of equations. The control strategy proposed relies on this analysis and it allows to impose an explicit level of damping in the platform pitch motion to the controller without changing the period of platform pitching. This explicit form of the damping in the platform pitch dynamics is a novelty of this work. The chosen strategy is, then, compared to the one which does not consider a platform pitch compensation. The latter neglects 65 the following phenomenon: the blade feather reduces the aerodynamic thrust and, thus, a part of the opposing force on the platform is neglected. The document is organized as follows. 2 Floating Offshore Wind Turbine and its controller The floating offshore wind turbine (FOWT) is represented by a system of two degrees of free The floating offshore wind turbine (FOWT) is represented by a system of two degrees of freedom, namely the rotor speed Ω The floating offshore wind turbine (FOWT) is represented by a system of two degrees of freedom, namely the rotor speed Ω and the platform pitch angle, Φ. Surge degree of freedom is not considered in this model. In fact, surge speed of the FOWT 75 can be neglected with respect to the speed at nacelle generated by the pitch motion of the platform. This is already mentioned in (Sarkar et al., 2020b), where the authors remarked that the dynamics of the surge motion is much slower than the one of the pitch. Hence, the surge can be considered as a static offset in the position of the wind turbine without any effects on the controller. Two control parameters, B (blade pitch) and τg (generator torque), and two external disturbances, V (wind speed) and W 80 (waves speed) are considered. For all the values that form a given operating point (namely Ω,Φ,B,Tg,V,W), the notation X = ¯x + x is adopted, with x being the small perturbation of a steady-state operating point ¯x. The model is, then, based on the two fundamental equations: 80 ˙Ωg = Ng Jr (Ta −NgTg) (1) 5 ˙Ωg = Ng Jr (Ta −NgTg) (1) 85 85 Jt ¨Φ + Dt ˙Φ + KtΦ = htFa + τw Jt ¨Φ + Dt ˙Φ + KtΦ = htFa + τw (2) (2) where Ωg is the generator speed, hereafter noted Ω, Ta and Tg are the aerodynamic and electric torque, Ng is the gearbox ratio and Jr is the rotor inertia. Jt is the total system moment of inertia about the pitch rotation, Dt is the natural damping coefficient (assumed constant), Kt is a spring-like restoring coefficient (mainly given by the mooring lines of the floating platform), ht is where Ωg is the generator speed, hereafter noted Ω, Ta and Tg are the aerodynamic and electric torque, Ng is the gearbox ratio and Jr is the rotor inertia. 2 Floating Offshore Wind Turbine and its controller Moreover, the hypothesis of ϕ being small allows one to remove the terms ∂τa ∂ϕ ϕ, ∂Fa ∂ϕ ϕ and ∂τw ∂ϕ ϕ. Note that Fa and τw are separated for clarity, which explains why dFa does not depend on a w perturbation. Moreover, the hypothesis of ϕ being small allows one to remove the terms ∂τa ∂ϕ ϕ, ∂Fa ∂ϕ ϕ and ∂τw ∂ϕ ϕ. ϕ ϕ ϕ Relative wind vr is the wind velocity in rotor reference frame, it is computed from v by: ϕ ϕ ϕ Relative wind vr is the wind velocity in rotor reference frame, it is computed from v by: Relative wind vr is the wind velocity in rotor reference frame, it is computed from v by: vr = v −ht ˙ϕ 105 (8) Under the assumption of ϕ being small, ht ˙ϕ represents the rotor fore-aft velocity in a fixed global reference frame. Equations 1 and 2 applied to small perturbations of a steady-state point can therefore be expressed in the linear form: Under the assumption of ϕ being small, htϕ represents the rotor fore aft velocity in a fixed global reference frame. Equations 1 and 2 applied to small perturbations of a steady-state point can therefore be expressed in the linear form: Equations 1 and 2 applied to small perturbations of a steady-state point can therefore be expressed in the linear form: ˙ω = Ng Jr ∂τa ∂ω ω + ∂τa ∂v (v −ht ˙ϕ) + ∂τa ∂β β −Ngτg ˙ω = Ng Jr ∂τa ∂ω ω + ∂τa ∂v (v −ht ˙ϕ) + ∂τa ∂β β −Ngτg (1’) v −ht ˙ϕ) + ∂τa ∂β β −Ngτg (1 (1’) Jt ¨ϕ + Dt ˙ϕ + Ktϕ = ht ∂Fa ∂ω ω + ∂Fa ∂v (v −ht ˙ϕ) + ∂Fa ∂β β + ∂τw ∂w w (2’) 110 ∂Fa ∂v (v −ht ˙ϕ) + ∂Fa ∂β β + ∂τw ∂w w (2’) (2’) Those coupled second order equations yield the following four dimensional state-space model: Those coupled second order equations yield the following four dimensional state-space model: ˙x = A0x + Bcuc + Bdud Where x = (θ, ˙θ,ϕ, ˙ϕ)T , θ = R ω (i.e. 2 Floating Offshore Wind Turbine and its controller Jt is the total system moment of inertia about the pitch rotation, Dt is the natural damping coefficient (assumed constant), Kt is a spring-like restoring coefficient (mainly given by the mooring lines of the floating platform), ht is where Ωg is the generator speed, hereafter noted Ω, Ta and Tg are the aerodynamic and electric torque, Ng is the gearbox ratio and Jr is the rotor inertia. Jt is the total system moment of inertia about the pitch rotation, Dt is the natural damping coefficient (assumed constant), Kt is a spring-like restoring coefficient (mainly given by the mooring lines of the floating platform), ht is the height of the rotor (approximately the tower length), Fa is the aerodynamic force flowing from the rotor to the system and 90 τw is the overturning moment given by the waves. 3 3 https://doi.org/10.5194/wes-2022-109 Preprint. Discussion started: 21 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/wes-2022-109 Preprint. Discussion started: 21 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/wes-2022-109 Preprint. Discussion started: 21 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. Once a steady-state operating point ¯x is reached, the same two equations can be applied to any small variations x around this operating point. Equations 1 and 2 applied on X can be written: ˙ω = Ng Jr (τa −Ngτg) (3) Jt ¨ϕ + Dt ˙ϕ + Ktϕ = htdFa + dτw (4) ˙ω = Ng Jr (τa −Ngτg) Jt ¨ϕ + Dt ˙ϕ + Ktϕ = htdFa + dτw 95 ˙ω = Ng Jr (τa −Ngτg) (3) (4) 95 The infinitesimal thrust and torques satisfy Tg = ¯τg +τg, Ta = ¯τa +τa, Fa = ¯ Fa +dFa and τw = ¯τw +dτw. By considering small perturbations of a steady-state operating point (given by Ω,Φ,B,Tg,V,W), it allows one to use the following linear forms: τa = ∂τa ∂ω ω + ∂τa ∂v vr + ∂τa ∂β β (5) dFa = ∂Fa ∂ω ω + ∂Fa ∂v vr + ∂Fa ∂β β (6) 100 dτw = ∂τw ∂w w (7) τa = ∂τa ∂ω ω + ∂τa ∂v vr + ∂τa ∂β β dFa = ∂Fa ∂ω ω + ∂Fa ∂v vr + ∂Fa ∂β β 100 dτw = ∂τw ∂w w (5) (6) (7) Note that Fa and τw are separated for clarity, which explains why dFa does not depend on a w perturbation. 2.1 Control model description 115 In this section the pitch controller model is described. In this section the pitch controller model is described. In this section the pitch controller model is described. The present control model considers ωr as the reference for ω and 0 as the reference for ˙ϕ. It is based on several SISO (single-input-single-output) feedback loops. It can be seen as a multi-SISO: – Proportional : βP = kP (Ω−Ωr) – Proportional : βP = kP (Ω−Ωr) – Integral : βI = kI R (Ω−Ωr) = kI(θ −tΩr) 120 – Integral : βI = kI R (Ω−Ωr) = kI(θ −tΩr) – Integral : βI = kI R (Ω−Ωr) = kI(θ −tΩr) – Beta platform pitch compensation : βcomp = kβ( ˙Φr −˙Φ) – Generator torque platform pitch compensation : τg,comp = kτg( ˙Φr −˙Φ) Controllers described by the literature considering the same compensations (Abbas et al., 2021; Stockhouse et al., 2021) aim at maintaining ω steady near its rated value by acting on the blade pitch β to variate the aerodynamic torque τa with g y y g p y q the opposite sign with respect to the rotor relative speed ω. However, this strategy neglects the following phenomenon: the 125 blade feather modifies the aerodynamic thrust Fa. Thus, a part of the opposing force on the platform is neglected. The strategy developed in this paper aims at minimizing ϕ variations with the constraint of maintaining a constant ω. Such a control strategy should reduce the loads on the structures (nacelle, tower and floater). Section 3 considers a full aero-hydro-servo-elastic model to verify this assumption. It is analysed the performance of the control strategy in a FOWT realistic environment reproduced by a numerical twin. 130 the opposite sign with respect to the rotor relative speed ω. However, this strategy neglects the following phenomenon: the 125 blade feather modifies the aerodynamic thrust Fa. Thus, a part of the opposing force on the platform is neglected. The strategy developed in this paper aims at minimizing ϕ variations with the constraint of maintaining a constant ω. Such a control strategy should reduce the loads on the structures (nacelle, tower and floater). Section 3 considers a full aero-hydro-servo-elastic model to verify this assumption. It is analysed the performance of the control strategy in a FOWT realistic environment reproduced by a numerical twin. 130 2 Floating Offshore Wind Turbine and its controller ˙θ = ω), uc = (β,τg)T and ud = (v,w)T , and : (9) ˙x = A0x + Bcuc + Bdud ˙x = A0x + Bcuc + Bdud (9) Where x = (θ, ˙θ,ϕ, ˙ϕ)T , θ = R ω (i.e. ˙θ = ω), uc = (β,τg)T and ud = (v,w)T , and : ud T , θ = R ω (i.e. ˙θ = ω), uc = (β,τg)T and ud = (v,w)T , and : ω (i.e. ˙θ = ω), uc = (β,τg)T and ud = (v,w)T , and : A0 =   0 1 0 0 0 Ng Jr ∂τa ∂ω 0 −ht Ng Jr ∂τa ∂v 0 0 0 1 0 ht Jt ∂Fa ∂ω −Kt Jt −1 Jt (Dt + h2 t ∂Fa ∂v )   Bc =   0 0 Ng Jr ∂τa ∂β − N2 g Jr 0 0 ht Jt ∂Fa ∂β 0   Bd =   0 0 Ng Jr ∂τa ∂v 0 0 0 ht Jt ∂Fa ∂v 1 Jt ∂τw ∂w   (10) (10) 4 4 https://doi.org/10.5194/wes-2022-109 Preprint. Discussion started: 21 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. 2.2 Global State-Space description For a FOWT, the objective ot the pitch control is to remain in the equilibrium operating point. It translates in: ¯ω = Ωr and ¯˙ϕ = ˙Φr = 0. This objective allows one to justify the linear form for of the global equations 1’ and 2’. For constant inputs ¯v and ¯w, this operating point is reached by the appropriated pitch (¯β) and electric torque ( ¯τg). For a FOWT, the objective ot the pitch control is to remain in the equilibrium operating point. It translates in: ¯ω = Ωr and ¯˙ϕ = ˙Φr = 0. This objective allows one to justify the linear form for of the global equations 1’ and 2’. For constant inputs ¯v and ¯w, this operating point is reached by the appropriated pitch (¯β) and electric torque ( ¯τg). 135 The control model is, here, introduced into the wind turbine state space description. For small perturbations of this steady- state operating point, the PI controller described previously becomes: – Proportional: (11) βP = kP ω – Integral: βI = kI Z ω = kIθ (12) 140 – Beta platform pitch compensation: βcomp = −kBeta ˙ϕ (13) 5 – Generator torque platform pitch compensation: τg,comp = −kτg ˙ϕ (14) https://doi.org/10.5194/wes-2022-109 Preprint. Discussion started: 21 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/wes-2022-109 Preprint. Discussion started: 21 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. – Generator torque platform pitch compensation: – Generator torque platform pitch compensation: τg,comp = −kτg ˙ϕ (14) τg,comp = −kτg ˙ϕ (14) (14) τg,comp = −kτg ˙ϕ Figure 1 shows the entire picture of the control model. This control strategy acts on the two dynamic systems, platform and 145 rotor. Hence, one can appreciate how the bottom-fixed scheme acting on the rotor (ω) with a proportional integral scheme is then corrected by the βcomp and the τg that depends on the platform pitch dynamics error. Figure 1 shows the entire picture of the control model. This control strategy acts on the two dynamic systems, platform and 145 rotor. Hence, one can appreciate how the bottom-fixed scheme acting on the rotor (ω) with a proportional integral scheme is then corrected by the βcomp and the τg that depends on the platform pitch dynamics error. 145 Figure 1. 2.3 Non minimum phase zeros analysis and resolution (negative damping on the control) 65 This section analysis the problem of negative damping by addressing the positions of the zeros of each component of G in the complex plane, i.e. the points where equation 21 is well defined but becomes: χA XT 0 · X = 0. (22) This translates the fact that s is a NMPZ if it exists a specific XT 0 , such as, for any value of U(s), the linear combination XT 0 N(s)U(s) gives XT 0 = 0. 170 Physically a XT 0 is equivalent to an infinitesimal shifting along a specific direction of a steady-state point that can not be obtained with any infinitesimal shifting of the input. This phenomenon is better illustrated case by case. For the rest of the section, an open loop control on β, is considered in order to highlight the NPMZ. Hence, βol is added to the multiple SISO as already described in 16. Since the feedback control on β can’t erase the NMPZ condition, to lighten the XT 0 N(s)U(s) gives XT 0 = 0. 170 Physically a XT 0 is equivalent to an infinitesimal shifting along a specific direction of a steady-state point that can not be obtained with any infinitesimal shifting of the input. This phenomenon is better illustrated case by case. Physically a XT 0 is equivalent to an infinitesimal shifting along a specific direction of a steady-state point that can not be obtained with any infinitesimal shifting of the input. This phenomenon is better illustrated case by case. For the rest of the section, an open loop control on β, is considered in order to highlight the NPMZ. Hence, βol is added to the multiple SISO as already described in 16. Since the feedback control on β can’t erase the NMPZ condition, to lighten the formulas, the notation β will be used instead of βol in this section. 175 formulas, the notation β will be used instead of βol in this section. 175 2.2 Global State-Space description Block diagram of the controller model The linear expression of uc = (β,τg)T as a function of x = (θ, ˙θ,ϕ, ˙ϕ)T is uc = K0x + uc,ol where: Figure 1. Block diagram of the controller model The linear expression of uc = (β,τg)T as a function of x = (θ, ˙θ,ϕ, ˙ϕ)T is uc = K0x + uc,ol where: The linear expression of uc = (β,τg)T as a function of x = (θ, ˙θ,ϕ, ˙ϕ)T is uc = K0x + uc,ol where: The linear expression of uc = (β,τg)T as a function of x = (θ, ˙θ,ϕ, ˙ϕ)T is uc = K0x + uc,ol where: K0 =  kI kP 0 −kβ 0 0 0 −kτg   K0 =  kI kP 0 −kβ 0 0 0 −kτg   (15) K0 =  kI kP 0 −kβ 0 0 0 −kτg   (15) (15) is the matrix of the control gains and uc,ol is an optional additional control (open loop) that can be considered. This is useful 0 to analyse the NMPZ in the next section. By replacing it in equation 9, it leads to: is the matrix of the control gains and uc,ol is an optional additional control (open loop) that can be considered. This is useful 150 to analyse the NMPZ in the next section. 2.2 Global State-Space description By replacing it in equation 9, it leads to: ˙x = (A0 + BcK0)x + Bcuc,ol + Bdud ˙x = (A0 + BcK0)x + Bcuc,ol + Bdud (16) ˙x = (A0 + BcK0)x + Bcuc,ol + Bdud (16) l matrix of the system of equations: Which leads to define the global matrix of the system of equations: define the global matrix of the system of equations: Which leads to define the global matrix of the system of equations: Which leads to define the global matrix of the system of equations: Which leads to define the global matrix of the system of equations: A = (A0 + BcK0) =   0 1 0 0 kI Ng Jr ∂τa ∂β Ng Jr ( ∂τa ∂ω + kP ∂τa ∂β ) 0 Ng Jr (−ht ∂τa ∂v −kβ ∂τa ∂β + kτgNg) 0 0 0 1 kI ht Jt ∂Fa ∂β ht Jt ( ∂Fa ∂ω + kP ∂Fa ∂β ) −Kt Jt −1 Jt (Dt + h2 t ∂Fa ∂v + kβht ∂Fa ∂β )   (17) (17) 6 https://doi.org/10.5194/wes-2022-109 Preprint. Discussion started: 21 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. The time domain system can be rewritten in the complex domain. Using the following notation, L {x(t)} = X, equation 16 155 translates into: 155 X = (sI −A)−1(BcU c,ol + BdU d). (18) By defining: B = h Bc \| Bd i , u =        βol τg,ol v w        B = h Bc \| Bd i , u =        βol τg,ol v w        (19) (19) and 160 G(s) = (sI −A)−1B = 1 det(sI −A)Com(sI −A)T B = 1 χA(s)N(s), (20) G(s) = (sI −A)−1B = 1 det(sI −A)Com(sI −A)T B = 1 χA(s)N(s), (20) it leads to: it leads to: χA(s)X(s) = N(s)U(s) (21) G(s) is a 4 × 4 matrix. Every component of which can be written as the quotient of a polynomial in s and χa(s). G(s) is a 4 × 4 matrix. Every component of which can be written as the quotient of a polynomial in s and χa(s). G(s) is a 4 × 4 matrix. Every component of which can be written as the quotient of a polynomial in s and χa(s). https://doi.org/10.5194/wes-2022-109 Preprint. Discussion started: 21 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/wes-2022-109 Preprint. Discussion started: 21 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/wes-2022-109 Preprint. Discussion started: 21 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. χA(s)ϕ(s) = N3,1(s)β(s), (23) χA(s)ϕ(s) = N3,1(s)β(s), (23) N3,1(s) = Jr Ng ∂Fa ∂β s2 + ∂τa ∂β ∂Fa ∂ω −∂Fa ∂β ∂τa ∂ω s 180 N3,1(s) = Jr Ng ∂Fa ∂β s2 + ∂τa ∂β ∂Fa ∂ω −∂Fa ∂β ∂τa ∂ω s (24) 0 ∂τa ∂β ∂Fa ∂ω −∂Fa ∂β ∂τa ∂ω s (2 (24) The condition for the NMPZ on β →ϕ control is that N3,1 has a root with a real part strictly positive. Assuming that β = βf, the fine pitch, the previous derivatives are all negative. Hence, the root research of N3,1 leads to: ∂τa ∂ω / ∂τa ∂β < ∂Fa ∂ω / ∂Fa ∂β (25) ∂τa ∂ω / ∂τa ∂β < ∂Fa ∂ω / ∂Fa ∂β (25) Intuitively, this corresponds to an operating point where τa is rather influenced by β and Fa is rather influenced by ω. This NMPZ does not depend on parameters of the platform, it is only related to WTG performances. However, the amplitude of the 185 phenomenon is related to the platform properties. It is to be noted that this NMPZ has never been highlighted in literature and the control model (with compensations of the platform motions) introduced in Section 2.1 in the feedback control loop does not prevent it. It results only from the characteristic of the FOWT system. Further works should focus on this phenomenon and introduce corrections to prevent it for any FOWT system. In absence of NMPZ, ie. eq. 25 being false, increasing β from a steady-state operating point (ie. setting d¨β > 0, d ˙β > 0 190 and dβ > 0) will always imply a reduction of ϕ. In presence of NMPZ, ie. if eq. 25 is true, the reduction or the increase of ϕ depends on the ratio between ¨β and ˙β. In absence of NMPZ, ie. eq. 25 being false, increasing β from a steady-state operating point (ie. setting d¨β > 0, d ˙β > 0 190 and dβ > 0) will always imply a reduction of ϕ. In presence of NMPZ, ie. if eq. 25 is true, the reduction or the increase of ϕ depends on the ratio between ¨β and ˙β. The latter only happens when eq. 2.3.1 ϕ-NMPZ: negative damping on ϕ control by β Gain equation in the Laplace domain for β →ϕ control is obtained by projecting 21 on the x = (0,0,ϕ,0) axis and considering only a β perturbation, ie. an input u = (β,0,0,0). The resulting equation is: 7 7 https://doi.org/10.5194/wes-2022-109 Preprint. Discussion started: 21 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. Set of parameters to show the NMPZ of eq. 25 eq. 25 false eq. 25 true ∂τa ∂v 2980.9 kN.s 3079 kN.s ∂Fa ∂v 354.8 kN.s.m−1 355.6 kN.s.m−1 ∂τa ∂ω −58597.1 kN.m.s.rad−1 −55499.5 kN.m.s.rad−1 ∂Fa ∂ω −5658.0 kN.s.rad−1 −5820.4 kN.s.rad−1 ∂τa ∂β −152347.8 kN.m.rad −160140.5 kN.m.rad ∂Fa ∂β −16052.2 kN.rad −15260 kN.rad Figure 2. ϕ and ω responses to a β-step input (at t = 10s): on the left, values (Table 1) are chosen so that eq. 25 is false: ϕ decreases. On the right, values (Table 1) are chosen so that the eq. 25 is true: ϕ increases even though β has step up. https://doi.org/10.5194/wes-2022-109 Preprint. Discussion started: 21 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/wes-2022-109 Preprint. Discussion started: 21 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. Table 1. Set of parameters to show the NMPZ of eq. 25 Table 1. Set of parameters to show the NMPZ of eq. 25 eq. 25 false eq. 25 true ∂τa ∂v 2980.9 kN.s 3079 kN.s ∂Fa ∂v 354.8 kN.s.m−1 355.6 kN.s.m−1 ∂τa ∂ω −58597.1 kN.m.s.rad−1 −55499.5 kN.m.s.rad−1 ∂Fa ∂ω −5658.0 kN.s.rad−1 −5820.4 kN.s.rad−1 ∂τa ∂β −152347.8 kN.m.rad −160140.5 kN.m.rad ∂Fa ∂β −16052.2 kN.rad −15260 kN.rad Figure 2. ϕ and ω responses to a β-step input (at t = 10s): on the left, values (Table 1) are chosen so that eq. 25 is false: ϕ decreases. On the right, values (Table 1) are chosen so that the eq. 25 is true: ϕ increases even though β has step up. Figure 2. ϕ and ω responses to a β-step input (at t = 10s): on the left, values (Table 1) are chosen so that eq. 25 is false: ϕ decreases. On the right, values (Table 1) are chosen so that the eq. 25 is true: ϕ increases even though β has step up. https://doi.org/10.5194/wes-2022-109 Preprint. Discussion started: 21 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. 25 is verified: increasing blade pitch reduces τa more than it increases Fa (because τa is rather influenced by β), thus ω increases and causes Fa to decrease (because Fa is rather influenced by ω) and then ϕ to The latter only happens when eq. 25 is verified: increasing blade pitch reduces τa more than it increases Fa (because τa is rather influenced by β), thus ω increases and causes Fa to decrease (because Fa is rather influenced by ω) and then ϕ to fl y β) a ( a fl y ) ϕ increase. If eq. 25 is not verified, increasing blade pitch β from a steady-state operating point always reduces platform pitch 195 ϕ. In practice, this effect can become an issue for a control algorithm mainly focused on ω stabilization since it generates unexpected platform dynamics. Figure 2 reproduces in the time domain ϕ and ω responses to a β-step input (at t = 10s): values (resumed in Table 1) are chosen so that eq. 25 is false: ϕ decreases. On the right, values are chosen so that the eq. 25 is true: ϕ increases even though β has step up. increase. If eq. 25 is not verified, increasing blade pitch β from a steady-state operating point always reduces platform pitch 195 ϕ. In practice, this effect can become an issue for a control algorithm mainly focused on ω stabilization since it generates unexpected platform dynamics. Figure 2 reproduces in the time domain ϕ and ω responses to a β-step input (at t = 10s): values (resumed in Table 1) are chosen so that eq. 25 is false: ϕ decreases. On the right, values are chosen so that the eq. 25 is true: ϕ increases even though β has step up. increase. If eq. 25 is not verified, increasing blade pitch β from a steady-state operating point always reduces platform pitch 195 ϕ. In practice, this effect can become an issue for a control algorithm mainly focused on ω stabilization since it generates unexpected platform dynamics. Figure 2 reproduces in the time domain ϕ and ω responses to a β-step input (at t = 10s): values (resumed in Table 1) are chosen so that eq. 25 is false: ϕ decreases. On the right, values are chosen so that the eq. 25 is true: ϕ increases even though β has step up. 8 Table 1. 2.3.2 ω-NMPZ: negative damping on ω control by β 200 215 However, the complete prevention of the problem can be obtained by several set of parameters that involves both the WTG, the floating platform and the control set-up. In fact, for this NMPZ, equation 14 of the control model described in Section 2.1 allows one to avoid NMPZ by choosing a well-suited value of kτg. This compensation has been already introduced by Stockhouse et al. (2021) with the formula: However, the complete prevention of the problem can be obtained by several set of parameters that involves both the WTG, the floating platform and the control set-up. In fact, for this NMPZ, equation 14 of the control model described in Section 2.1 allows one to avoid NMPZ by choosing a well-suited value of kτg. This compensation has been already introduced by Stockhouse et al. (2021) with the formula: kτg = mτg ht Ng ∂τa ∂v , mτg ∈[0,1] (29) 220 kτg = mτg ht Ng ∂τa ∂v , mτg ∈[0,1] 220 (29) kτg = mτg t Ng a ∂v , mτg ∈[0,1] (29) 220 The authors notice however it usually needs to be saturated because of turbine generator design constraints. In absence of NMPZ, i.e. eq. 28 being false, increasing β from a steady-state operating point will always imply reducing ω. In order to visualize this NMPZ, Figure 3 shows ω responses to a β-step input (at t = 10s). On the left, parameters (Table 2) are chosen so that the condition eq. 28 is false: ω decreases. On the right, parameters are chosen so that condition eq. 28 is true: at first ω increases even though β has step up. 225 ors notice however it usually needs to be saturated because of turbine generator design constraints. y g g In absence of NMPZ, i.e. eq. 28 being false, increasing β from a steady-state operating point will always imply reducing ω. In order to visualize this NMPZ, Figure 3 shows ω responses to a β-step input (at t = 10s). On the left, parameters (Table 2) are chosen so that the condition eq. 28 is false: ω decreases. On the right, parameters are chosen so that condition eq. 28 is true: at first ω increases even though β has step up. 225 Table 2. Set of parameters to show the NMPZ of eq. 28 Table 2. Set of parameters to show the NMPZ of eq. 28 eq. 28 false eq. 2.3.2 ω-NMPZ: negative damping on ω control by β 200 Gain equation in the Laplace domain for β →ω control is given by: χA(s) ω(s) = N2,1(s) β(s) (26) χA(s) ω(s) = N2,1(s) β(s) (26) N2,1(s) = Jt ht ∂τa ∂β s3 + Dt ht ∂τa ∂β + ht ∂τa ∂β ∂Fa ∂v −∂Fa ∂β ∂τa ∂v + kτgNg ∂Fa ∂β s2 + Kt ht ∂τa ∂β s (27) Hence the condition for NMPZ on β →ω control is: 5 N2,1(s) = Jt ht ∂τa ∂β s3 + Dt ht ∂τa ∂β + ht ∂τa ∂β ∂Fa ∂v −∂Fa ∂β ∂τa ∂v + kτgNg ∂Fa ∂β s2 + Kt ht ∂τa ∂β s (27) 205 205 Hence the condition for NMPZ on β →ω control is: Hence the condition for NMPZ on β →ω control is: h2 t ∂Fa ∂v − ∂τa ∂v −kτg Ng ht ∂Fa ∂β ∂τa ∂β ! < −Dt (28) https://doi.org/10.5194/wes-2022-109 Preprint. Discussion started: 21 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. This corresponds with an operating point where τa is rather influenced by v and Fa is rather influenced by β. In presence of NMPZ, i.e. if eq. 28 is true, the sign of dω depends on the choice of d¨β, d ˙β and dβ. Intuitively, the latter only happens when eq. 28 is verified: increasing blade pitch will reduce Fa more than it increases τa (because Fa is rather influenced by β), thus ˙ϕ will decrease and cause relative wind vr = v −ht ˙ϕ to increase. As τa is rather influenced by v, this will reduce ω in the end. 210 In practice, this effect can become an issue if a ω control algorithm obtains the opposite result than what was expected. Because of this issue, literature has concerned the NMPZ phenomenon on β →ω control and suggested several control corrections. Due to the nature of this phenomenon, any correction concerning β control, introduced in eq. 13, can’t prevent this NMPZ. However, detuning the PI controller (by lowering kP and kI gains) or using the β platform pitch compensation dβcomp = kβ ˙ϕ as suggested in (Abbas et al., 2021), can mitigate the effect of NMPZ when eq. 28 is true. 215 dβcomp = kβ ˙ϕ as suggested in (Abbas et al., 2021), can mitigate the effect of NMPZ when eq. 28 is true. 2.3.2 ω-NMPZ: negative damping on ω control by β 200 28 true ∂τa ∂v 2980.9 kN.s 2838 kN.s ∂Fa ∂v 354.8 kN.s.m−1 303.0 kN.s.m−1 ∂τa ∂ω −58597.1 kN.m.s.rad−1 −59428.7 kN.m.s.rad−1 ∂Fa ∂ω −5658.0 kN.s.rad−1 −6282.9 kN.s.rad−1 ∂τa ∂β −152347.8 kN.m.rad −133058.7 kN.m.rad ∂Fa ∂β −16052.2 kN.rad −18247.0 kN.rad 10 Figure 3. ω responses to a β-step input (at t = 10s). On the left, parameters (Table 2) are chosen so that the condition eq. 28 is false: ω decreases. On the right, parameters are chosen so that condition eq. 28 is true: at first ω increases even though β has step up. 2 3 3 C l i f NMPZ l i https://doi.org/10.5194/wes-2022-109 Preprint. Discussion started: 21 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. Figure 3. ω responses to a β-step input (at t = 10s). On the left, parameters (Table 2) are chosen so that the condition eq. 28 is false: ω decreases. On the right, parameters are chosen so that condition eq. 28 is true: at first ω increases even though β has step up. Figure 3. ω responses to a β-step input (at t = 10s). On the left, parameters (Table 2) are chosen so that the condition eq. 28 is false: ω decreases. On the right, parameters are chosen so that condition eq. 28 is true: at first ω increases even though β has step up. 2.3.3 Conclusion of NMPZ analysis: Comparison between Figures 2 and 3 enlightens what really happens after a step input, with and without NMPZ: at the be- ginning both ω and ˙ϕ always decrease just after the step. However, when both NMPZ conditions eq. 25 and eq. 28 are false, those tendencies don’t change. Conversely, when eq. 25 is true, we observe that \| ˙ω\| is so big that ˙ϕ jumps into positive values. Similarly, when eq. 28 is true, we observe that \| ˙ϕ\| is so big that ω jumps (only for a short time) into positive values. NMPZ, as we have seen in the examples, can cause important shifts and unexpected behaviors for both ω and ϕ. Comparison between Figures 2 and 3 enlightens what really happens after a step input, with and without NMPZ: at the be- ginning both ω and ˙ϕ always decrease just after the step. However, when both NMPZ conditions eq. 25 and eq. 28 are false, those tendencies don’t change. Conversely, when eq. 25 is true, we observe that \| ˙ω\| is so big that ˙ϕ jumps into positive values. Similarly, when eq. 28 is true, we observe that \| ˙ϕ\| is so big that ω jumps (only for a short time) into positive values. NMPZ, as 230 we have seen in the examples, can cause important shifts and unexpected behaviors for both ω and ϕ. The NMPZ β →ϕ doesn’t depend on above defined parameters: the model in Section 2.1 does not give solution to always prevent it, but condition eq. 25 forecasts which operating points it affects. On the other hand, a wise choice of τg avoids β →ω NMPZ, which is the main reason why this compensation has already been introduced by Stockhouse et al. (2021) and in the we have seen in the examples, can cause important shifts and unexpected behaviors for both ω and ϕ. The NMPZ β →ϕ doesn’t depend on above defined parameters: the model in Section 2.1 does not give solution to always prevent it, but condition eq. 25 forecasts which operating points it affects. On the other hand, a wise choice of τg avoids β →ω NMPZ, which is the main reason why this compensation has already been introduced by Stockhouse et al. (2021) and in the control model in Section 2.1. 2.3.3 Conclusion of NMPZ analysis: 235 In order to complete the analysis of NMPZ phenomena related to FOWT system, a hypothetical situation where both condi- tions eq. 25 and eq. 28 are true has been simulated and reported in Figure 4. At first, the dynamics are always the same: both ˙ϕ and ω decrease, but soon they both diverge because of the negative damping. It is to be noted that, kP /kI corrections (without compensations τg and kβ) can delay this divergence but can not avoid it. 11 Table 3. Set of parameters to show the instability given by NMPZs of eq. 25 and eq. 28. eq. 25 and eq. 28 true ∂τa ∂v 3105.0 kN.s ∂Fa ∂v 293.0 kN.s.m−1 ∂τa ∂ω −51356.5 kN.m.s.rad−1 ∂Fa ∂ω −7150.0 kN.s.rad−1 ∂τa ∂β −148063.0 kN.m.rad ∂Fa ∂β −16543.6 kN.rad Figure 4. an hypothetical situation where both conditions eq. 25 and eq. 28 are true. At first, both ˙ϕ and ω decrease, but soon they both diverge because of the negative damping (kP /kI corrections can delay this divergence but can not avoid it). https://doi.org/10.5194/wes-2022-109 Preprint. Discussion started: 21 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/wes-2022-109 Preprint. Discussion started: 21 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. Table 3. Set of parameters to show the instability given by NMPZs of eq. 25 and eq. 28. Table 3. Set of parameters to show the instability given by NMPZs of eq. 25 and eq. 28. eq. 25 and eq. 28 true ∂τa ∂v 3105.0 kN.s ∂Fa ∂v 293.0 kN.s.m−1 ∂τa ∂ω −51356.5 kN.m.s.rad−1 ∂Fa ∂ω −7150.0 kN.s.rad−1 ∂τa ∂β −148063.0 kN.m.rad ∂Fa ∂β −16543.6 kN.rad Figure 4. an hypothetical situation where both conditions eq. 25 and eq. 28 are true. At first, both ˙ϕ and ω decrease, but soon they both diverge because of the negative damping (kP /kI corrections can delay this divergence but can not avoid it). Figure 4. an hypothetical situation where both conditions eq. 25 and eq. 28 are true. At first, both ˙ϕ and ω decrease, but soon they both diverge because of the negative damping (kP /kI corrections can delay this divergence but can not avoid it). 2.4 Damping analysis 240 The study of the damping is related to χA(s) = det(sI −A). The explicit form of χA is: χA is: 250 250 250 χA(s) = χrot(s)χplt(s) + Nght JrJt s (kps + kI)∂Fa ∂β ht ∂τa ∂v − Jr Ng ∂Fa ∂β (kP s + kI) + ∂Fa ∂ω kβ ∂τa ∂β + kτgNg (30) (30) where: where: where: χrot(s) = s2 −Ng Jr ∂τa ∂ω s −Ng Jr ∂τa ∂β (kP s + kI) χplt(s) = s2 + 1 Jt (Dt + h2 t ∂Fa ∂v + kβht ∂Fa ∂β )s + Kt Jt (31) χrot(s) = s2 −Ng Jr ∂τa ∂ω s −Ng Jr ∂τa ∂β (kP s + kI) χplt(s) = s2 + 1 Jt (Dt + h2 t ∂Fa ∂v + kβht ∂Fa ∂β )s + Kt Jt (31) The term in square parenthesis represents the coupling term between the dynamics of the platform (ϕ) and the dynamics of the rotor (ω). 5 The term in square parenthesis represents the coupling term between the dynamics of the platform (ϕ) and the dynamics of the rotor (ω). the rotor (ω). 255 In this coupled form, it is complicated to explicit the damping of the system. In the next paragraph, under some hypothesis, the coupled system can be separated in two second order systems, one related to the rotor dynamics ω and the other one related to the floating dynamics ϕ. In particular, for the latter, it is possible to define a damping for the floating platform and obtain an explicit form for the compensation term kβ related to the imposed damping. 2.4 Damping analysis 240 In section 2.3 the issue of NMPZ, ie. the issue of negative damping in the control/input side of the equation, is analysed. The influence of the gains kP , kI, kβ and kτg on the damping of the system (cf. Section 2.3) is investigated within the analytical framework set in the previous sections. The goal is to optimize (or tune) the stability of ω and ϕ responses to an external (v and w) disturbance. In other words, the goal is to obtain an explicit expression of the damping of the FOWT system with respect to In section 2.3 the issue of NMPZ, ie. the issue of negative damping in the control/input side of the equation, is analysed. The influence of the gains kP , kI, kβ and kτg on the damping of the system (cf. Section 2.3) is investigated within the analytical framework set in the previous sections. The goal is to optimize (or tune) the stability of ω and ϕ responses to an external (v and w) disturbance. In other words, the goal is to obtain an explicit expression of the damping of the FOWT system with respect to the control parameters, kP , kI, kβ and kτg, such that, for an imposed level of damping, one can obtain a value of the control 245 parameters. This is a powerful result for the floating wind community and a novelty of this work with respect to the existing literature. the control parameters, kP , kI, kβ and kτg, such that, for an imposed level of damping, one can obtain a value of the control 245 parameters. This is a powerful result for the floating wind community and a novelty of this work with respect to the existing literature. 12 https://doi.org/10.5194/wes-2022-109 Preprint. Discussion started: 21 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/wes-2022-109 Preprint. Discussion started: 21 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. Considering the whole system, with both degrees of freedom ω and ϕ and their coupling, χA(s) of equation 18 in the complex domain leads to the equation 21. The study of the damping is related to χA(s) = det(sI −A). The explicit form of χA is: Considering the whole system, with both degrees of freedom ω and ϕ and their coupling, χA(s) of equation 18 in the complex domain leads to the equation 21. e kI ≤0, νrot and ζrot would be imaginary according to the formulas above. Grot would be no longer a band-pass filter. 2.4.1 Simplified analysis of rotor dynamics: 260 The 275 corresponding filter Grot is a second order band-pass filter with cutoff angular frequency νrot 1. The above formulas enable one to obtain explicitly kI and kP . They are well known: several controllers, such as ROSCO (Abbas et al., 2021), suggest to define: Thus, when all interactions with platform pitch are neglected, the rotor behaves like a second order oscillatory system. The 275 corresponding filter Grot is a second order band-pass filter with cutoff angular frequency νrot 1. 275 275 The above formulas enable one to obtain explicitly kI and kP . The above formulas enable one to obtain explicitly kI and kP . eral controllers, such as ROSCO (Abbas et al., 2021), suggest to define: They are well known: several controllers, such as ROSCO (Abbas et al., 2021), suggest to defin \|kI\| = ν2 rot Ng Jr ∂τa ∂β and \|kP \| = Ng Jr ∂τa ∂ω + 2ζrotνrot Ng Jr ∂τa ∂β (37) 2.4.2 Simplified analysis of platform dynamics: 0 Ng Jr ∂τa ∂ω + 2ζrotνrot Ng Jr ∂τa ∂β (37) form dynamics: \|kI\| = ν2 rot Ng Jr ∂τa ∂β and \|kP \| = Ng Jr ∂τa ∂ω + 2ζrotνrot Ng Jr ∂τa ∂β = Ng Jr ∂τa ∂ω + 2ζrotνrot Ng Jr ∂τa ∂β (37) (37) Jr ∂β Jr ∂β 2.4.2 Simplified analysis of platform dynamics: 2.4.1 Simplified analysis of rotor dynamics: 260 Defining a damping coefficient (or a damping ratio) requires to reduce the global system to a second order oscillatory system. Equations 16 couples rotor and platform pitch dynamics, they hence involve a 4th order polynomial expression. In order to deal with rotor dynamics independently of the platform, it is supposed: ht ˙ϕ ≪v (32) ht ˙ϕ ≪v (32) For large FOWT systems, this hypothesis is, generally, respected. It implies: 265 For large FOWT systems, this hypothesis is, generally, respected. It implies: 265 For large FOWT systems, this hypothesis is, generally, respected. It implies: 265 Ngkτg ˙ϕ ≪∂τa ∂v v ∂τa ∂β kβ ˙ϕ ≪∂τa ∂v v Ngkτg ˙ϕ ≪∂τa ∂v v ∂τa ∂β kβ ˙ϕ ≪∂τa ∂v v Ngkτg ˙ϕ ≪∂τa ∂v v ∂τa ∂β kβ ˙ϕ ≪∂τa ∂v v (33) (33) Under such assumption, the linear form of equation 1 becomes: ˙ω = Ng Jr ∂τa ∂ω ω + ∂τa ∂v v + ∂τa ∂β β −τg (1”) 13 13 https://doi.org/10.5194/wes-2022-109 Preprint. Discussion started: 21 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. and the control is described by the PI controller: ˙β = kP ˙ω + kIω, such that the resulting Laplace transform equation is and the control is described by the PI controller: ˙β = kP ˙ω + kIω, such that the resulting Laplace transform equation is and the control is described by the PI controller: ˙β = kP ˙ω + kIω, such that the resulting Laplace transform equation is ω(s) = Grot(s)v(s) (34) 0 ω(s) = Grot(s)v(s) 270 (34) where, considering a kI > 0, Grot(s) = ∂τa ∂v s s2 −Ng Jr ∂τa ∂ω s −Ng Jr ∂τa ∂β (kP s + kI) , i.e. Grot(jν) = 1 1 + j 2ζrot ν νrot −νrot ν −∂τa ∂v Ng Jr ∂τa ∂ω + Ng Jr ∂τa ∂β kP (35) with: νrot = s −Ng Jr ∂τa ∂β kI , ζrot = − Ng Jr ∂τa ∂ω + Ng Jr ∂τa ∂β kP 2 q −Ng Jr ∂τa ∂β kI a kI , ζrot = − Ng Jr ∂τa ∂ω + Ng Jr ∂τa ∂β kP 2 q −Ng Jr ∂τa ∂β kI t = − g Jr ∂τa ∂ω + g Jr ∂τa ∂β kP 2 q −Ng Jr ∂τa ∂β kI (36) (36) Thus, when all interactions with platform pitch are neglected, the rotor behaves like a second order oscillatory system. = (sI −Aplt)−1Bd 2.4.2 Simplified analysis of platform dynamics: 280 39 gives: (42) ϕ(s) = Gplt,1,1(s)v(s) + Gplt,1,2(s)w(s), 295 with: with: (Gplt,1,1,Gplt,1,2)(s) = ht Jt ∂Fa ∂v s2+ 1 Jt (Dt+h2 t ∂Fa ∂v +kβht ∂Fa ∂β )s+ Kt Jt , 1 Jt ∂τw ∂w s2+ 1 Jt (Dt+h2 t ∂Fa ∂v +kβht ∂Fa ∂β )s+ Kt Jt , (43) i.e.: (Gplt,1,1,Gplt,1,2)(jν) = 1 1 − ν νplt 2 + 2jζplt ν νplt ht Kt ∂Fa ∂v , 1 Kt ∂τw ∂w , (44) (Gplt,1,1,Gplt,1,2)(s) = ht Jt ∂Fa ∂v s2+ 1 Jt (Dt+h2 t ∂Fa ∂v +kβht ∂Fa ∂β )s+ Kt Jt , 1 Jt ∂τw ∂w s2+ 1 Jt (Dt+h2 t ∂Fa ∂v +kβht ∂Fa ∂β )s+ Kt Jt , (43) (43) (Gplt,1,1,Gplt,1,2)(s) = Jt ∂v s2+ 1 Jt (Dt+h2 t ∂Fa ∂v +kβht ∂Fa ∂β )s+ Kt Jt , Jt ∂w s2+ 1 Jt (Dt+h2 t ∂Fa ∂v +kβht ∂Fa ∂β )s+ Kt Jt , (43) i.e.: i.e.: i.e.: (Gplt,1,1,Gplt,1,2)(jν) = 1 1 − ν νplt 2 + 2jζplt ν νplt ht Kt ∂Fa ∂v , 1 Kt ∂τw ∂w , (44) ν r Kt ζ 1 D + h2 ∂Fa + k h ∂Fa (45) 00 (Gplt,1,1,Gplt,1,2)(jν) = 1 1 − ν νplt 2 + 2jζplt ν νplt ht Kt ∂Fa ∂v , 1 Kt ∂τw ∂w , (44) 1 √KtJt Dt + h2 t ∂Fa ∂v + kβht ∂Fa ∂β (45) νplt = r Kt Jt , ζplt = 1 2√KtJt Dt + h2 t ∂Fa ∂v + kβht ∂Fa ∂β 300 νplt = r Kt Jt , ζplt = 1 2√KtJt Dt + h2 t ∂Fa ∂v + kβht ∂Fa ∂β (45) 300 (45) Thus, when all interactions with rotor dynamics are neglected, the platform behaves like a second order oscillatory system. The corresponding filter Gplt is a second order low-pass filter with cutoff angular frequency defined by νplt and damping ratio defined by ζplt. Thus, when all interactions with rotor dynamics are neglected, the platform behaves like a second order oscillatory system. The corresponding filter Gplt is a second order low-pass filter with cutoff angular frequency defined by νplt and damping ratio defined by ζplt. 2In (Abbas et al., 2021), βcomp is defined as in (Stockhouse et al., 2021) but with the convention βcomp = kfloat ˙ϕ so that kfloat = −kβ = ht ∂τa ∂v / ∂τa ∂β is negative. 2.4.2 Simplified analysis of platform dynamics: 280 2.4.2 Simplified analysis of platform dynamics: 280 Similarly to what is done in the previous paragraph, here the global system 1 is reduced to a second order oscillatory system that allows us to have a better understanding of platform dynamics. Considering kP = kI = 0 and assuming: kI = 0 and assuming: Considering kP = kI = 0 and assuming: Considering kP = kI = 0 and assuming: ∂Fa ∂ω ω << ∂Fa ∂v vr + ∂Fa ∂β β vr + ∂Fa ∂β β ∂Fa ∂ω ω << ∂Fa ∂v vr + ∂Fa ∂β β (38) (38) The latter is the condition to decouple the global system. It enables to consider ϕ response independently of ω, and as a second 285 order oscillatory system’s degree of freedom. The resulting Laplace transform equation is: The latter is the condition to decouple the global system. It enables to consider ϕ response independently of ω, and as a second 285 order oscillatory system’s degree of freedom. The resulting Laplace transform equation is:  ϕ ˙ϕ  (s) = Gplt(s)ud(s) (39)  ϕ ˙ϕ  (s) = Gplt(s)ud(s) (39) ud =  v w  : the input array is reduced because only the damping in the output side is analysed and it is not necessary for this to consider any additional control input.  to consider any additional control input. Gplt(s) = (sI −Aplt)−1Bd (40) 290 Gplt(s) = (sI −Aplt)−1Bd 290 (40) 1In case kI ≤0, νrot and ζrot would be imaginary according to the formulas above. Grot would be no longer a band-pass filter. 14 Aplt =  0 1 −Kt Jt −1 Jt (Dt + h2 t ∂Fa ∂v + kβht ∂Fa ∂β )   (41) Aplt is the bottom-right part of A defined in 17. Looking at the ϕ degree of freedom, eq. mp is defined as in (Stockhouse et al., 2021) but with the convention βcomp = kfloat ˙ϕ so that kfloat = −kβ = ht ∂τa ∂v / ∂τ ∂ 2.5.1 Expected effect on platform dynamics Figure 5 shows the second order low-pass filter Gplt. In other words, it is how ζplt value can affect the damping of platform oscillations. 315 315 It can be observed that ζplt has a significant effect on the damping of platform oscillations only for angular frequencies ν ≈νplt,natural. Yellow vertical band in figure 5 shows the interval of angular frequencies Idamped, arbitrarily defined by: Idamped = νplt,natural √ 2 , √ 2νplt,natural i Idamped = νplt,natural √ 2 , √ 2νplt,natural Idamped = νplt,natural √ 2 , √ 2νplt,natural (48) Idamped = νplt,natural √ 2 , √ 2νplt,natural (48) (48) that are directly damped when ζplt increases. Therefore, it is to be expected that ζplt-fixed strategy will be well fit to reduce platform motion and tower loads when their variations happen at an angular frequency ν ∈Idamped. that are directly damped when ζplt increases. Therefore, it is to be expected that ζplt-fixed strategy will be well fit to reduce platform motion and tower loads when their variations happen at an angular frequency ν ∈Idamped. Figure 5. Bode diagram of second order low-pass filter Gplt Figure 5. Bode diagram of second order low-pass filter Gplt Figure 5. Bode diagram of second order low-pass filter Gplt 2.5 Artificial damping of the platform : ζplt-fixed strategy By knowing the features of the FOWT, one can impose a given level of damping and obtain an explicit expression for the kβ: 305 kβ = 1 ht ∂Fa ∂β 2 p KtJtζplt −Dt −h2 t ∂Fa ∂v kβ = 1 ht ∂Fa ∂β 2 p KtJtζplt −Dt −h2 t ∂Fa ∂v (46) (46) The strategy is such that kβ is a negative number instead of what is proposed in the literature. In (Stockhouse et al., 2021), βcomp = −kβ ˙ϕ is introduced in order to erase at first order the coupling between platform and rotor dynamics, and therefore kβ is positive, defined by: kβ = −ht ∂τa ∂v /∂τa ∂β 310 (47) 2In (Abbas et al., 2021), βcomp is defined as in (Stockhouse et al., 2021) but with the convention βcomp = kfloat ˙ϕ so that kfloat = −kβ = ht ∂τa ∂v / ∂τa ∂β is negative. 15 https://doi.org/10.5194/wes-2022-109 Preprint. Discussion started: 21 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. 2.5.2 Expected effect on rotor dynamics 320 Thus, if the characteristic time of platform dynamics is small enough, the equation truncated at first order is valid and it is to be expected that, at least for some tunings of the PI controler, ζplt-fixed strategy would increase rotor speed variations. 335 3 Numerical tests with time domain simulations In this section, it is analysed how the new control strategy described in the previous section affects platform and rotor dynamics, and especially the impact on tower loads and rotational speed. The reference is the control strategy without compensation, with kβ = 0. The ζplt-fixed strategy has been implemented in the ROSCO environment (ROSCO, 2021), replacing the existing pitch 340 control. The rest of the controller remains basically the same. In the next, ζplt-fixed strategy is compared to the one without compensation, named reference. The only difference between the two terms of comparison concerns the kβ. For the ζplt-fixed strategy, it is given by 45. For the reference, it is kβ = 0. The ζplt-fixed strategy has been implemented in the ROSCO environment (ROSCO, 2021), replacing the existing pitch 340 control. The rest of the controller remains basically the same. In the next, ζplt-fixed strategy is compared to the one without compensation, named reference. The only difference between the two terms of comparison concerns the kβ. For the ζplt-fixed strategy, it is given by 45. For the reference, it is kβ = 0. The ζplt-fixed strategy has been implemented in the ROSCO environment (ROSCO, 2021), replacing the existing pitch 340 control. The rest of the controller remains basically the same. In the next, ζplt-fixed strategy is compared to the one without compensation, named reference. The only difference between the two terms of comparison concerns the kβ. For the ζplt-fixed strategy, it is given by 45. For the reference, it is kβ = 0. 2.5.2 Expected effect on rotor dynamics 320 In this part the first order effect of ζplt-fixed strategy on the rotor dynamics is analysed. The state space representation of the FOWT dynamics is given by equation 16. By considering 0 input (ie. uc,ol = ud = 0), this leads to the following linear equation, truncated at first order: ˙ω = ¨θ = A2,1θ + A2,2 ˙θ + A2,4 ˙ϕ, (49) where 325 where 325 325 A2,4 = Ng Jr −ht ∂τa ∂v −kβ ∂τa ∂β + kτgNg = Ng Jr −ht ∂τa ∂v + ∂τa ∂β ht ∂Fa ∂β Dt + h2 t ∂Fa ∂v −2 p KtJtζplt + kτgNg ! . (50) 16 https://doi.org/10.5194/wes-2022-109 Preprint. Discussion started: 21 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/wes-2022-109 Preprint. Discussion started: 21 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. Moreover, the following inequalities are verified for an above-rated operating point: ht ∂τa ∂v −kτgNg ≥0 ∂τa ∂β ∂Fa ∂β > 0 1 2√KtJt Dt + h2 t ∂Fa ∂v ≤ζplt ht ∂τa ∂v −kτgNg ≥0 ∂τa ∂β ∂Fa ∂β > 0 1 2√KtJt Dt + h2 t ∂Fa ∂v ≤ζplt ht ∂τa ∂v −kτgNg ≥0 ∂τa ∂β ∂Fa ∂β > 0 1 2√KtJt Dt + h2 t ∂Fa ∂v ≤ζplt (51) (51) First inequality comes from 29 (notice that in section 3, it is considered τg = 0). Third inequality is a consequence of the assumption that ζplt-fixed strategy aims at increasing the damping of the platform. This implies that: 330 First inequality comes from 29 (notice that in section 3, it is considered τg = 0). Third inequality is a consequence of the assumption that ζplt-fixed strategy aims at increasing the damping of the platform. This implies that: 330 ∂ ∂ζplt \|A2,4\| > 0 (52) meaning that the first order coupling between platform dynamics and rotor dynamics will increase when ζplt increases if ζplt-fixed strategy is applied. Thus, if the characteristic time of platform dynamics is small enough, the equation truncated at first order is valid and it is to be expected that, at least for some tunings of the PI controler, ζplt-fixed strategy would increase rotor speed variations. 35 meaning that the first order coupling between platform dynamics and rotor dynamics will increase when ζplt increases if ζplt-fixed strategy is applied. 3.2 Tuning of the PI controller Values of kP , kI, kβ are continuously updated following the explicit expression given in equations 36 and 45, then low-pass filtered. This means that globally, for each of these test cases and each set of parameters, kP , kI, kβ have almost fixed values. After some tests, for all the considered simulations, ζrot = 0.6 and νrot = 0.01 are chosen for the PI controller’s tuning. This choice ensures that most of the wave spectrum (which peaks at T ≈11 s, ie. ν ≈0.57 rads−1) and platform dynamics natural angular frequency (νplt ≈0.22 rad.s−1) fall outside of Grot pass-band. In the next sections, several values of platform damping are analyzed for the ζplt-fixed strategy and compared to the reference strategy for different test case. 3.1 Test cases The simulation tool used is OpenFast v2.4.0 (https://github.com/OpenFAST/openfast) and the FOWT model considered is the 345 IEA 15 MW wind turbine mounted over the UMaine VolturnUS-S semi-submersible floater (Allen et al., 2020). Initially simple constant wind and monochromatic waves are testes in order to verify the analytical developments of the previous section. Then a test case more representative of the industrial design of FOWT is considered by testing a DLC1.1. For the latter, simulation consider only 1 seed of 3600 seconds with aligned wind and irregular waves. For this time simulation, this is statistically equivalent to 600 seconds and 6 seeds. 350 The simulation tool used is OpenFast v2.4.0 (https://github.com/OpenFAST/openfast) and the FOWT model considered is the 345 IEA 15 MW wind turbine mounted over the UMaine VolturnUS-S semi-submersible floater (Allen et al., 2020). Initially simple constant wind and monochromatic waves are testes in order to verify the analytical developments of the previous section. Then a test case more representative of the industrial design of FOWT is considered by testing a DLC1.1. For the latter, simulation consider only 1 seed of 3600 seconds with aligned wind and irregular waves. For this time simulation, this is statistically equivalent to 600 seconds and 6 seeds. 350 The simulation tool used is OpenFast v2.4.0 (https://github.com/OpenFAST/openfast) and the FOWT model considered is the 345 IEA 15 MW wind turbine mounted over the UMaine VolturnUS-S semi-submersible floater (Allen et al., 2020). Initially simple constant wind and monochromatic waves are testes in order to verify the analytical developments of the previous section. Then a test case more representative of the industrial design of FOWT is considered by testing a DLC1.1. For the latter, simulation consider only 1 seed of 3600 seconds with aligned wind and irregular waves. For this time simulation, this is statistically equivalent to 600 seconds and 6 seeds. 350 17 17 https://doi.org/10.5194/wes-2022-109 Preprint. Discussion started: 21 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. mental conditions for the numerical test cases. 3.3 Still wind and monochromatic wave For the still wind and monochromatic wave condition, two ζplt are tested: ζplt = 0.1 and ζplt = 0.25. The corresponding quality factors are: Q = 5 and Q = 2, respectively. Thus, the platform is expected to behave like an under-damped second 360 order oscillatory system. Table 6 states external conditions for test cases with still wind and monochromatic wave. Hereafter, results are plotted over time are drawn for a 100 seconds time interval in a simulation on a long period of time, so that the operating point is reached. When necessary for a better understanding, results are reported for a longer interval. able 4. Environmental conditions for the numerical test cases. case wind speed V (ms−1) wave period Tp (s) wave height Hw (m) (1) 11 11 1.5 (2) 11 28.75 1.5 (3) 22 11 1.5 (4) 22 28.75 1.5 Table 5 gives the mean value of kβ for test cases (1) to (4). Cases (1), (2) and cases (3), (4) are gathered together as they use the same mean value of kβ. 365 Table 5 gives the mean value of kβ for test cases (1) to (4). Cases (1), (2) and cases (3), (4) are gathered together as they use the same mean value of kβ. 365 case ζplt = 0.10 ζplt = 0.25 reference (1) and (2) kβ = −8.6 kβ = −42.7 kβ = 0.0 (3) and (4) kβ = −7.4 kβ = −34.8 kβ = 0.0 18 18 Figure 6. Platform pitch Φ (deg) for a monochromatic wave of period 11s (test case (1) on the left and (3) on the right). https://doi.org/10.5194/wes-2022-109 Preprint. Discussion started: 21 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/wes-2022-109 Preprint. Discussion started: 21 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. Figure 6. Platform pitch Φ (deg) for a monochromatic wave of period 11s (test case (1) on the left and (3) on the right). Figure 6. Platform pitch Φ (deg) for a monochromatic wave of period 11s (test case (1) on the left and (3) on the right). Figure 7. Platform pitch Φ (deg) for a monochromatic wave of period 28.75s (test case (2) on the left and (4) on the right). Figure 7. Platform pitch Φ (deg) for a monochromatic wave of period 28.75s (test case (2) on the left and (4) on the right). 3.3 Still wind and monochromatic wave Figures 6 and 7 show the forced oscillations of the platform when it is subjected to a monochromatic wave of period 11s (which corresponds to the realistic fundamental period of a wave) and 28.75s (which is the natural period of the platform). As shown by the analytical development, increasing ζplt reduces platform oscillations, especially when the wave period is close to the natural period of the platform (see diagram 5). This is shown in Figures 8 and 9 where the density of occurrence of each 370 value of tower bottom reaction moment is reported. For the monochromatic wave with period 11s the damping of the ζplt-fixed strategy is less evident. More floating wind test cases are reported in the next section. Figures 6 and 7 show the forced oscillations of the platform when it is subjected to a monochromatic wave of period 11s (which corresponds to the realistic fundamental period of a wave) and 28.75s (which is the natural period of the platform). As shown by the analytical development, increasing ζplt reduces platform oscillations, especially when the wave period is close to the natural period of the platform (see diagram 5). This is shown in Figures 8 and 9 where the density of occurrence of each 370 value of tower bottom reaction moment is reported. For the monochromatic wave with period 11s the damping of the ζplt-fixed strategy is less evident. More floating wind test cases are reported in the next section. to the natural period of the platform (see diagram 5). This is shown in Figures 8 and 9 where the density of occurrence of each 370 value of tower bottom reaction moment is reported. For the monochromatic wave with period 11s the damping of the ζplt-fixed strategy is less evident. More floating wind test cases are reported in the next section. 19 Figure 8. Tower base moment (MN.m) densities for a monochromatic wave of period 11s (test case (1) on the left and (3) on the right). https://doi.org/10.5194/wes-2022-109 Preprint. Discussion started: 21 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/wes-2022-109 Preprint. Discussion started: 21 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. Figure 8. Tower base moment (MN.m) densities for a monochromatic wave of period 11s (test case (1) on the left and (3) on the right). Figure 8. 3.3 Still wind and monochromatic wave Tower base moment (MN.m) densities for a monochromatic wave of period 11s (test case (1) on the left and (3) on the right). Figure 8. Tower base moment (MN.m) densities for a monochromatic wave of period 11s (test case (1) o Figure 9. Tower base moment (MN.m) densities for a monochromatic wave of period 28.75s (test case (2) on the left and (4) on the right). Figure 9. Tower base moment (MN.m) densities for a monochromatic wave of period 28.75s (test case (2) on the left and (4) on the right). Figure 9. Tower base moment (MN.m) densities for a monochromatic wave of period 28.75s (test case (2) on Figure 10. Rotor speed (rpm) evolution over time for test cases (1) on the left and (2) on the right Figure 10. Rotor speed (rpm) evolution over time for test cases (1) on the left and (2) on the right 20 Figure 11. Rotor speed (rpm) evolution over time for test cases (3) on the left and (4) on the right. https://doi.org/10.5194/wes-2022-109 Preprint. Discussion started: 21 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/wes-2022-109 Preprint. Discussion started: 21 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/wes-2022-109 Preprint. Discussion started: 21 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. Figure 11. Rotor speed (rpm) evolution over time for test cases (3) on the left and (4) on the right. Figure 11. Rotor speed (rpm) evolution over time for test cases (3) on the left and (4) on the right. ζplt-fixed strategy’s effect on rotor dynamics is not easily described by a second order linear equation: it involves the coupling between platform and rotor dynamics, which was analysed at first order in 2.5. From this analysis, ζplt-fixed strategy was ζplt-fixed strategy’s effect on rotor dynamics is not easily described by a second order linear equation: it involves the coupling between platform and rotor dynamics, which was analysed at first order in 2.5. From this analysis, ζplt-fixed strategy was expected to increase the coupling between platform and rotor dynamics for a short characteristic time. In Figure 10 and 11 it 75 can be observed that for a short characteristic time (11s) ζplt-fixed strategy increases rotor speed variations, but not for a longer characteristic time, such as 28.75s (for which it behaves slightly better than kβ = 0 strategy). 3.3 Still wind and monochromatic wave expected to increase the coupling between platform and rotor dynamics for a short characteristic time. In Figure 10 and 11 it 375 can be observed that for a short characteristic time (11s) ζplt-fixed strategy increases rotor speed variations, but not for a longer characteristic time, such as 28.75s (for which it behaves slightly better than kβ = 0 strategy). To conclude this part of the tests: β-compensation strategies perform very differently depending on the oscillatory frequency of the platform: To conclude this part of the tests: β-compensation strategies perform very differently depending on the oscillatory frequency of the platform: – For angular frequencies ν ∈Idamped = h νplt,natural √ 2 , √ 2νplt,natural i , ζplt-fixed strategy is very effective when it comes 380 to the damping of platform oscillations, as seen in paragraph 2.5 (cf. figure 5). The tests highlight that ζplt-fixed strategy is reducing both tower loads and rotor speed variations in turbulent wind conditions. – For angular frequencies outside the previous set, ζplt-fixed strategy is less effective for damping platform oscillations. Tower loads reduction by ζplt-fixed strategy is therefore barely visible, whereas rotor speed variations are actually am- plified, especially when comparing this strategy to reference strategy. 385 385 3.4 DLC1.2 tests blades are fully deformable. As already done for the previous test cases, the ζplt-fixed strategy is compared to the referen 395 strategy, kβ = 0. The level of damping imposed to the platform is ζplt-fixed = 0.1. This value is found to be the most interesting to be test for this floater and WTG configuration. Other tests with higher values of imposed damping show less interesting results. T choice of the right ζplt for each FOWT system is important and demands for some iterations before concluding. Table 6. Environmental conditions for DLC 1.2. Time sim [s] w.speed [m.s−1] w. condition Tp [s] Hs [m] γ waves dir. 3600 12.0 −24.0 Normal turbulence B 11.0 1.5 2.0 co-linear Figure 12. evolution of kβ for some of the simulations. The level of damping imposed to the platform is ζplt-fixed = 0.1. This value is found to be the most interesting to be tested for this floater and WTG configuration. Other tests with higher values of imposed damping show less interesting results. The choice of the right ζplt for each FOWT system is important and demands for some iterations before concluding. Table 6. Environmental conditions for DLC 1.2. Figure 12. evolution of kβ for some of the simulations. Figure 12. evolution of kβ for some of the simulations. Figure 12 shows how kβ evolves for some cases of the simulation pool. The below-rated behaviour is more dynamic than 400 the over-rated, where the floating feedback is more stable. As remarked in 2.5, the kβ for the ζplt-fixed is a negative value. From the time series of the tower bottom moment, a rainflow algorithm is used to count the cycles following ASTM norma- tive (ASTM, 2017). The DEL is obtained by using a Wohler’s curve with a single slope of exponent m = 3.0. Platform pitch, power, rotor speed, blade pitch, tower load and tower DEL results of the simulations for the comparison are resumed in Figure 400 13. The ζplt-fixed strategy reduces platform pitch motion for all wind speeds. As expected this is done by the coupling with the 405 rotor and the use of the blade pitch. The increase in the average value of the pitch explains the slight decrease in rotor speed for the above rated wind speeds. The generator power is also affected, slightly increased for lower wind speeds and slightly decreased for higher wind speeds (around +/ −1%). 13. 3.4 DLC1.2 tests Tests presented hereafter are more representative of what is done during design or verification of offshore wind structure. They are inspired by the DLC 1.2, for normal power production in normal turbulence and normal sea state, as described in IEC standards. This kind of load case aim at assessing the fatigue design criteria. Kaimal’s turbulence model is considered following IEC 61400 v.3 for a Wind Turbine of turbulence type B, for average wind speeds from 12 ms−1 to 24 ms−1, as 390 described in Table 6. The wind box is generated by TurbSim tool developed by NREL. For the waves, JONSWAP distributions are considered with significant wave height Hs = 1.5m, wave period Tp = 11.0s and γ = 2.0. Wind and waves are considered aligned in the same direction. All the degrees of freedom of the floating platform are allowed, including the surge motion. In other terms, the numerical twin reproduces, with the accuracy of the chosen model, the actual motion of the FOWT. Tower and Tests presented hereafter are more representative of what is done during design or verification of offshore wind structure. They are inspired by the DLC 1.2, for normal power production in normal turbulence and normal sea state, as described in IEC standards. This kind of load case aim at assessing the fatigue design criteria. Kaimal’s turbulence model is considered following IEC 61400 v.3 for a Wind Turbine of turbulence type B, for average wind speeds from 12 ms−1 to 24 ms−1, as 390 described in Table 6. The wind box is generated by TurbSim tool developed by NREL. For the waves, JONSWAP distributions are considered with significant wave height Hs = 1.5m, wave period Tp = 11.0s and γ = 2.0. Wind and waves are considered aligned in the same direction. All the degrees of freedom of the floating platform are allowed, including the surge motion. In other terms, the numerical twin reproduces, with the accuracy of the chosen model, the actual motion of the FOWT. Tower and 21 https://doi.org/10.5194/wes-2022-109 Preprint. Discussion started: 21 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. blades are fully deformable. As already done for the previous test cases, the ζplt-fixed strategy is compared to the reference 95 strategy, kβ = 0. blades are fully deformable. As already done for the previous test cases, the ζplt-fixed strategy is compared to the reference 395 strategy, kβ = 0. 3.4 DLC1.2 tests The ζplt-fixed strategy reduces platform pitch motion for all wind speeds. As expected this is done by the coupling with the 405 rotor and the use of the blade pitch. The increase in the average value of the pitch explains the slight decrease in rotor speed for the above rated wind speeds. The generator power is also affected, slightly increased for lower wind speeds and slightly decreased for higher wind speeds (around +/ −1%). 22 https://doi.org/10.5194/wes-2022-109 Preprint. Discussion started: 21 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. Loads in the tower are reduced and fatigue Design Equivalent Load (DEL) also. This is remarkable for around rated speed. For high wind speeds, the gain is less evident. In average there is a gain around 15% of the DEL. Table 7 shows, for the 10 410 ms−1 case, a deeper comparison by reporting the statistics of the quantities of interest extracted from this simulation. The difference in the minimum value of the moment at the tower base is remarkable: passing from 80.97 MNm for the reference up to 165.40 MNm for the ζplt-fixed strategy. This is also clear in the difference of the standard deviations. The amplitudes of the oscillations of this load are reduced. It is interesting to analyse Figure 14. This figure reports a deeper analysis of the fatigue damage. In fact, the stress in the 415 tower bottom section is obtained by considering the design proposed by UMaine in (Allen et al., 2020). Then, an offshore Wohler’s curve is considered with two slopes in the log-log domain: m = 3.0 for loads with less than 1.0 million cycles and m = 5.0 for loads with higher number of cycles. Those are typical values proposed by DNV for offshore steel structures. This analysis leads to obtain an estimation of the 25 years damage at the tower bottom. The gain is much more evident than the DEL. It is interesting to analyse Figure 14. This figure reports a deeper analysis of the fatigue damage. In fact, the stress in the 415 tower bottom section is obtained by considering the design proposed by UMaine in (Allen et al., 2020). Then, an offshore Wohler’s curve is considered with two slopes in the log-log domain: m = 3.0 for loads with less than 1.0 million cycles and m = 5.0 for loads with higher number of cycles. 3.4 DLC1.2 tests Those are typical values proposed by DNV for offshore steel structures. This analysis leads to obtain an estimation of the 25 years damage at the tower bottom. The gain is much more evident than the DEL. This is due to the second slope, m = 5.0, which amplifies the changes in the load amplitudes. Offshore WTG in production are 420 mostly subjected to a very high number of cycles of small amplitudes. This figure shows also the effect of the turbulence on the fatigue. In fact, looking at the reference, up to 12 ms−1, the shape of the damage distribution follows the one of the thrust curve. However, since the turbulence is a percentage of the average wind speed, from 16 ms−1, the damage starts increasing again. Table 7. Statistics for results concerning the case with mean wind speed at 10 ms−1. For each quantity of interest, there is the comparison of the minimum, maximum, mean and standard deviation values produced by the ζplt-fixed control strategy and the reference. ζplt = 0.1 kβ = 0 ζplt = 0.1 kβ = 0 ζplt = 0.1 kβ = 0 ζplt = 0.1 kβ = 0 min min mean mean max max st.d. st.d PtfmPitch [deg] 2.84 1.09 4.55 4.56 5.62 5.91 0.51 0.59 TwrBsM [MNm] 165.40 80.97 366.66 367.79 493.80 505.10 43.20 46.82 GenPwr [MW] 7.85 5.49 12.09 12.01 15.15 15.19 1.29 1.47 BldPitch [deg] 0 0 0.218 0.176 6.651 8.33 0.394 0.729 RotSpeed [rpm] 5.23 5.34 6.90 6.92 7.90 8.00 0.61 0.60 The coupling between platform pitch and rotor dynamics is increased. Hence, an increase in the pitch utilization is expected. 425 A specific fatigue analysis of the pitch bearing is realized by following (Shan et al., 2021), where three methods to evaluate the fatigue of the pitch bearing are compared leading to comparable results. The second method is implemented here in order to quantify the increment in the pitch fatigue caused by the ζplt-fixed strategy with respect to the reference. The bearing life is inversely proportional to the cube of the bearing loading. From the overturning moment acting on the bearing, the equivalent loading at N revolutions of the pitch bearing is given by: 430 The coupling between platform pitch and rotor dynamics is increased. Hence, an increase in the pitch utilization is expected. 3.4 DLC1.2 tests 425 A specific fatigue analysis of the pitch bearing is realized by following (Shan et al., 2021), where three methods to evaluate the fatigue of the pitch bearing are compared leading to comparable results. The second method is implemented here in order to quantify the increment in the pitch fatigue caused by the ζplt-fixed strategy with respect to the reference. The bearing life is inversely proportional to the cube of the bearing loading. From the overturning moment acting on the bearing, the equivalent loading at N revolutions of the pitch bearing is given by: 430 425 Meq = X i ∆βi M 3 i N !1/3 (53) Meq = X i ∆βi M 3 i N !1/3 (53) (53) 23 https://doi.org/10.5194/wes-2022-109 Preprint. Discussion started: 21 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. 13. Comparison results for the DLC1.2 for the UMaine floater with IEA15MW WTG. The imposed level of damping in the platfor ics for the ζplt-fixed strategy is 0.10. Outputs show statistics for platform pitch; blade pitch; Tower bending moment, max and damag ent load; rotor speed and generator power. Figure 13. Comparison results for the DLC1.2 for the UMaine floater with IEA15MW WTG. The imposed level of damping in the platform dynamics for the ζplt-fixed strategy is 0.10. Outputs show statistics for platform pitch; blade pitch; Tower bending moment, max and damage equivalent load; rotor speed and generator power. where i is the time step of the simulation. The discrete integral considers the product of the time series of the overturning moment Mi and the blade pitch variation ∆βi, over the entire simulation. To take into account the fact that, for each wind 24 Figure 14. Fatigue cumulative damage at tower bottom by using rainflow counting and linear Miner’s rule. The damage is obtained consider- ing the tower base design proposed by the UMaine, a Wohler’s curve bi-linear with m = 3.0 up to 106 cycles and m = 5.0 after, as proposed by DNV for Offshore steel. The probability of occurrence of each wind is equal, without any weibull distribution. https://doi.org/10.5194/wes-2022-109 Preprint. Discussion started: 21 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/wes-2022-109 Preprint. Discussion started: 21 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. Figure 14. Fatigue cumulative damage at tower bottom by using rainflow counting and linear Miner’s rule. Figure 15. Increment in the use of the pitch bearing given by the ζplt-fixed strategy with respect to the reference. The increment is expressed in terms of % of Meq (eq. 53) https://doi.org/10.5194/wes-2022-109 Preprint. Discussion started: 21 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. Figure 15. Increment in the use of the pitch bearing given by the ζplt-fixed strategy with respect to the reference. The increment is expressed in terms of % of Meq (eq. 53) Figure 15. Increment in the use of the pitch bearing given by the ζplt-fixed strategy with respect to the reference. The increment is expressed in terms of % of Meq (eq. 53) 4 Conclusions The first part of this paper presents the analysis of the NMPZ related to the system of equations describing the dynamcs of a 440 floating offshore wind turbine (FOWT). The equation of the rotor dynamics and the one of the platform dynamics are analysed in the complex domain to explicit the conditions leading to their respective NMPZs. One of those NMPZ, the instability given by the blade pitch on the rotor dynamics, is already known in literature and a compensation already exists to avoid it. The other one, the instability given by the blade pitch to the platform dynamics, is a novelty in the community. The effects of the The first part of this paper presents the analysis of the NMPZ related to the system of equations describing the dynamcs of a 440 floating offshore wind turbine (FOWT). The equation of the rotor dynamics and the one of the platform dynamics are analysed in the complex domain to explicit the conditions leading to their respective NMPZs. One of those NMPZ, the instability given by the blade pitch on the rotor dynamics, is already known in literature and a compensation already exists to avoid it. The other one, the instability given by the blade pitch to the platform dynamics, is a novelty in the community. The effects of the NMPZs are analysed on two analytical test cases: at the beginning both ω and ˙ϕ always converge to the right solutions just after 445 the first steps. When both NMPZ conditions are not verified, those tendencies don’t change. However, when the ˙ϕ-NMPZ is verified, \|ω\| becomes so big that ˙ϕ jumps into unexpected values without converging to the expected solution. Similarly, when the ω-NMPZ condition is verified, \| ˙ϕ\| becomes so big that ω oscillates before converging to the expected solution. NMPZs can cause important shifts and unexpected behaviors for both ω and ϕ. In the second part of the paper, the damping analysis is further investigated proposing a new strategy control for FOWT, 450 named ζplt-fixed. This strategy is based on a compensation parameter kβ proportional to the platform pitch velocity. It considers the coupling between the rotor dynamics and the floating platform dynamics. The idea behind this control strategy is to activate the blade pitch to damp the platform motions. 3.4 DLC1.2 tests The damage is obtained consider- ing the tower base design proposed by the UMaine, a Wohler’s curve bi-linear with m = 3.0 up to 106 cycles and m = 5.0 after, as proposed by DNV for Offshore steel. The probability of occurrence of each wind is equal, without any weibull distribution. Figure 14. Fatigue cumulative damage at tower bottom by using rainflow counting and linear Miner’s rule. The damage is obtained consider- ing the tower base design proposed by the UMaine, a Wohler’s curve bi-linear with m = 3.0 up to 106 cycles and m = 5.0 after, as proposed by DNV for Offshore steel. The probability of occurrence of each wind is equal, without any weibull distribution. speed, the mean blade pitch is different, the 90 degrees of the pitch range are divided in 30 sectors, each one corresponding to speed, the mean blade pitch is different, the 90 degrees of the pitch range are divided in 30 sectors, each one corresponding to a different zone of the bearing. This corresponds to consider a tooth function in the integral of eq. 53 and it is well explained in 435 Figure 11 of (Shan et al., 2021). In Figure 15, it is reported the increase in Meq given by the ζplt-fixed strategy with respect to the reference. The increase in the use of the pitch bearing is estimated in a factor of 1.5 in average, with peaks of 2 around the rated wind speed. This is an aspect of the control strategy to be considered and it is an axis of improvement for future works. 25 4 Conclusions An explicit expression linking kβ to ζplt (damping ratio imposed to the platform) is obtained by deriving a second order filter from the equation of the platform dynamics. In the second part of the paper, the damping analysis is further investigated proposing a new strategy control for FOWT, 450 named ζplt-fixed. This strategy is based on a compensation parameter kβ proportional to the platform pitch velocity. It considers the coupling between the rotor dynamics and the floating platform dynamics. The idea behind this control strategy is to activate the blade pitch to damp the platform motions. An explicit expression linking kβ to ζplt (damping ratio imposed to the platform) is obtained by deriving a second order filter from the equation of the platform dynamics. This is different with respect to already existing strategies based on platform pitch compensation which aims at decoupling 455 rotor and platform dynamics. This difference is underlined by the values of kβ, which is negative for the new control strategy, This is different with respect to already existing strategies based on platform pitch compensation which aims at decoupling 455 rotor and platform dynamics. This difference is underlined by the values of kβ, which is negative for the new control strategy, 26 https://doi.org/10.5194/wes-2022-109 Preprint. Discussion started: 21 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. while it is positive for the ones existing in literature. For each FOWT system, some iterations are necessary in order to find the optimum value for ζplt. The performances of the ζplt-fixed strategy are tested analytically and numerically by considering an OpenFAST numerical twin of the Umaine IEA15MW FOWT. For a test representative of the DLC1.2, the ζplt-fixed strategy allows to reduce the loads at the tower foundation interface for all the considered wind speeds, without significant losses in 460 terms of power production. The damage analysis shows a remarkable gain in terms of fatigue lifetime. The blade pitch use slightly increases remaining in the bounds of a standard controller limitations. while it is positive for the ones existing in literature. For each FOWT system, some iterations are necessary in order to find the optimum value for ζplt. The performances of the ζplt-fixed strategy are tested analytically and numerically by considering an OpenFAST numerical twin of the Umaine IEA15MW FOWT. 4 Conclusions For a test representative of the DLC1.2, the ζplt-fixed strategy allows to reduce the loads at the tower foundation interface for all the considered wind speeds, without significant losses in 460 terms of power production. The damage analysis shows a remarkable gain in terms of fatigue lifetime. The blade pitch use slightly increases remaining in the bounds of a standard controller limitations. This work highlights the importance of defining proper controller strategies for FOWT in order to reduce loads on the structure or improve the performances and, then, it aims at helping the industry to achieve the objective in terms of LCOE reduction. 465 460 This work highlights the importance of defining proper controller strategies for FOWT in order to reduce loads on the structure or improve the performances and, then, it aims at helping the industry to achieve the objective in terms of LCOE reduction. 465 Author contributions. Matteo Capaldo has contributed for the original idea of the new control strategy, the development of the numerical twin and the numerical tests and he is the main contributor for the paper editing. Paul Mella developed the mathematical framework, he has contributed for the original idea of the new control strategy and he has contributed for the paper editing Competing interests. The authors declare there are not competing interests in this work. Competing interests. The authors declare there are not competing interests in this work. 27 27 https://doi.org/10.5194/wes-2022-109 Preprint. Discussion started: 21 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/wes-2022-109 Preprint. Discussion started: 21 December 2022 c⃝Author(s) 2022. CC BY 4.0 License. References 470 Abbas, N., Zalkind, D., Pao, L. & A., W.: A Reference open-source controller for fixed and floating offshore wind turbines, Wind Energy Science, 7, 53–73, https://doi.org/10.5194/wes-7-53-2022, 2021. 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https://openalex.org/W3016393960	https://hal.archives-ouvertes.fr/hal-02895473/document	English	null	A semi‐objective circulation pattern classification scheme for the semi‐arid Northeast Brazil	International journal of climatology	2,020	cc-by	13,129	A semi-objective circulation pattern classification scheme for the semi-arid Northeast Brazil Patrick Laux, Brian Böker, Eduardo Sávio Martins, Francisco Vasconcelos Junior, Vincent Moron, Tanja Portele, Christof Lorenz, Andreas Philipp, Harald Kunstmann To cite this version: Patrick Laux, Brian Böker, Eduardo Sávio Martins, Francisco Vasconcelos Junior, Vincent Moron, et al.. A semi-objective circulation pattern classification scheme for the semi-arid Northeast Brazil. International Journal of Climatology, 2021, 41 (1), pp.51-72. 10.1002/joc.6608. hal-02895473 A semi-objective circulation pattern classification scheme for the semi-arid Northeast Brazil Distributed under a Creative Commons Attribution 4.0 International License K E Y W O R D S bias correction, circulation pattern classification, Northeast Brazil, precipitation, SANDRA, statistical downscaling Patrick Laux1,2 \| Brian Böker2 \| Eduardo Sávio Martins3 \| Francisco das Chagas Vasconcelos Junior3 \| Vincent Moron4 \| Tanja Portele1 \| Christof Lorenz1 \| Andreas Philipp2 \| Harald Kunstmann1,2 Patrick Laux1,2 \| Brian Böker2 \| Eduardo Sávio Martins3 \| Francisco das Chagas Vasconcelos Junior3 \| Vincent Moron4 \| Tanja Portele1 \| Christof Lorenz1 \| Andreas Philipp2 \| Harald Kunstmann1,2 1Institute of Meteorology and Climate Research (IMK-IFU), Campus Alpin, Karlsruhe Institute of Technology (KIT), Garmisch-Partenkirchen, Germany 2Institute of Geography, University of Augsburg, Augsburg, Germany 3Ceará Institute for Meteorology and Water Resources (FUNCEME), Fortaleza, Brazil Correspondence Correspondence Patrick Laux, Institute of Meteorology and Climate Research (IMK-IFU), Campus Alpin, Karlsruhe Institute of Technology (KIT), Garmisch-Partenkirchen, Germany. Email: patrick.laux@kit.edu Abstract The semi-arid Northeast Brazil (NEB) is just recovering from a very severe water cri- sis induced by a multiyear drought. With this crisis, the question of water resources management has entered the national political agenda, creating an opportunity to better prepare the country to deal with future droughts. In order to improve climate predictions, and thus preparedness in NEB, a circulation pattern (CP) classification algorithm offers various options. Therefore, the main objective of this study was to develop a computer aided CP classification based on the Simulated ANnealing and Diversified RAndomization clustering (SANDRA) algorithm. First, suitable predic- tor variables and cluster domain setting are evaluated using ERA-Interim reanalyses. It is found that near surface variables such as geopotential at 1,000 hPa (GP1,000) or mean sea level pressure (MSLP) should be combined with horizontal wind speed at the upper 700 hPa level (UWND700). A 11-cluster solution is favoured due to the trade-offs between interpretability of the cluster centroids and the explained vari- ances of the predictors. Second, occurrence and transition probabilities of this 11-cluster solution of GP1,000 and UWND700 are analysed, and typical CPs, which are linked to dry and wet conditions in the region are identified. The suitability of the new classification to be potentially applied for statistical downscaling or CP- conditional bias correction approach is analysed. The CP-conditional cumulative density functions (CDFs) exhibit discriminative power to separate between wet and dry conditions, indicating a good performance of the CP approach. 4Aix-Marseille University, CNRS, IRD, INRAE, Coll. de France, CEREGE, Aix-en-Provence, France 4Aix-Marseille University, CNRS, IRD, INRAE, Coll. de France, CEREGE, Aix-en-Provence, France HAL Id: hal-02895473 https://hal.science/hal-02895473v1 Submitted on 19 Apr 2021 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License Received: 21 December 2019 Revised: 10 April 2020 Accepted: 14 April 2020 Published on: 4 May 2020 DOI: 10.1002/joc.6608 Received: 21 December 2019 Revised: 10 April 2020 Accepted: 14 April 2020 Published on: 4 May 2020 DOI: 10.1002/joc.6608 R E S E A R C H A R T I C L E Funding information Funding information Seasonal water re-sources management in semi-arid regions: Transfer of regionalized global information to practice (SaWaM), Grant/Award Number: 02WGR1421A Int J Climatol. 2021;41:51–72. wileyonlinelibrary.com/journal/joc 1 \| INTRODUCTION is characterized by recurrent droughts (Marengo et al., 2017), and just now still recovering from a series of drought years from 2012 to 2018 (Pilz et al., 2019). This has recently led to a severe water crisis, not only in NEB, The semi-arid northeast of Brazil (NEB) is one of the most densely populated dryland regions in the world. It This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. © 2020 The Authors. International Journal of Climatology published by John Wiley & Sons Ltd on behalf of the Royal Meteorological Society. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, dis medium, provided the original work is properly cited and is not used for commercial purposes. © 2020 The Authors International Journal of Climatology published by John Wiley & Sons Ltd on behalf of the Royal Meteorologica wileyonlinelibrary.com/journal/joc Int J Climatol. 2021;41:51–72. 52 LAUX ET AL. LAUX ET AL. 52 but also the large metropolitan areas of S~ao Paulo, Belo Horizonte, and Rio de Janeiro. surface temperature (SST) anomaly patterns in the Atlan- tic, the Pacific, and to a lesser extent the Indian Ocean (Ward and Folland, 1991). Likewise, in Hastenrath (2012), drought occurrence in NEB has been linked to SST anomalies in the eastern Pacific. The El Niño Southern Oscillation (ENSO) as well as the northern tropical Atlantic region (i.e., the Tropical Atlantic SST Dipole) is influencing the location of the Intertropical Convergence Zone (ITCZ), which is known to be the main source of rain during the rainy season in this region. The NEB can be characterized by a high-rainfall vari- ability, that is, on interannual and intraannual scale, but also on decadal scale (Zhou and Lau, 2001). According to Kousky (1979), eastern coastal regions may receive even more than 2000 mm/year, while some interior valleys receive less than 400 mm/year. Particu- larly in these arid regions, the year-to-year variability is higher. They show that frontal systems, which penetrate the southern parts of the NEB, play a crucial role for December–January precipitation in the southern part, but also for fall and winter precipitation along the coast. 1 \| INTRODUCTION Often, it is distinguished also between “circulation to environment” and “environ- ment to circulation” approaches (e.g., Dayan et al., 2012). In the former, the classification is performed without consider- ation of the target variable, whereas the latter accounts for the target variable(s) during the classification. Both approaches are suitable tools in synoptic climatology, and they can be applied both to analyse the link between atmo- spheric circulation types derived from CP classifications and surface variables such as, precipitation, temperature, or discharge (e.g., Bárdossy et al., 2002; Bárdossy, 2010; Wypych et al., 2018; Bednorz et al., 2019). Such links exist even when the local scale climate is driven by mesoscale events such as convective systems, since these are condi- tional on the synoptic conditions (Goodess and Jones, 2002). Other authors established links between atmospheric conditions or Sea Surface Temperature (SST) patterns and droughts (e.g., Burgdorf et al., 2019), and dem- onstrated the usability of their classification for drought prediction. Due to the existence of such links, CP classifica- tions have been originally developed for weather forecasting. manual (subjective) and computer assisted (objective or mostly semi-objective) methods. Often, it is distinguished also between “circulation to environment” and “environ- ment to circulation” approaches (e.g., Dayan et al., 2012). In the former, the classification is performed without consider- ation of the target variable, whereas the latter accounts for the target variable(s) during the classification. Both approaches are suitable tools in synoptic climatology, and they can be applied both to analyse the link between atmo- spheric circulation types derived from CP classifications and surface variables such as, precipitation, temperature, or discharge (e.g., Bárdossy et al., 2002; Bárdossy, 2010; Wypych et al., 2018; Bednorz et al., 2019). Such links exist even when the local scale climate is driven by mesoscale events such as convective systems, since these are condi- tional on the synoptic conditions (Goodess and Jones, 2002). Other authors established links between atmospheric conditions or Sea Surface Temperature (SST) patterns and droughts (e.g., Burgdorf et al., 2019), and dem- onstrated the usability of their classification for drought prediction. Due to the existence of such links, CP classifica- tions have been originally developed for weather forecasting. Most approaches are developed for the mid-latitudes, where distinct high- and low-pressure systems are distin- guished. 1 \| INTRODUCTION A relatively large body of literature exists for Cen- tral Europe (e.g., James, 2007; Philipp et al., 2007, 2010) or the Mediterranean region (e.g., Corte-Real et al., 1995; Goodess and Palutikof, 1998; Trigo and DaCamara, 2000; Fernández-González et al., 2012). Within the framework of the European Cooperation in Science and Technology (COST) action 733 Harmonization and Applications of Weather Types Classifications for European Regions, a bunch of different objective and subjective weather type classifica- tions for Europe have been collected and provided for cli- mate science community (Philipp et al., 2007), and a classification catalogue has been derived for the European domain (Huth et al., 2008; Philipp et al., 2010). Only few studies exist for tropical and subtropical regions. Amongst them are the study of Ngarukiyimana et al. (2018) for Rwanda in East Africa, Moron et al. (2008) for the Senegal in West Africa, and Robertson et al. (2004) for the NEB. In the latter, a non-homogeneous hidden Mar- kov model was used to downscale daily precipitation occur- rence at 10 stations in NEB, using GCM simulations of seasonal-mean large-scale precipitation, obtained with his- torical sea surface temperatures prescribed globally. It was concluded that their model provides a useful tool for under- standing the statistics of rainfall occurrence at stations with respect to the large-scale atmospheric patterns, and for pro- ducing local-scale daily rainfall series for impact studies. Although GCMs are often strongly biased in prognos- tic variables such as precipitation, it is widely accepted that GCMs are able to appropriately reflect the large-scale circulation (e.g., Sunyer et al., 2015). This forms the basis for statistical downscaling of hydrometeorological target variables, nowadays, the most widely used application of CPs (e.g., Wetterhall et al., 2007; Bárdossy, 2010; Bárdossy and Pegram, 2011). Apart from dynamical downscaling by using process-based regional climate models (RCMs), in statistical downscaling the large-scale synopic information, represented by CPs is used to derive hydrometeorological variables of interest (e.g., precipita- tion, temperature, and discharge) on local scales. More- over, CP classification can be applied for model evaluation and validation, for example, to evaluate the performance of GCMs or RCMs due to their representation of the large-scale information (e.g., Dafka et al., 2018; Prein et al., 2019). 1 \| INTRODUCTION Such frontal systems lower the surface pressure at low latitudes, and thus favour the southward movement of the equatorial trough zone. Figure 1 illustrates such interannual variability by showing precipitation anoma- lies for three selected years. Heavy precipitation deficits up to 1,000 mm can be seen during the recent drought period for the years 2012 and 2014, whereas precipitation excess is exemplary shown for the year 1985. According to Costa et al. (2016), most of the drought and wet anomalies could be linked to anomalous states of El Niño Southern Oscillation (ENSO), but not neces- sarily linked to specific El Niño or La Niña events. In a later study, Costa et al. (2018) identified a multi-annual relationship between the state of the sea surface tempera- ture (SST) of the Atlantic and Pacific oceans and anoma- lous hydrological variability in NEB. Thereby, the northern Tropical Atlantic conditions were shown to play an important role in modulating the long-term variability of the hydrological response of the basins, while only extreme ENSO anomalies seemed to affect the rainy season. The high-rainfall variability on the different temporal scales poses significant challenges for water resources management in the region. Even in years with average rainfall amounts, such as in 2017 and 2018, NEB was not able to overcome its water scarcity due to the high-spatial variability of the rainfall in the region. In these years, the largest reservoirs could not be filled up by more than 10% of its capacity. Such large-scale synoptic information, represented by general circulation models (GCMs) can be used for predic- tion purposes in many water resources management appli- cations. One possibility to implement the large-scale synoptic information is to classify the atmospheric state into circulation patterns or weather types, hereinafter referred to as circulation patterns (CPs) (e.g., Fernández-González et al., 2012). In general, one can distinguish between Intraannual or seasonal precipitation variability dur- ing the wet season of the interior NEB is impacted by sea FIGURE 1 Wet precipitation anomaly of 1985 (left) and dry precipitation anomalies of 2012 (middle) and 2014 (right) over the northeast region of Brazil (kriging of observation data, 1981–2010) FIGURE 1 Wet precipitation anomaly of 1985 (left) and dry precipitation anomalies of 2012 (middle) and 2014 (right) over the northeast region of Brazil (kriging of observation data, 1981–2010) 53 LAUX ET AL. 53 manual (subjective) and computer assisted (objective or mostly semi-objective) methods. 2 \| STUDY REGION relatively high-spatiotemporal variability of precipitation (see Figure 3). The figure shows the monthly precipita- tion climatology for Três Marias, Sobradinho, Aracaju (from the South to the North). The upper SF basin, repre- sented by Três Marias, provides the highest rainfall amounts to the SF river basin, the main season lasts from October to February. The main generating rainfall system is the incursion of cold fronts, that is, the South Atlantic Convergence Zone. For the northernmost area, the lower SF basin, represented by Aracuju, the main season peaks from may to July. This region is affected by the the move- ment of the ITCZ. The middle SF basin, as depicted by Sobradinho, has an intermediate precipitation climatol- ogy. Besides the onset of the rainy season, the overall rainfall amounts decrease from the upper to the lower SF basin. During the dry season (June–August), significant amounts of rains (and thus streamflow) can be observed across the SF basin due to convective systems and pene- trating cold fronts. For this reason, it should be noted that water resources management in the SF basin requires weather and climate forecasts not only for the rainy season, but for the entire year. This need is also cor- roborated by the fact that water from the SF River is We selected the S~ao Francisco (SF) river basin to high- light the complex climatology and specific challenges with respect to water resources management in NEB, which helps to explain our setup for the classification in Section 3. The SF basin (Figure 2) covers an area of approxi- mately 630,000 km2, and has its sources in the southeast of Brazil, in the state of Minas Gerais. From there, the SF river flows approximately 2,700 km until it meets the Atlantic Ocean in the northeastern region. Starting in Minas Gerais, it crosses the states of Bahia, Pernambuco, Alagoas and Sergipe. The SF river basin partly covers also the states of Goiás and the Federal District Brasilia. The large size of SF river basin (it corresponds approximately to the size of France) and the fact that it is an interstate basin makes water resources management a particularly challenging task. 1 \| INTRODUCTION Due to the above mentioned physical link between synoptic-scale patterns such as ENSO and the hydrological situation in the NEB, the main objective of this study is to develop a computer aided circulation pattern (CP) classifica- tion approach for applications in water resources manage- ment. For this reason, it is aimed at a throughout evaluation based on different predictor variables as well as domain set- tings for the classification with respect to its ability to dis- criminate between dry and wet conditions in the NEB. Thus, it may provide the base for many applications in water resources management in the region. The paper is structured as follows: in the following Section 2, we elucidate more detailed climatic features of the study region within NEB. We selected the S~ao Fran- cisco (SF) river basin to highlight the complex climatol- ogy and the specific challenges for water resources management. In Section 3, the SANDRA CP classifica- tion method, the Markov Chain approach as well as the data for the classification and the validation used in this study are explained. Section 4 is subdivided into the description of the classification and the selected features of the classified CPs, that is, Occurrence Probability of CPs, Persistence of CPs, Transition Probability of CPs, Wetness Indices of CPs, and the CDF of CPs, followed by Section 5. Another very recent application is the identification and correction of the spatio-temporal bias structures in RCMs (Le Roux et al., 2019). The RCMs may have largely different performances depending on the season and the prevailing large-scale atmospheric circulation (Laux et al., 2011; Wetterhall et al., 2012). The inclusion of conditional infor- mation about the state of the atmospheric circulation, for example, as CP time series, obtained by classification algo- rithms may thus help to improve the bias correction. An overview of different classification algorithms, distin- guished between subjective, objective, and mixed approaches can be found in Huth et al. (2008). LAUX ET AL. LAUX ET AL. 54 2 \| STUDY REGION The CPs are finally linked to local weather conditions by calculating the wetness index and the CP-conditional CDFs, based on observation data LAUX ET AL. 55 FIGURE 4 Flow chart of applied methods and data. The applied work flow (from top to bottom) is indicated by the arrows, the main methods are given in the grey shaded boxes, that is, the 55 LAUX ET AL. 55 FIGURE 3 The monthly precipitation climatology for Três Marias, Sobradinho and Aracaju, derived from GPCP, representing the upper-, middle-, and lower-SF basin, respectively. The location of the stations can be obtained from Figure 2 FIGURE 3 The monthly precipitation climatology for Três Marias, Sobradinho and Aracaju, derived from GPCP, representing the upper-, middle-, and lower-SF basin, respectively. The location of the stations can be obtained from Figure 2 FIGURE 3 The monthly precipitation climatology for Três Marias, Sobradinho and Aracaju, derived from GPCP, representing the upper-, middle-, and lower-SF basin, respectively. The location of the stations can be obtained from Figure 2 FIGURE 3 The monthly precipitation climatology for Três FIGURE 3 The monthly precipitation climatology for Três Marias, Sobradinho and Aracaju, derived from GPCP, representing the upper-, middle-, and lower-SF basin, respectively. The location of the stations can be obtained from Figure 2 diverted to the north of the more semi-arid NEB, in order to improve water availability there. In particular, seasonal climate forecasts for the forthcoming months can help decision makers in water resources manage- ment in such semi-arid environments (e.g., Siegmund et al., 2015). 3 \| DATA AND METHODS FIGURE 4 Flow chart of applied methods and data. The applied work flow (from top to bottom) is indicated by the arrows, the main methods are given in the grey shaded boxes, that is, the SANDRA CP classification and the Markov chain approach. Both methods are applied based on ERA-Interim data. The CPs are finally linked to local weather conditions by calculating the wetness index and the CP-conditional CDFs, based on observation data FIGURE 4 Flow chart of applied methods and data. The FIGURE 4 Flow chart of applied methods and data. The applied work flow (from top to bottom) is indicated by the arrows, the main methods are given in the grey shaded boxes, that is, the SANDRA CP classification and the Markov chain approach. Both methods are applied based on ERA-Interim data. The CPs are finally linked to local weather conditions by calculating the wetness index and the CP-conditional CDFs, based on observation data Figure 4 provides a work flow of the procedure applied in this study. The work flow (from top to bottom) is indi- cated by the arrows, the main methods are given in the grey shaded boxes, that is, (i) the SANDRA CP Classifica- tion and (ii) the Markov Chain approach. Both methods are applied based on ERA-Interim data. The CPs are finally linked to local weather conditions (see Table 1) by calculating the wetness index and the CP-conditional CDFs, based on observation data. In the following the data are described first, followed by the methods. used cycle 31r2 of ECMWF's Integrated Forecast System (IFS). Data are provided on a reduced Gaussian grid with approximately uniform horizontal 79 km spacing (approximately 0.75 of latitude and longitude), at 60 vertical levels (with top level at 0.1 hPa). A detailed description can be found in Dee et al. (2011). ERA- Interim is a reanalysis product, frequently used for validation purposes and known to be skillful in rep- resenting atmospheric processes (Lin et al., 2014). Note that we did not use the more recent high-resolution (approximately 31 km) ERA5 product by intention, since the higher resolution would have tremendously increased the computational demands without expected significantly improved results. This is due to the fact that the spatial variability in the upper-level atmo- spheric fields (as usually applied for CP classifications) is relatively low. 2 \| STUDY REGION The precipitation regime in the SF basin is complex as it is modulated by different meteorological systems, such as the South Atlantic Convergence Zone in the southernmost part of the basin and ITCZ, leading to a FIGURE 2 The S~ao Francisco (SF) river basin in NEB. The figure is obtained from https://de.m. wikipedia.org/wiki/Datei:S~ao_ Francisco_basin_map.png, released under the licence CC BY-SA 4.0 FIGURE 2 The S~ao Francisco (SF) river basin in NEB. The figure is obtained from https://de.m. wikipedia.org/wiki/Datei:S~ao_ Francisco_basin_map.png, released under the licence CC BY-SA 4.0 FIGURE 2 The S~ao Francisco (SF) river basin in NEB. The figure is obtained from https://de.m. wikipedia.org/wiki/Datei:S~ao_ Francisco_basin_map.png, released under the licence CC BY-SA 4.0 FIGURE 4 Flow chart of applied methods and data. The applied work flow (from top to bottom) is indicated by the arrows, the main methods are given in the grey shaded boxes, that is, the SANDRA CP classification and the Markov chain approach. Both methods are applied based on ERA-Interim data. The CPs are finally linked to local weather conditions by calculating the wetness index and the CP-conditional CDFs, based on observation data 55 diverted to the north of the more semi-arid NEB, in order to improve water availability there. In particular, seasonal climate forecasts for the forthcoming months can help decision makers in water resources manage- ment in such semi-arid environments (e.g., Siegmund et al., 2015). 3 \| DATA AND METHODS Figure 4 provides a work flow of the procedure applied in this study. The work flow (from top to bottom) is indi- cated by the arrows, the main methods are given in the grey shaded boxes, that is, (i) the SANDRA CP Classifica- tion and (ii) the Markov Chain approach. Both methods are applied based on ERA-Interim data The CPs are FIGURE 3 The monthly precipitation climatology for Três Marias, Sobradinho and Aracaju, derived from GPCP, representing the upper-, middle-, and lower-SF basin, respectively. The location of the stations can be obtained from Figure 2 FIGURE 4 Flow chart of applied methods and data. The applied work flow (from top to bottom) is indicated by the arrows, the main methods are given in the grey shaded boxes, that is, the SANDRA CP classification and the Markov chain approach. Both methods are applied based on ERA-Interim data. 3.1.1 \| ERA-Interim reanalyses The ERA-Interim project, initiated in 2006, aimed to provide a bridge between ECMWF's previous reanalysis, that is, ERA-40 (1957–2002), and the next-generation extended reanalysis envisaged at ECMWF (ERA5). Improvements compared to ERA-40 mainly comprised the representation of the hydrological cycle, the quality of the stratospheric circulation, and the handling of biases and changes in the observing system. The ERA- Interim atmospheric model and reanalysis system has LAUX ET AL. LAUX ET AL. 56 56 TABLE 1 Station IDs, coordinates, and missing values (%) Station ID Name Lat Lon Missing values 101 JACOBINA −11.2 −40.5 7.7 102 REMANSO −9.6 −42.1 3.8 110 JURAMENTO −16.8 −43.7 12.2 114 IBIRITE −20.0 −44.0 13.9 115 SAO MATEUS −18.7 −39.9 9.5 120 JOAO PINHEIRO −17.7 −46.2 11.2 127 ITABAIANINHA −11.1 −37.8 17.0 135 BOM JESUS DO PIAUI −9.1 −44.1 14.9 161 BOM JESUS DA LAPA −13.3 −43.4 15.3 166 PAULISTANA −8.1 −41.1 14.6 171 MONTE AZUL −15.2 −42.9 12.4 179 POSSE −14.1 −46.4 8.8 182 BARRA −11.1 −43.2 10.5 183 CAETITE −14.1 −42.5 17.4 184 LENCOIS −12.6 −41.4 15.1 202 PAO DE ACUCAR −9.8 −37.4 17.3 203 DIAMANTINA −18.2 −43.6 12.8 207 PESQUEIRA −8.4 −36.8 9.3 244 PROPRIA −10.2 −36.8 8.7 248 MONTES CLAROS −16.7 −43.8 20.0 255 VALE DO GURGUEIA CRISTIANO CASTRO −8.4 −43.7 13.0 256 JANAUBA −15.8 −43.3 8.7 265 PIRAPORA −17.4 −44.9 18.2 290 CORRENTINA −13.3 −44.6 7.6 297 JANUARIA −15.4 −44.0 6.4 309 ITAMARANDIBA −17.9 −42.9 7.8 334 UNAI −16.4 −46.9 7.5 364 ITUACU −13.8 −41.3 14.1 467 BURITIS −15.5 −46.4 17.7 744 PATROCINIO −19.0 −47.0 14.5 TABLE 1 Station IDs, coordinates, and missing values (%) other meteorological state agencies such as Meteorology and Water Resource Center of Ceara State (FUNCEME) in northeastern Brazil). FUNCEME has collected and provided the data for this study, and has been responsible to quality check and correct the data. The predictor variables used for the CP classification (see Table 2) are daily ERA-Interim data at 12:00 UTC. The number of grid cells used in the classification experi- ments depends on the domain size, for the final classifi- cation setup in the domain 10N–30S and 60–20W, 54 × 54 grid cells have been used. From the provided observation data, a small subset of 30 stations within the domain 8–21S and 47–36W have been pre-selected for evaluating the CP classification on local scale. 3.1.1 \| ERA-Interim reanalyses The selection has been made based on the fraction of allowed missing values not exceeding 20% during the period 1980 to 2016. The location of the selected stations and the fraction of missing values (%) is shown in Figure 5. The exact coordinates and station names are given in Table 1. 3.1.2 \| Observation data The precipitation data used in this study consists of mete- orological stations from INMET (National Institute for Meteorology), ANA (National Water Agency) and various LAUX ET AL. TABLE 2 Explained cluster variance (ECV) [0, …, 1] of the performed SANDRA classification experiments using the domain 10N–30S and 60–20W and geopotential height (GP) in 1,000, 700, 500, and 300 hPa), mean sea level pressure (MSLP), and horizontal wind component (UWND) in 700 hPa (upper part) Number CPs 5 6 7 8 9 10 11 12 13 14 15 16 Predictor variables GP1,000 0.69 GP700 0.61 GP500 0.61 GP300 0.64 GP1,000 + GP700 0.60 GP1,000 + GP500 0.54 GP1,000 + GP300 0.57 (MSLP + UWND700) 0.65 0.67 0.68 0.69 0.70 0.71 0.72 (GP1,000 + UWND700) 0.61 0.63 0.65 0.67 0.68 0.69 0.70 0.71 0.72 0.72 0.73 0.73 (GP1,000 + UWND700) 10N–30S and 60–10W 0.67 (GP1,000 + UWND700) 10N–30S and 60–0W 0.65 (GP1,000 + UWND700) 10N–30S and 60W–10E 0.65 (GP1,000 + UWND700) 15N–35S and 65–15W 0.59 (GP1,000 + UWND700) 10N – 40S and 60–10W 0.59 (GP1,000 + UWND700) 10N–40S and 70–10W 0.56 Note: Domain experiments by using GP in 1,000 hPa and UWND in 700 hPa with different domain extensions (lower part). TABLE 2 Explained cluster variance (ECV) [0, …, 1] of the performed SANDRA classification experiments using the domain 10N–30S and 60–20W and geopotential height (GP) in 1,000, 700, 500, and 300 hPa), mean sea level pressure (MSLP), and horizontal wind component (UWND) in 700 hPa (upper part) ts by using GP in 1,000 hPa and UWND in 700 hPa with different domain extensions (lower part). Note: Domain experiments by using GP in 1,000 hPa and UWND in 700 hPa with different domain extensions (lower part). performance of the simulated annealing algorithm com- pared to the conventional k-means classification (e.g., Lutz et al., 2012). The latter has no strategy to avoid of getting stuck in local optima during the optimization procedure. Simulated annealing has been designed to bet- ter approximate the global optimum, that is, it does not converge to a local optimum which cannot be left any- more during the optimization procedure (Philipp et al., 2010; Bárdossy et al., 2015). The method allows data objects to be assigned to a wrong cluster during the iteration process, meaning that the object is not necessar- ily assigned to its closest cluster centroid. 3.1.2 \| Observation data Each object can be classified into a wrong cluster, if the acceptance proba- bility P is larger than a random number between 0 and 1, where P is given by: In semi-arid NEB, improved knowledge of the expected precipitation amounts a few months ahead may help water managers for decision support. CP classification can poten- tially contribute in various ways: it can be used as a tool for statistical downscaling of GCMs, or as a CP-conditional bias correction tool for GCMs and RCMs on various time scales (short-, seasonal-, and long-term scales). The SANDRA CP classification approach can be used therefore and is described in the following subsection. Next, the setup for the classification for the study region is given, followed by a description of the data used in this study. Note that the period for the circulation pattern classi- fication as well as for the analyses of the results is split into a calibration period (1980–2009) and an indepen- dent, but shorter validation period (2010–2016). P=exp EDold −EDnew T , ð1Þ ð1Þ 3.2.1 \| SANDRA circulation pattern classification where where ED= ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ x1 −x2 k k2 q : ð2Þ ð2Þ Simulated ANnealing and Diversified RAndomization clustering (SANDRA) cluster analysis, developed by Philipp et al. (2007), is applied for the NEB for the first time. It has been selected due to the improved All data objects x are normalized before the Euclidean distance ED is calculated. EDold is the ED between the 8 LAUX ET AL. LAUX ET AL. 58 FIGURE 5 Map showing the station ID numbers and the number of missing values (%) for each station. The exact coordinates and number of missing values can be obtained from Table 1 FIGURE 5 Map showing the station ID numbers and the number of missing values (%) for each station. The exact coordinates and number of missing values can be obtained from Table 1 data object and the old cluster, EDnew is the ED to the potentially new cluster, T is a control parameter (the annealing temperature) that is reduced after each itera- tion step by a constant factor a, the so-called cooling rate: run with the best ECV is chosen as final result of the SANDRA clustering. The final classification setup is obtained by evaluating the performance of multiple runs with different predictor variables (i.e., predictor screening), different cluster num- bers, and different domain locations (Table 2, upper part). An 11-cluster-solution of both, the horizontal wind at 700 hPa (UWND700) and the geopotential at 1,000 hPa (GP1,000) has been selected as suitable solution. An 11-cluster-solution meets the predefined threshold of 0.7 (corresponding to 70%) of the ECV while keeping the number of classes relatively small. A too large number of classes is expected to reduce the sample size for certain CPs and would thus hinder to derive robust results in the CP-conditional analyses. The ECV in relation to an increasing number of classes is describing a logarithmic function. An increase of the cluster numbers from 11 to 19 would result in an increase of ECV from 0.7 to 0.75 only (Figure 6). Note that a classification based on the mean sea level pressure (MSLP) combined with the UWND700 would result in very similar performances. Ti+1 =a×Ti, ð3Þ ð3Þ where i is the iteration step. The initial value of T is auto- matically chosen for each run by the used clustering soft- ware and its typical value is around 108–109. 3.2.1 \| SANDRA circulation pattern classification A constant value of 0.99 is used for the cooling rate a for all iteration steps. With the above algorithm and setting of values, the initial iteration steps of a single classification run are often characterized by wrong assignments due to the high-annealing temperature value. As annealing temper- ature decreases in the following iteration steps, the prob- ability of wrong assignments decreases accordingly. During the final iteration steps reassignments mostly take place if the resulting distance between object and cluster centroid is smaller than the current distance. The process of reassignments repeats until no further changes, neither wrong nor right assignments, are taking place. The limit of iterations is infinite, thus the process repeats until convergence is reached. Then, the algorithm calculates the explained cluster variance (ECV) of the current clustering for comparison of different runs. The classification has been tested using different domain settings for the two predictand variables GP1,000 and UWND700 and a 10-cluster solution (see Table 2, lower part). Based on the performed domain settings, the domain between 10N–30S and 60–20W is found to deliver good results. The classification is repeated a predefined number of times with randomized starting partitions of the data (called diversified randomization, here: 1,000 times) to overcome issues of the simulated annealing algorithm with respect to the optimization starting position. The In SANDRA, the number of clusters used for the clas- sification has to be predefined. The selection of the num- bers of clusters depends on different criteria and can be understood as a trade-off between the interpretability of LAUX ET AL. 59 3.2.2 \| Markov chains the cluster centroids and the overall explained cluster variance (ECV) of the predictor field(s). The ECV is defined as: The occurrence and sequence of CPs can be modelled as a Markov chain, which is a stochastic model describing the sequence of possible events in which the probability of each event depends only on the state attained in the previous event. A Markov Chain can be used for describ- ing systems that follow a chain of linked events such as, a sequence of rainfall events or, as used here, a sequence of CPs. A Markov chain consists of a finite set of states (e.g., dry or wet, different CPs), whose transitions, that is, the change from a certain CP state into another (e.g., from CP1 to CP2) occurring with certain (transition) probabilities. Markov chains are frequently applied in modelling and statistical downscaling precipitation (e.g., Robertson and Smyth, 2003; Laux et al., 2009; Greene et al., 2011). In this study, occurrence probabili- ties of the CPs are calculated. Moreover, their persis- tences and transition probabilities and their transition pathways are derived. Graphically, a Markov chain can be represented by a directed graph, which illustrates tran- sition probabilities and pathways. The states are repre- sented by vertices, and transitions are described by arrows. The Markov chain is strongly connected if there is a directed path from each vertex to every other vertex (e.g., Gansner et al., 1993). ECV=1−WSS TSS , ð4Þ ð4Þ where WSS is the so-called within-cluster sum of square, and TSS is the total sum of squares. WSS is calculated as follows: WSS= X k i=1 X x∈Ci x−zi k k2, ð5Þ ð5Þ where x denotes all data objects who belong to the Clus- ter Ci, zi refers to the ith corresponding cluster centroid, and k denotes the number of clusters. More technical information about the SANDRA classification is given in Philipp et al. (2007, 2010) and Hoffmann and Schlünzen (2013). A small number of clusters increase the interpretabil- ity while reducing the fraction of the ECV. Higher cluster numbers may also increase the possibility to better link the prevailing circulation with unusual conditions, such as dry or wet conditions. 3.2.2 \| Markov chains FIGURE 6 Explained variance of joint horizontal wind component at 700 hPa and geopotential (GP) at 1,000 hPa as a function of the cluster number FIGURE 6 Explained variance of joint horizontal wind component at 700 hPa and geopotential (GP) at 1,000 hPa as a function of the cluster number LAUX ET AL. LAUX ET AL. 60 3.2.3 \| Linking CPs to precipitation is representing normal, that is, approximately average conditions. In order to study the impact of the CP on the weather state (i.e., wet or dry), the wetness index Iwet (Bárdossy, 2010) is applied. It is defined as follows: In order to study the impact of the CP on the weather state (i.e., wet or dry), the wetness index Iwet (Bárdossy, 2010) is applied. It is defined as follows: Another possibility to link the CPs to the weather state is to analyse the CP-conditional cumulative density functions (CDFs). This indicates whether or not the dif- ferent CPs have discriminative power to describe the weather state, and thus can be applied in applications such as bias correction or statistical downscaling. Iweti CP ð Þ= zd,i CP ð Þ zd,i , ð6Þ ð6Þ where zd,i(CP) is the normalized daily (d) precipitation amount of a certain CP at station i and zd,i is the overall mean daily precipitation amount at station i, irrespectively of the prevailing CP. The wetness index thus offers one possibility to compare the clusters with respect to their precipitation amount (wetness) between different locations (stations). A value higher than unity of any given CP means that the CP is much wetter than the average precipitation, and a value significantly smaller than unity that the CP is much drier than the average precipitation at this sta- tion. In turn, a value around unity means that the CP 4 \| RESULTS In the following, the derived CPs are grouped according to their major atmospheric conditions. The CP are then described according to their major characteristics and their relation to wet and dry states in NEB, as analysed based on the wetness index Iwet. Then, the occurrence fre- quencies, the persistences, and the transition pathways and -probabilities of the CPs are analysed. Finally, the value of the classification is discussed with respect to the development of a CP-conditional bias correction. FIGURE 7 Cluster centroids of geopotential (GP) at 1,000 hPa (m2s−2) of the selected 11-cluster- solution FIGURE 7 Cluster centroids of geopotential (GP) at 1,000 hPa (m2s−2) of the selected 11-cluster- solution 61 LAUX ET AL. 61 4.1 \| Circulation patterns (CPs) of the cluster solution maritime High and continental ridge (group B), and a CP with a High pressure bridge (group C). The clusters can then be further subdivided according to the magnitude/ degree of the derived groups (Table 3) as well as in com- bination with UWND700, as shown in Figure 8. Thus, in this study, we combined atmospheric information from lower (1,000 hPa) and middle troposphere (700 hPa), which is often seen as more credible for, for example, determining convective events, caused by high reaching clouds (e.g., Dayan et al., 2012). Since the grouping is The classification scheme is based on the dominant cen- ters of GP1,000 within the domain (Figure 7), which are virtually existing only in the southern, that is, the sub- tropical part of the domain. The CPs can be grouped into three major groups (A, B, C), corresponding to a CP with a clear contrast between a maritime High- and a conti- nental Low-pressure system (group A), a CP with a TABLE 3 Unique character combination consisting of a 6-character string to label and characterize the derived CPs based on the GP1,000 field Position in name string Abbreviations 1. & 4. m: Maritime c: Continental b: Bridge 2. & 5. H: High L: Low E: Extension 3. & 6. w: Weak m: Moderate s: Strong Note: Positions 1 & 4 define the position of the Centre of action, 2 & 5 the group or type of the Centre of action, and 3 & 6 their degree/mag- nitude. As an example, mHscLm means maritime High strong, continental Low moderate. TABLE 3 Unique character combination consisting of a 6-character string to label and characterize the derived CPs based on the GP1,000 field Position in name string Abbreviations 1. & 4. m: Maritime c: Continental b: Bridge 2. & 5. H: High L: Low E: Extension 3. & 6. w: Weak m: Moderate s: Strong Note: Positions 1 & 4 define the position of the Centre of action, 2 & 5 the group or type of the Centre of action, and 3 & 6 their degree/mag- nitude. As an example, mHscLm means maritime High strong, continental Low moderate. 4.1 \| Circulation patterns (CPs) of the cluster solution acter combination consisting of a 6-character string to label and characterize the derived CPs based on the TABLE 3 Unique character combination consisting of a 6-character string to label and characterize the GP fi ld Note: Positions 1 & 4 define the position of the Centre of action, 2 & 5 the group or type of the Centre of action, and 3 & 6 their degree/mag- nitude. As an example, mHscLm means maritime High strong, continental Low moderate. Note: Positions 1 & 4 define the position of the Centre of action, 2 & 5 the group or type of the Centre of action, and 3 & 6 their degree/mag- nitude. As an example, mHscLm means maritime High strong, continental Low moderate. FIGURE 8 Cluster centroids of horizontal wind speed at 700 hPa (ms−1) of the selected 11-cluster- solution FIGURE 8 Cluster centroids of horizontal wind speed at 700 hPa (ms−1) of the selected 11-cluster- solution FIGURE 8 Cluster centroids of horizontal wind speed at 700 hPa (ms−1) of the selected 11-cluster- solution 62 LAUX ET AL. 62 4.1.1 \| Group a: CP With a clear contrast between a maritime high- and a continental low-pressure system There is a moderate westerly flow in front of the south Brazil- ian coast and over southern Brazil as well as a moder- ate easterly flow over the Amazon and the Equatorial Atlantic. Similarly to CP3, CP5 indicates wet condi- tions (WIs >> 1, see Figure 9e). • CP5 (mHwcLs, maritime High weak, continental Low strong): CP5 (Figures 7e and 8e) is characterized by a subtropical High shifted far into the South Atlantic and a strong continental Low over Southwest Brazil as well as low pressure in front of the coast of southern Brazil. In general, the magnitude of the GP1,000 fields are low in magnitude and only poorly differentiated over the Amazon and the Equatorial Atlantic. There is a moderate westerly flow in front of the south Brazil- ian coast and over southern Brazil as well as a moder- ate easterly flow over the Amazon and the Equatorial Atlantic. Similarly to CP3, CP5 indicates wet condi- tions (WIs >> 1, see Figure 9e). • CP3 (mHmcLs, maritime High moderate, continental Low strong): CP3 (Figures 7c and 8c) is characterized by a relatively weak subtropical High over the South Atlantic and a strong continental Low over Southwest Brazil. Like for CP1, only marginally differentiated GP1,000 fields prevail over the Amazon and the equato- rial Atlantic. Overall, the horizontal wind component is moderate and the westerly flows are shifted to the Southwest of the continental region, the easterly flows prevail over the Amazon and the equatorial Atlantic. CP3 indicates rather wet conditions in NEB (WIs > 1), see Figure 9c. • CP3 (mHmcLs, maritime High moderate, continental Low strong): CP3 (Figures 7c and 8c) is characterized by a relatively weak subtropical High over the South Atlantic and a strong continental Low over Southwest Brazil. Like for CP1, only marginally differentiated GP1,000 fields prevail over the Amazon and the equato- rial Atlantic. Overall, the horizontal wind component is moderate and the westerly flows are shifted to the Southwest of the continental region, the easterly flows prevail over the Amazon and the equatorial Atlantic. CP3 indicates rather wet conditions in NEB (WIs > 1), see Figure 9c. • CP6 (mHmcLw, maritime High moderate, continental Low weak): CP6 (Figures 7f and 8f) is characterized by a moderately pronounced subtropical High shifted to the South Atlantic and a weak continental Low over Southwest Brazil. Note: Frequencies >20% are bolded. 4.1.1 \| Group a: CP With a clear contrast between a maritime high- and a continental low-pressure system based on the atmospheric conditions and does not include the local weather situation, it cannot be fully avoided that wet and dry conditions can occur within a certain group. Subsequent calculations are done based on the individual 11 CPs. • CP1 (mHscLm, maritime High strong, continental Low moderate): CP1 (Figures 7a and 8a) is characterized by a strong subtropical High over the South Atlantic, which extends to the coastal regions of Central Brazil In the following, the assignment of the 11 CPs to the three groups is described and each CP is briefly characterized: FIGURE 9 Annual consideration of the wetness index (WI) for 30 selected stations based on the selected 11-cluster-solution FIGURE 9 Annual consideration of the wetness index (WI) for 30 selected stations based on the selected 11-cluster-solution 63 LAUX ET AL. 63 • CP5 (mHwcLs, maritime High weak, continental Low strong): CP5 (Figures 7e and 8e) is characterized by a subtropical High shifted far into the South Atlantic and a strong continental Low over Southwest Brazil as well as low pressure in front of the coast of southern Brazil. In general, the magnitude of the GP1,000 fields are low in magnitude and only poorly differentiated over the Amazon and the Equatorial Atlantic. There is a moderate westerly flow in front of the south Brazil- ian coast and over southern Brazil as well as a moder- ate easterly flow over the Amazon and the Equatorial Atlantic. Similarly to CP3, CP5 indicates wet condi- tions (WIs >> 1, see Figure 9e). and a continental Low over Southwest Brazil. Only marginally differentiated GP1,000 fields prevail over the Amazon and the equatorial Atlantic. The maximum westerly flow of the UWND700 is in the coastal region of South Brazil and the maximum easterly flow is in the coastal region of the Amazon. This CP is linked with wetness indices WIs ≈1 or smaller in NEB, which leads to rather dry to average rainfall amounts (Figure 9a). • CP5 (mHwcLs, maritime High weak, continental Low strong): CP5 (Figures 7e and 8e) is characterized by a subtropical High shifted far into the South Atlantic and a strong continental Low over Southwest Brazil as well as low pressure in front of the coast of southern Brazil. In general, the magnitude of the GP1,000 fields are low in magnitude and only poorly differentiated over the Amazon and the Equatorial Atlantic. Note: Frequencies >20% are bolded. TABLE 4 Occurrence frequencies of CPs during the year of the selected 11-cluster-solution for the calibration period 1980–2009 and the validation period 2010–2016 (in brackets) 4.1.2 \| Group B: CP With a maritime high and a continental ridge • CP2 (mHwcLw), maritime High weak, continental Low weak: CP2 (Figures 7b and 8b) CP2 shows WIs >> 1 and leads to wet conditions in NEB (Figure 9b). • CP2 (mHwcLw), maritime High weak, continental Low weak: CP2 (Figures 7b and 8b) CP2 shows WIs >> 1 and leads to wet conditions in NEB (Figure 9b). • CP7 (mHscEs, maritime High strong, continental Exten- sion strong: CP7 (Figures 7g and 8g) is characterized by a pronounced subtropical High over the South Atlantic extending to continental continental South Brazil. The magnitude of the GP1,000 field is high, but poorly differentiated over the Amazon and the Equatorial Atlantic. The westerly flow is weak and shifted to the South. The easterly flow has a pronounced maximum over the Amazon. This CP, in general, can be linked to dry conditions (Figure 9g). • CP10 (bHs, bridge High strong): CP10 (Figures 7j and 8j) can be described by a strong high-pressure bridge with its two maximums over the South Atlantic and TABLE 5 Average persistence (days) of the CPs for the selected 11-cluster-solution for the calibration period 1980–2009 and the validation period 2010–2016 (values for the dry and the wet season are in brackets) • CP8: mHmcEm, maritime High moderate, continental Extension moderate: CP8 (Figures 7h and 8h) capti- vates with a moderately pronounced subtropical exten- ding to continental continental South Brazil. The magnitude of the GP1,000 is relatively high, but only poorly differentiated over the Amazon, the Equatorial Atlantic and Southwest Brazil. CP8 is linked to normal (average) conditions in NEB (WIs ≈1), see Figure 9h. CP Calibration (1980–2009) Validation (2010–2016) (wet, dry) (wet, dry) CP1 1.9 2.1 (1.7, 2.0) (2.1 2.1) CP2 1.9 2.0 (1.9, 1.7) (2.0, 1.9) CP3 2.1 2.2 (2.1, 1.7) (2.3, 1.7) CP4 1.6 1.8 (1.3, 1.6) (1.4, 1.8) CP5 2.6 2.9 (2.7, 1.8) (3.0, 2.0) CP6 1.7 1.9 (1.9, 1.4) (2.1, 1.4) CP7 2.8 2.6 (−, 2.7) (−, 2.6) CP8 1.8 1.8 (1.6, 1.8) (1.8, 1.7) CP9 2.4 2.2 (1.5, 2.4) (1.3, 2.2) CP10 2.0 2.0 (1.3, 2.0) (1.0, 2.0) CP11 2.0 1.9 (2.1, 1.6) (2.0, 1.6) • CP9: mHscEm, maritime High strong, continental Extension moderate: CP9 (Figures 7i and 8i) is charac- terized by a pronounced subtropical High with the maximum over the South Atlantic and a high-pressure ridge over Central Brazil. 4.1.3 \| Group C: CP with a high-pressure bridge and a weak maximum of the easterly flow prevails in the region of the Amazonian coast. CP6 shows WIs > 1, and thus rather wet conditions in NEB (Figure 9f). • CP4 (bHw, bridge High weak): CP4 (Figures 7d and 8d) is characterized by a moderate subtropical High over the South Atlantic and a pronounced continental High over Southwest Brazil. There is a tendency for a high- pressure bridge formation over the coast of Central Brazil and a lower GP1,000 in the coastal region of South Brazil. Overall, only moderate and poorly differ- entiated GP1,000 fields over the Amazon and Equatorial Atlantic prevail. The maximum of the westerly flows is shifted to the South Atlantic and the easterly flow is moderate and has its maximum over the eastern equa- torial Atlantic. CP4 can be linked with dry conditions (Figure 9d). 4.1.1 \| Group a: CP With a clear contrast between a maritime high- and a continental low-pressure system The GP1,000 fields are low in magni- tude and only poorly differentiated over the Amazon and the Equatorial Atlantic. The maximum of the westerly flow is in front of the coast of southern Brazil, TABLE 4 Occurrence frequencies of CPs during the year of the selected 11-cluster-solution for the calibration period 1980–2009 and the validation period 2010–2016 (in brackets) TABLE 4 Occurrence frequencies of CPs during the year of the selected 11-cluster-solution for the calibration period 1980–2009 and the validation period 2010–2016 (in brackets) J F M A M J J A S O N D CP1 4.4 5.7 3.2 4.7 8.5 13.3 9.9 14.6 20.7 23.8 11.4 4.5 (9.7) (7.7) (5.9) (5.3) (6.5) (7.8) (8.3) (9.3) (11.2) (12.8) (12.7) (12.2) CP2 9.8 13.3 25.4 21.6 11.8 0.6 0.2 0.1 2.1 6.6 9.6 11.8 (7.8) (8.4) (11.7) (14.4) (13.0) (11.3) (9.6) (8.4) (7.7) (7.4) (8.2) (8.1) CP3 24.6 18.4 12.9 10.4 4.8 0.3 0.6 0.8 2.0 13.8 27.7 30.3 (26.3) (31.6) (25.3) (21.5) (17.8) (15.2) (13.1) (11.5) (10.4) (10.6) (12.0) (13.5) CP4 0.3 2.0 1.2 5.6 19.2 12.8 8.2 8.4 13.0 5.2 1.8 0.1 (0.5) (0.5) (1.7) (3.3) (7.2) (7.6) (7.7) (7.8) (8.0) (7.9) (7.4) (6.8) CP5 24.9 15.3 14.5 5.9 2.3 0.4 0.0 0.1 1.0 5.3 17.1 24.5 (14.7) (12.8) (11.6) (9.1) (7.3) (6.1) (5.2) (4.5) (4.1) (4.5) (5.6) (7.6) CP6 22.4 21.2 13.2 8.9 5.3 3.8 4.0 2.3 5.6 10.6 12.2 16.8 (25.3) (19.8) (19.1) (15.9) (13.4) (12.0) (10.5) (9.3) (8.8) (8.8) (8.6) (9.5) CP7 0.0 0.0 0.0 0.0 3.0 11.0 21.9 17.3 7.4 0.8 0.0 0.0 (0.0) (0.0) (0.0) (0.0) (0.7) (3.2) (6.1) (7.8) (8.1) (7.2) (6.6) (6.0) CP8 1.2 2.2 2.8 9.8 20.4 19.0 11.0 10.2 16.8 13.0 3.0 1.5 (1.4) (0.7) (2.8) (4.4) (8.3) (9.7) (9.5) (9.4) (9.9) (10.1) (9.5) (8.8) CP9 0.1 0.0 0.1 0.3 5.0 19.1 23.4 30.4 18.7 7.1 0.9 0.2 (0.5) (0.2) (0.3) (0.2) (0.8) (2.9) (6.8) (9.5) (10.8) (10.4) (9.6) (8.8) CP10 0.0 0.0 0.0 0.9 4.1 17.0 20.2 14.9 7.3 1.2 0.1 0.0 (0.0) (0.0) (0.0) (0.0) (0.4) (3.2) (5.1) (6.8) (6.7) (6.1) (5.6) (5.1) CP11 12.3 21.8 26.7 32.0 15.5 2.7 0.5 0.9 5.4 12.8 16.2 10.2 (13.8) (18.3) (21.5) (25.8) (24.6) (21.1) (18.1) (15.9) (14.3) (14.2) (14.2) (13.6) Note: Frequencies >20% are bolded. LAUX ET AL. 64 4.2.1 \| Occurrence probability of CPs In the 11-cluster-solution, it is found that the occurrence of certain CPs heavily depends on the time of the year (Table 4). While some of the CPs predominantly occur during the rainy season (CP2, CP3, CP5, CP6, CP11), others like CP1, CP4, CP7, CP8, CP9, CP10 predomi- nantly occur during the dry season. CP7, CP9, and CP10 are typical dry season patterns and occur nearly 4.2 \| Seasonal consideration of selected features of the CPs Overall, the mean persistence of the classified CPs is about 2.1 days with a standard deviation of 0.4 days for both, the calibration and the validation period (Table 5). This indicates that the obtained results are robust for independent periods. In semi-arid regions such as the NEB with a distinct dry and rainy season during the year, one would expect a cer- tain level of discriminative power of the classification based on a seasonal consideration. The applied classifica- tion is thus being separately analysed for the rainy season from November to April, and the dry season from May to October by considering the occurrence probability, the persistence as well as the spatial distribution of the wet- ness index: However, the persistences vary for the dry and the wet season. Average over all CPs, the differences of the mean persistences between dry and wet season is about 0.5 days. This is true for both, the calibration and the val- idation period. However, there are large discrepancies for different CPs. While the differences can be approximately 0.9 days (for calibration period, and even 1.0 day for the validation period) for CP5 and CP9, only small differ- ences can be found for CP2 and CP8 (0.2 days for calibra- tion, 0.1 days for validation). 4.1.2 \| Group B: CP With a maritime high and a continental ridge In general, the GP1,000 is rela- tively high, but poorly differentiated over the Amazon, the Equatorial Atlantic and Southwest Brazil. The westerly flows are moderate with their maximums shifted to Southwest Brazil. The easterly flows are pro- nounced and have their maximums over the Amazon. CP9 is linked with anomalous dry conditions (WIs << 1, see (Figure 9i). • CP11: mHwcEw, maritime High weak, continental Extension weak: CP11 (Figures 7k and 8k) shows a sub- tropical High over the South Atlantic, extended to con- tinental southern Brazil. In general, the GP1,000 fields shows low values and is only poorly differentiated over the Amazon, the Equatorial Atlantic and Southwest Brazil. The westerly flow is weak only and the maxi- mum is shifted to the South Atlantic. The easterly flow is moderate with its maximum shifted to the Equato- rial Atlantic. CP11 brings wet conditions to NEB (Figure 9k). LAUX ET AL. 65 Southwest Brazil. The GP1,000 has relatively high values, but is poorly differentiated over the Amazon and the Equatorial Atlantic. The westerly flows are strong and have their maximum in front of the coast of South Brazil. The easterly flow is also strong with its maximum over the Amazon. For most of the stations, CP10 is linked with weakly dry conditions (Figure 9j). exclusively during the dry season. CP5 virtually does not occur during the dry season. This indicates that the iden- tified cluster solution based on the atmospheric fields is potentially able to discriminate between dry and wet states in NEB. 4.2.3 Based on Markov chains, the directed graph of the CPs of the 11-cluster-solution is calculated using the transition matrices (Figure 10). This figure represents the state changes from one CP to another for the whole year as FIGURE 10 Directed graph of the selected 11-cluster-solution w/o discrimination between wet and dry season. The arrows represent the transitions from a certain CP to another, and the colours of the arrows represent the corresponding transition probabilities of o ry g FIGURE 10 Directed graph of the selected 11-cluster-solution w/o discrimination between wet and dry season. The arrows represent the transitions from a certain CP to another, and the colours of the arrows represent the corresponding transition probabilities FIGURE 10 Directed graph of the selected 11-cluster-solution w/o discrimination between wet and dry season. The arrows represent the transitions from a certain CP to another, and the colours of the arrows represent the corresponding transition probabilities LAUX ET AL. LAUX ET AL. 66 66 well as the corresponding transition probabilities, coded as colours of the arrows. It can be seen that the graph is strongly connected, that is, most of the states (CPs) are connected to each other. On the other hand, it can be seen that some of the transition probabilities from some CPs to others are increased. This includes, for example, the transitions from CP4 to CP8, from CP7 to CP9, or from CP10 to CP7. This goes along with physically plausi- ble transitions, that is, sequences from one CP to another. The transitions from CP4 to CP8 and CP10 to CP7 are essentially comparable shifts of patterns. In both cases a high-pressure bridge pattern transitions into a single but large high-pressure field pattern. The main difference between these transitions is the intensity of the starting high-pressure bridge and the resulting single high- pressure field. CP4 is essentially a less intense CP10 and CP8 is a less intense CP7, so the CP4 to CP8 transition can simply be considered as equal to the CP10 to CP7 transition under less intense pressure fields. The CP7 to CP9 transition reflects a change from the strongest single FIGURE 11 Directed graph of the selected 11-cluster-solution for the wet (top) and the dry season (bottom). The arrows represent the transitions from a certain CP to another, and the colours of the arrows represent the corresponding transition probabilities FIGURE the selected the wet (top) (bottom). 4.2.3 CP7 does not occur and CP10 shows a highly vari- able pattern, which is related to the very small number of 4.2.3 Th transitions fr another, and arrows repre transition pr FIGURE 11 Directed graph of the selected 11-cluster-solution for the wet (top) and the dry season (bottom). The arrows represent the transitions from a certain CP to another, and the colours of the arrows represent the corresponding transition probabilities 67 LAUX ET AL. 67 high-pressure field pattern into a less intense version of itself. Since all of these before mentioned transitions are from one high-pressure state into another and occur in an area considered as part of the subtropical high- pressure area resulting from the tropical circulation, the increased transition probabilities are considered as plau- sible. A direct transition into a non-high pressure CP would imply the occurrence of a sudden change of the whole tropical circulation, which can be considered as a low-probability event. The CP10 to CP7 and the CP7 to CP9 transitions described above are a sequence of high- probability transitions. Due to their high-occurrence probabilities and the fact that these CPs are considered as dry CPs (with a low WI) this sequences in particular might be a valuable for future research on drought prediction. The directed graphs differ significantly for the differ- ent seasons (Figure 11). For the wet season (Figure 11, top), the graph is less connected compared to that of the dry season (Figure 11, bottom). This is due to the fact that CP7 virtually does not occur during the wet season. In addition, some of the transitions occur only in one FIGURE 12 Dry season consideration of the wetness index (WI) for 30 selected stations based on the selected 11-cluster-solution FIGURE 12 Dry season consideration of the wetness index (WI) for 30 selected stations based on the selected 11-cluster-solution LAUX ET AL. 68 LAUX ET AL. 68 direction. This is the case for the transition from CP10 to CP8 as well as from CP9 to CP3. Figures 12 and 13 depict the WIs for 30 different stations in NEB, separately for the dry and the wet season in the calibration period. One can see that CP4 shows extraordi- nary high values of WI (WI >> 1) during the dry season, but also enhanced values for CP2 and CP5, and for fewer stations also for CP11. During the wet season, on the other hand, CP4 leads to very dry conditions for all sta- tions. 4.2.4 \| Wetness indices of CPs The wetness indices (WIs) without discrimination between wet and dry season is already described in com- bination with the occurrence of the different CPs. FIGURE 13 Wet season consideration of the wetness index (WI) for 30 selected stations based on the selected 11-cluster-solution. Please note that CP7 (shown in subplot g) does not occur during the wet season FIGURE 13 Wet season consideration of the wetness index (WI) for 30 selected stations based on the selected 11-cluster-solution. Please note that CP7 (shown in subplot g) does not occur during the wet season RE 14 Empirical ive distribution function of cted 11-cluster-solution for trarily selected precipitation the region AL. 69 FIGURE 14 Empirical cumulative distribution function of the selected 11-cluster-solution for one arbitrarily selected precipitation gauge in the region LAUX ET AL. 69 LAUX ET AL. this study, we followed a typical “circulation to environ- ment” approach, in which the CP classification (here: the SANDRA algorithm) is performed as a separate step that typify significant modes of the atmospheric circulation. After the classification, the CPs are linked to environmental phenomena at surface, such as heavy precipitation events causing high-runoff episodes (e.g., Prudhomme and Genevier, 2011; Wypych et al., 2018; Bednorz et al., 2019) or droughts (e.g., Dayan et al., 2012; Burgdorf et al., 2019). frequencies during the rainy season. In this case, few rainfall occurrences may cause very high WIs. For the remaining CPs, WIs slightly larger than unity are found. For both, the dry and the wet season, no clear spatial pat- terns of the stations across the NEB as, for example, related to topography for any given CP is found. The results are very similar and could thus be confirmed for the validation period (not shown). Although the classification algorithm per se is objec- tive, a lot of subjective decisions have to be met and may heavily impact on the results. The decisions comprise the selection of suitable predictor variables, the domain size and -location, the number of cluster centroids, and others. Therefore, tremendous efforts have been made for predictor screening and domain settings, making this a valuable base for further studies in NEB. For NEB, the only available study on circulation pattern classifications the authors are aware of is Robertson et al. (2004). 4.2.5 \| CDFs of CPs The required level of discriminative power of the identi- fied CPs is assessed for one arbitrarily selected precipita- tion gauge in the region. The empirical cumulative density function is shown for the different CPs for the annual consideration (Figure 14). It can be seen that wet CPs (e.g., CP2, CP3, CP5, CP6, CP11) exhibit a different shape compared to the remaining CPs that are linked to dry situations. This, in combination with different bias correction approaches, such as quantile mapping, poten- tially allows to derive transfer functions with a higher performance. It is stressed that also the grouping of the CPs is based on subjective decisions, and these decisions are based on the atmospheric conditions exclusively, which means that the observed weather states at surface have not been considered. However, the discriminative power of the resulting groups confirmed their potential applicability: it 4.2.4 \| Wetness indices of CPs In our study, for instance, it is found that near surface pressure patterns, such as MSLP and GP, reveal better classifica- tion performances, expressed as total explained cluster variances, when combined with upper level variables, such as the horizontal wind component. 5 \| DISCUSSION AND CONCLUSIONS A semi-objective circulation pattern classification has been performed and analysed for the semi-arid NEB region. In 70 LAUX ET AL. LAUX ET AL. 70 is found that group A (CPs 1, 2, 3, 5, 6) is the wettest (WI = 1.19 on average for all stations), followed by group B (CPs 7, 8, 9, 11) with an average WI = 0.82, and group C (CPs 4, 10), with an average WI = 0.70 (not shown). Other grouping approaches, for example, by considering the wetness index would possibly lead to different groups. For the case of this study, CP1 could be (re-)grouped into group B and CP11 into group A, thus increasing the dif- ferences in the WIs between both groups and better dis- criminating between dry and wet conditions in NEB. In this study, however, it is intentionally decided to group due to the atmospheric conditions exclusively. Moreover, it is worth mentioning that the grouping has been per- formed only to aid interpretability of the results, all con- siderations for potential applications are done based on the 11 individual CPs. combined with upper level variables, such as the horizontal wind component in 700 hPa. The choice of the low-level vari- able, that is, if the GP1,000 or the MSLP is applied, does not affect the overall classification performance. is found that group A (CPs 1, 2, 3, 5, 6) is the wettest (WI = 1.19 on average for all stations), followed by group B (CPs 7, 8, 9, 11) with an average WI = 0.82, and group C (CPs 4, 10), with an average WI = 0.70 (not shown). Other grouping approaches, for example, by considering the wetness index would possibly lead to different groups. For the case of this study, CP1 could be (re-)grouped into group B and CP11 into group A, thus increasing the dif- ferences in the WIs between both groups and better dis- criminating between dry and wet conditions in NEB. In this study, however, it is intentionally decided to group due to the atmospheric conditions exclusively. Moreover, it is worth mentioning that the grouping has been per- formed only to aid interpretability of the results, all con- siderations for potential applications are done based on the 11 individual CPs. ACKNOWLEDGEMENTS The study was mainly funded by the BMBF project Sea- sonal water resources management in semi-arid regions: Transfer of regionalized global information to practice (SaWaM, project number: 02WGR1421A). The clustering software has been produced in the EU funded COST Action 733 program and is available under: http:// cost733.geo.uni-augsburg.de (last access: 10/2019). We would also like to thank the ECMWF for providing the ERA-Interim reanalysis data as well as FUNCEME for the precipitation observations. The authors also thank the IT team of the KIT/IMK-IFU for providing access to and for maintaining the HPC-environment. Finally, we wish to thank the two anonymous reviewers for their valuable comments and suggestions. In addition to that, the CP classification can also be used to study drought dynamics in the region. Global warming may not only amplify drought duration and severity, but possibly changing drought dynamics in com- plex ways (Burgdorf et al., 2019). Although droughts are recurrent phenomena (Marengo et al., 2017), the recent multi-annual drought from 2012 to 2018 might be an indication for a change in the drought dynamics in NEB. Since droughts in NEB are known to be linked to the SST anomaly patterns in the Atlantic, the Pacific, and to a lesser extent in the Indian Ocean (e.g., Ward and Folland, 1991; Hastenrath, 2012; Costa et al., 2018), future works can focus on drought prediction using CP classification based on SST as predictor. Due to the strong link between SST anomalies and MSLP patterns, the presented SANDRA classification can also be tested for other domains covering also parts of the Pacific or the Indian Ocean. As suggested by the results of this study, near surface pressure patterns, such as MSLP or GP, reveal better classification performances, when 5 \| DISCUSSION AND CONCLUSIONS The development of statistical downscaling or CP- conditional bias correction approaches for NEB is beyond the scope of this paper, however, will be subject of further studies. Different characteristics with respect to occurrence, persistence, and transition probability during the dry and the wet season are promising indicators for discriminative power of the identified CPs. Therefore, it will be evaluated to which extend the occurrence- and transition probabilities as well as persistences of the CPs may help to improve statisti- cal downscaling- and bias correction approaches. Finally, another classification strategy following the “environment to circulation” approach can be applied, in which the CP classification is considered along a specific environment- based criteria set for a particular phenomenon, such as a drought or a flood (Dayan et al., 2012). An example is the classification of Bárdossy and Pegram (2012). The findings of this study open various avenues for applications in water resources management in NEB, such as a tool for statistical downscaling of GCMs based on CPs, or a CP-conditional bias correction algorithm of RCM out- put. Therefore, one may capitalize on the fact that a typical “wet” CP has a different precipitation distribution in time and space compared to a “dry” CP, which has been demon- strated in this study. This may finally lead to more efficient and robust model corrections when compared to those using the full distribution for correction. A correction based on separate months does usually implicitly accounts for this, however, this may not be a suitable solution when the same transfer functions are applied in future climate impact studies since the timing of seasons might shift (Wetterhall et al., 2012). As such, a CP-conditional bias correction approach can be seen as one potential solution to partly overcome the caveats due to the stationary assumptions, which exist for other bias correction approaches. 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https://openalex.org/W2103985998	https://link.springer.com/content/pdf/10.1186/1556-276X-8-390.pdf	English	null	CuO hollow nanosphere-catalyzed cross-coupling of aryl iodides with thiols	Nanoscale research letters	2,013	cc-by	4,879	© 2013 Woo et al.; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. CuO hollow nanosphere-catalyzed cross-coupling of aryl iodides with thiols Hyunje Woo1, Balaji Mohan1, Eunjung Heo1, Ji Chan Park2, Hyunjoon Song3 and Kang Hyun Park1* Open Access Open Access Abstract New functionalized CuO hollow nanospheres on acetylene black (CuO/AB) and on charcoal (CuO/C) have been found to be effective catalysts for C-S bond formation under microwave irradiation. CuO catalysts showed high catalytic activity with a wide variety of substituents which include electron-rich and electron-poor aryl iodides with thiophenols by the addition of two equivalents of K2CO3 as base in the absence of ligands. Keywords: Microwave; Copper oxide; Acetylene black; Heterogeneous; Ullmann coupling of thiophenols with aryl halides. Previously, iron [6], nickel [7,8], palladium [9,10], cobalt [11], and copper-based [12-16] catalytic systems have been reported for this purpose. Even though significant improvements have been made, appropriate techniques are still needed for the synthesis of diaryl thioethers. To date, metal and metal oxide nanoparticles have often been used as metal catalysts because of their physical and chemical stability. In addition, the advantage of nanoparticles including large surface area and heterogeneous nature make them ap- plicable to a broad range of scientific fields and functions such as the immobilization of biomolecules [17], catalysis of organic [18-23] and electrochemical reactions [17], use in electrochemical sensors and biosensors [17], enhance- ment of electron transfer [17], labeling of biomolecules [17], and synthesis of nanofluids [24], antibacterial mate- rials [25], photocatalysts [25,26], solar cells [27], and so on. Among the various available metal oxide nanopar- ticles, two copper oxides (Cu2O, CuO) have been studied for use in p-type semiconductor materials with narrow band gaps. This is because copper oxides are less expen- sive, recyclable, and non-toxic and have suitable optical and electronic properties [28-32]. Thus, as part of the effort to find new catalytic systems and better understand the role of transition metal nanoparticles in organic transformations, we report herein the use of CuO hol- low nanoparticles as catalysts for efficient syntheses of diaryl thioethers. These CuO hollow nanoparticles have advantages in terms of large-scale synthesis and uniform shape compared to previous reported CuO nanoparticles [33,34]. In recent times, microwave-irradiated organic * Correspondence: chemistry@pusan.ac.kr 1Department of Chemistry and Chemistry Institute for Functional Materials, Pusan National University, Busan 609-735, Korea Full list of author information is available at the end of the article Woo et al. Nanoscale Research Letters 2013, 8:390 http://www.nanoscalereslett.com/content/8/1/390 Woo et al. Nanoscale Research Letters 2013, 8:390 http://www.nanoscalereslett.com/content/8/1/390 General Reagents were purchased from Aldrich Chemical Co. (St. Louis, MO, USA) and Strem Chemical Co. (Bischheim, France) and used as received. Reaction products were analyzed by the literature values of known compounds. CuO, CuO/AB, and CuO/C were characterized by trans- mission electron microscopy (TEM) (Philips F20 Tecnai operated at 200 kV, KAIST, Amsterdam, the Netherlands). Samples were prepared by placing a few drops of the corresponding colloidal solution on carbon-coated copper grids (Ted Pellar, Inc., Redding, CA, USA). The X-ray Preparation of Cu2O nanocubes p 2 Poly(vinylpyrrolidone) (PVP, Aldrich, Mw 55,000; 5.3 g), dissolved in 45 mL of 1,5-pentanediol (PD, Aldrich, 96%), was heated to 240°C under inert conditions. Then, 4.0 mmol of Cu(acac)2 (Strem, 98%), dissolved in 15 mL of PD, was injected into the hot PVP solution at 240°C, and the mixture was stirred for 15 min at the same temperature. The resulting colloidal dispersion was cooled to room temperature, and the product was separated by adding 150 mL of acetone, with centrifugation at 8,000 rpm for 20 min. The precipitates were washed with ethanol several times and re-dispersed in 50 mL of ethanol. Background Sulfur-containing aromatic compounds, notably aryl sulfides and their derivatives, are prominent in fields such as biological, pharmaceutical, and materials fields. In particular, their use in synthesizing biologically and pharmaceutically important organosulfur compounds such as HIV protease inhibitors [1] (Viracept, Nelfinavir Mesylate, AG 1343), LFA-1/ICAM-1 antagonists [2], and arylthioindoles [3] (potent inhibitors of tubulin assem- bly) is still not fully understood by synthetic chemists. In general, molecules containing one or more carbon- sulfur bonds can be used as molecular precursors for the synthesis of new materials [4]. However, compared to C-N and C-O bonds, the transition metal-catalyzed C(aryl)-S bond formation has not been well studied. This bond formation is thought to be partial because of the formation of an S-S coupled product and a concurrent deactivation of the metal catalyst due to the strong coordinative and adsorptive properties of sulfur, which can decrease catalytic activity [5]. General methods for C-S cross-coupling involve the condensation of aryl halides with thiols and, usually, require temperatures greater than 200°C. These methods also require strongly basic, toxic, high-boiling, polar solvents, namely HMPA, quinolone, or N,N-dimethylacetamide. In order to cir- cumvent these complications, a meticulous effort has been focused on the development of transition metal-catalyzed * Correspondence: chemistry@pusan.ac.kr 1Department of Chemistry and Chemistry Institute for Functional Materials, Pusan National University, Busan 609-735, Korea Full list of author information is available at the end of the article Woo et al. Nanoscale Research Letters 2013, 8:390 http://www.nanoscalereslett.com/content/8/1/390 Page 2 of 7 diffractometer (XRD) patterns were recorded on a Rigaku D/MAX-RB (12 kW; Shibuya-ku, Japan) diffractometer. The copper loading amounts were measured by induct- ively coupled plasma atomic emission spectroscopy (ICP- AES). Elemental compositions of CuO/AB were obtained using energy-dispersive X-ray spectroscopy (EDS) (550i, IXRF Systems, Inc., Austin, TX, USA). reactions have become increasingly popular as valuable alternatives to the use of conductive heating for promoting chemical reactions. Besides, improved yields within short reaction time were observed. Microwave activation, as a non-conventional energy source, is becoming a very popular and valuable technique in organic synthesis, as evidenced by the increasing number of annual publica- tions on this topic. In continuation of our previous reports [35], we discovered that microwave irradiation can even accelerate the Ullmann coupling of activated aryl iodides and thiophenols. Synthesis of CuO hollow nanostructures An appropriate concentration of aqueous ammonia solu- tion was added to 25 mL of the Cu2O cube dispersion in Figure 1 TEM images of (a, b) CuO hollow nanospheres; (c) XRD pattern; (d) size distribution diagram of CuO hollow nanospheres. Figure 1 TEM images of (a, b) CuO hollow nanospheres; (c) XRD pattern; (d) size distribution diagram of CuO hollow nanospheres. Woo et al. Nanoscale Research Letters 2013, 8:390 http://www.nanoscalereslett.com/content/8/1/390 Page 3 of 7 washed with ethanol thoroughly and dried in a vacuum oven at room temperature. ethanol (16 mM with respect to the precursor concen- tration). The mixture was subjected to stirring at room temperature for 2 h. The volume and concentration of the aqueous ammonia solution used for each structure were 1.0 mL and 14.7 M, respectively, for hollow cubes; 2.0 mL and 7.36 M, respectively, for hollow spheres; and 6.0 mL and 2.45 M for urchin-like particles, respectively. For shape optimization of the hollow spheres, a 3.68-M aque- ous ammonia solution was used. After the reaction, the products were collected by centrifugation at 6,000 rpm for 20 min. Synthesis procedures of CuO/AB and CuO/C The acetylene carbon black (STREM, 99.99%, 1.2 g) was mixed with 100 mL of the CuO hollow nanosphere dis- persion in ethanol (17.0 mM), and the reaction mixture was sonicated for 1 h at room temperature. After 1 h, the product CuO/AB was washed with ethanol several times and vacuum dried at room temperature. For the synthesis of CuO/C, the mixture solution of charcoal (0.8 g) and 50.0 mL of CuO hollow nanosphere dispersion in ethanol (50.0 mM) was refluxed for 4 h. After 4 h, the black suspension was cooled to room temperature and precipitated by centrifugation. The product CuO/C was General procedure for cross-coupling of aryl halides with thiophenol Into a 10-mL glass vial, 4.0 mg of CuO/AB and CuO/C, iodobenzene (0.11 mL, 1.0 mmol), thiophenol (0.11 mL, 1.1 mmol), and solvent (5.0 ml) were placed. The reaction mixture was irradiated with a microwave stove (MAS II, Sineo Microwave Chemistry Technology Co., Ltd., Shanghai, China) for 10 to 30 min. After reaction, the vial was cooled to RT. The solution was then filtered, concentrated under reduced pressure, and characterized by Gas chromatography–mass spectrometry (GC-MS) spectra. Yields were based on the amount of iodobenzene used in each reaction. Synthesis procedures of CuO/AB and CuO/C Results and discussion Catalyst characterization To optimize the reaction, several experiments were performed by varying solvent, reaction time, and reac- tion temperature and using either hollow nanospherical CuO, CuO/C, or CuO/AB as the catalyst. First, 5.0 mol% of hollow nanospherical CuO/C in DMF were used at a temperature of 120°C, and diphenyl thioether was obtained with 49% conversion (entry 1, Figure 3). CuO hollow nanoparticles were used as a catalyst to compare the catalytic activity with supported CuO catalysts and showed 75% conversion (entry 2, Figure 3). Quantity of Results and discussion Catalyst characterization The CuO hollow nanostructures were prepared by a con- trolled oxidation of Cu2O nanocubes using an aqueous ammonia solution according to a method in the literature [36]. The Cu2O nanocubes (average edge size of 50 nm) were converted to CuO hollow nanospheres by addition of ammonia solution (2.0 mL, 3.7 M) into Cu2O colloidal Figure 2 TEM images and EDS spectrum. TEM images of (a, b) CuO/AB. TEM image of (c) CuO/C, and the scale bar represents 200 nm. EDS spectrum of (d) CuO/AB. Figure 2 TEM images and EDS spectrum. TEM images of (a, b) CuO/AB. TEM image of (c) CuO/C, and the scale bar represents 200 nm. EDS spectrum of (d) CuO/AB. Woo et al. Nanoscale Research Letters 2013, 8:390 http://www.nanoscalereslett.com/content/8/1/390 Page 4 of 7 Scheme 1 Proposed mechanism for synthesis of aryl thioethers. solution by a dissolution-precipitation process. The TEM images in Figure 1a,b show monodisperse CuO hollow nanospheres that are composed of needle-like branches. The average size of these CuO hollow nanospheres was measured to be 103 ± 8 nm (Figure 1d). The CuO hollow nanospheres were analyzed using XRD analysis (Figure 1c). Two main peaks were present in the XRD patterns of the CuO hollow nanospheres that could be assigned to the reflections of the (002)/(11–1) and (111)/(200) planes in the CuO phase (JCPDS no. 48–1548). Immobilization of CuO hollow nanospheres on ace- tylene black (CuO/AB) was performed by sonication for 1 h at room temperature. The TEM images in Figure 2a, c show well-dispersed CuO/AB and CuO/C, maintaining their original size and structure. ICP-AES confirmed the content of copper metal on the acetylene black. EDS spectrum in Figure 2d showed that hollow CuO nanoparticles were immobilized on acetylene black. The X-ray photoelectron spectroscopy data at the energy regions of the Cu bands confirm that the elements of the three different shapes are only Cu(II). The peaks at 933.8 and 953.7 eV correspond to Cu 2p3/2 and Cu 2p1/2 bands, and the other two signals, at 943.8 and 962.4 eV, are the shakeup satellites, which are characteristic of d9 Cu(II) compounds [37]. Scheme 1 Proposed mechanism for synthesis of aryl thioethers. Ullmann coupling is already reported [38]. Scheme 1 shows a proposed mechanism for synthesis of aryl thioethers. Ullmann reaction of aryl halides with thiols catalyzed by CuO hollow nanoparticles Initially, the reaction of iodobenzene with thiophenol was chosen as a model reaction. Reaction mechanism about A Conv. (%)a A B 1 5 mol%-CuO/C 120 1000 10 DMF 49 36 64 2 5 mol%-CuO 120 1000 20 DMF 75 41 59 3 1 mol%-CuO/C 120 1000 20 DMF 61 71 29 4 2.5 mol%-CuO/C 120 1000 20 DMF 80 48 52 5 5 mol%-CuO/C 120 1000 20 DMF 81 48 52 6 Charcoalb 120 1000 20 DMF 47 1 99 7 5 mol%-CuO nanopowderc 120 1000 20 DMF 61 52 48 8 5 mol%-CuO/AB 120 1000 20 DMF 66 d 35 65 9 5 mol%-CuO/AB 120 1000 20 DMF 61 28 72 10 5 mol%-CuO/AB 120 1000 20 DMSO 60 90 10 11 5 mol%-CuO/AB 80 1000 10 MeCN 99 0 100 12 5 mol%-CuO/AB 180 1000 20 DMSO 75 16 84 13 5 mol%-CuO/AB 60 1000 20 DMSO 75 54 46 14 5 mol%-CuO/AB 120 1000 30 DMSO 66 82 18 15 5 mol%-CuO/AB 120 1000 60 DMSO 95 100 0 B Selectivity (%) Entry Catalyst Temp. ( oC) MW power (W) Time (min) Solvent I SH S S S + + Figure 3 Ullmann coupling reaction of iodobenzene with thiophenol. Woo et al. Nanoscale Research Letters 2013, 8:390 http://www.nanoscalereslett.com/content/8/1/390 Page 5 of 7 condition. The catalytic activities of both catalysts were almost equivalent, and 61% conversion was obtained (entry 9, Figure 3). Interestingly, when the solvent was changed to dimethyl sulfoxide (DMSO), diphenyl thioether was dominant under the same conditions (entry 10, Figure 3). At a temperature of 80°C and a reaction time of 10 min, >% conversion of diphenyl disulfide was achieved in the presence of MeCN (entry 11, Figure 3). There was no difference in the conversion between reaction tempera- tures of 180°C and 60°C (entries 12 and 13, Figure 3). When the reaction time was increased to 30 min, the conversion was slightly increased and the selectivity of diphenyl thioether was decreased (entry 14, Figure 3). We found that selectivity was dependent on several factors such as solvent used (entries 9 to 11, Figure 3), quantity of thiophenol (entries 8 and 9, Figure 3), reaction temperature (entries 12 to 14, Figure 3), and reaction time (entries 10 and 14, Figure 3). catalyst was also checked to observe the catalytic activity of CuO/C catalyst. Ullmann reaction of aryl halides with thiols catalyzed by CuO hollow nanoparticles There was no difference in conversion between 2.5 and 5 mol% of the catalyst (entries 3 to 5, Figure 3). When the reaction time was increased to 20 min, 81% conversion was achieved under the same conditions but with slight deviation in selectivity (entry 5, Figure 3). Only charcoal catalyst showed less catalytic activity and selectivity (entry 6, Figure 3). We tried one reaction using commercially available CuO nanopowder as catalyst. CuO nanopowder exhibited less catalytic ac- tivity than CuO/C catalyst although there is no surfactant in CuO nanopowder (entries 5 and 7, Figure 3). Our CuO hollow nanostructure showed better catalytic activity because of a high surface area. Conversion of 66% was achieved with the use of two equivalent thiophenols (2.2 mmol), and the amount of diphenyl disulfide in- creased due to homocoupling reaction as expected (entry 8, Figure 3). Next, the catalytic activity of the hollow nanospherical CuO/AB was compared with that of the hollow nanospherical CuO/C catalyst at the same The versatilities of our nanocatalyst were investigated by performing Ullmann coupling reactions of various A B A B 0 100 5 95 66 34 0 100 0 100 99 1 67 33 Selectivity (%) 27 Entry Substrate Conv. (%)a 1 20 3 4 5 19 79 81 2 6 31 7 46 I MeO I NH2 I O2N I HO I CH3 I I Br X I SH S S S X + + 5mol%, K2CO3(2eq) DMSO, 20min,120oC S NH2 S S NH2 S MeO S S MeO S O2N S S O2N S O H S S O H S CH3 S S CH3 S S S S Br S S Br Figure 4 CuO/AB-catalyzed Ullmann coupling reaction with various substrates. Competing interests 15. Rout L, Saha P, Jammi S, Punniyamurthy T: Efficient copper(I)-catalyzed C–S cross coupling of thiols with aryl halides in water. Eur J Org Chem 2008, 4:640–643. 15. Rout L, Saha P, Jammi S, Punniyamurthy T: Efficient copper(I)-catalyzed C–S cross coupling of thiols with aryl halides in water. Eur J Org Chem 2008, 4:640–643. The authors declare that they have no competing interests. Authors’ contributions h 16. Sperotto E, van Klink GPM, de Vries JG, van Koten G: Ligand-free copper-catalyzed C-S coupling of aryl iodides and thiols. J Org Chem 2008, 73:5625–5628. 16. Sperotto E, van Klink GPM, de Vries JG, van Koten G: Ligand-free copper-catalyzed C-S coupling of aryl iodides and thiols. J Org Chem 2008, 73:5625–5628. The manuscript was written through the contributions of all authors (HW, MB, EH, JCP, HS, and KHP). All authors read and approved the final manuscript. 17. Luo X, Morrin A, Killard AJ, Smyth MR: Application of nanoparticles in electrochemical sensors and biosensors. Electroanalysis 2006, 18:319–326. 17. Luo X, Morrin A, Killard AJ, Smyth MR: Application of nanoparticles in electrochemical sensors and biosensors. Electroanalysis 2006, 18:319–326. Received: 17 July 2013 Accepted: 12 September 2013 Published: 17 September 2013 Received: 17 July 2013 Accepted: 12 September 2013 Published: 17 September 2013 References 1. 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Acknowledgement This work was supported by a 2-year Research Grant of Pusan National University and National Research Foundation of Korea (NRF) through the Human Resource Training Project for Regional Innovation. 18. Harsha Vardhan Reddy K, Prakash Reddy V, Shankar J, Madhav B, Anil Kumar BSP, Nageswar YVD: Copper oxide nanoparticles catalyzed synthesis of aryl sulfides via cascade reaction of aryl halides with thiourea. Tetrahedron Lett 2011, 52:2679–2682. Conclusions In conclusion, CuO hollow nanospheres were synthe- sized by controlled oxidation of Cu2O nanocubes using aqueous ammonia solutions. Ullmann coupling reac- tions of aryl iodide with thiols were conducted to check the respective catalytic activities of CuO, CuO/ AB, and CuO/C hollow nanosphere catalysts under microwave irradiation. Various diaryl thioethers were obtained from electron-deficient aryl iodides, while diaryl disulfide was produced from electron-rich aryl iodides. 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Figure 4 CuO/AB-catalyzed Ullmann coupling reaction with various substrates. Woo et al. Nanoscale Research Letters 2013, 8:390 http://www.nanoscalereslett.com/content/8/1/390 Page 6 of 7 substrates under optimized reaction conditions. The reactions of substrates with electron-rich and electron- poor groups on the iodobenzene resulted in different yields and selectivities of the cross-coupling products (Figure 4). When the electron-rich substrates were used, more than 95% selectivity for diphenyl disulfide was obtained due to a homocoupling reaction of thiophenol although only a low yield of product was obtained in this case (entries 1, 2, 4, and 5, Figure 4). On the contrary, only 79% conversion was obtained in the case of electron- poor substituents such as 1-iodo-4-nitro-benzene, and the selectivity for product (A) was increased to 66% (entry 3, Figure 4). 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https://openalex.org/W1978460383	https://eprint.ncl.ac.uk/fulltext.aspx?url=204224/92582EBA-3F9E-4160-9F63-4D770E055763.pdf&pub_id=204224	English	null	The Challenges of Mitochondrial Replacement	PLOS genetics	2,014	cc-by	2,164	Copyright: © 2014 Chinnery 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. Chinnery PF, Craven L, Mitalipov S, Stewart JB, Herbert M, Turnbull DM. The Challenges of Mitochondrial Replacement. PLoS Genetics 2014, 10(4), e1004315. DOI link to article: http://dx.doi.org/10.1371/journal.pgen.1004315 Patrick F. Chinnery1,2, Lyndsey Craven1, Shoukhrat Mitalipov3, James B. Stewart4, Mary Herbert1,5, 1 Patrick F. Chinnery1,2, Lyndsey Craven1, Shoukhrat Mitalipov3, James B. Stewart4, Mary Herbert1,5, Douglass M. Turnbull1* 1 Wellcome Trust Centre for Mitochondrial Research, Institute for Ageing and Health, Newcastle University, Newcastle upon Tyne, United Kingdom, 2 Institute of Genetic Medicine, Newcastle University, Central Parkway, Newcastle upon Tyne, United Kingdom, 3 Division of Reproductive & Developmental Sciences, Oregon National Primate Research Center, Oregon Health & Science University, Beaverton, Oregon, United States of America, 4 Max Planck Institute for Biology of Ageing, Cologne, Germany, 5 Newcastle Fertility Centre, International Centre for Life, Newcastle University, Newcastle upon Tyne, United Kingdom The studies in macaques are also highly relevant to the risks proposed in humans associated with mitochondrial replace- ment. There are now multiple reports of the health status of the offspring born after mitochondrial replacement, and all have shown no difference between these off- spring and controls [6,7,15]. As highlight- ed in the reports, the macaques used for these experiments were not, as suggested by the authors of the recent commentary [10], highly genetically related, but some were from divergent subspecies with extensive differences in the rhesus ma- caque genome [6]. Thus, the experiments using the animal model closest to man have not shown any adverse effects from mitochondrial transfer. drial and nuclear genomes has not been given due weight. Should we therefore stop further clinical developments in this area with immediate effect? New advances in medicine often raise challenges, and none more so than those involving the manipulation of human oocytes and embryos. Issues around clin- ical need and ethical considerations must be taken into account, as well as the safety of the proposed technique. The discussion around the proposed mitochondrial re- placement techniques to prevent the transmission of mitochondrial DNA dis- ease has perhaps raised more challenges than most [1]. The authors raise an interesting evolu- tionary argument that the human mito- chondrial genome co-evolves with the nuclear genome in females, raising the possibility of a conflict with the paternal nuclear genome. They suggest Leber’s hereditary optic neuropathy (LHON) and male infertility could be potential exam- ples of this in humans [10]. Firstly, LHON is not a male-limited disease as they suggest [11]. Patrick F. Chinnery1,2, Lyndsey Craven1, Shoukhrat Mitalipov3, James B. Stewart4, Mary Herbert1,5, 1 The disorder affects ,10% of women carrying specific mtDNA mu- tations, and although there is increased penetrance in males, strenuous efforts have failed to identify a nuclear modifier gene to date, and the increased penetrance in men could simply reflect the absence of oestrogens [12]. As regards male infertility, there is no convincing evidence in man that inherited variants of mtDNA are at all relevant in the general population [13,14]. Indeed it is interesting that even in male patients with pathogenic mitochondrial DNA mutations, such as LHON, reduced fertility has not been reported to be a major clinical feature. Mitochondrial DNA diseases are both common and, in their severest forms, devastating [2]. There are limited treat- ments available for these patients, and those that are successful are focused on treating complications such as epilepsy and cardiac disease [3]. Mitochondrial DNA diseases are transmitted maternally, and for families carrying these mutations, a major, and justifiable, desire is to have unaffected children. For some women, preimplantation or prenatal diagnosis may be helpful [4,5], but for other women, these techniques will not result in disease- free offspring and the only options avail- able are either oocyte donation or mito- chondrial replacement at the oocyte or zygote stage. The need for this technique for these families is well established, as are the experimental methods that are re- quired for mitochondrial replacement [6– 8]. The major scientific concerns for those of us working in the field revolve around safety and efficacy. Some studies in laboratory mice have proposed a nuclear DNA–mitochondrial DNA interaction, but there are others that have reported no defect despite the use of very divergent genomes [16–18]. It is important to recognise that these studies, and those in invertebrates, have been performed on highly inbred species (often inbred over thousands of genera- tions) and the relevance to human populations must be questioned. Most human populations are outbred with In the United Kingdom, the Human Fertilisation and Embryology Authority (HFEA) recently considered the safety issues after extensive expert and public consultation [9]. This independent group of scientists reviewed all the evidence and concluded that mitochondrial replacement techniques have the potential to be used for patients with mitochondrial DNA disease, although further experiments are required before introduction into clinical practice, to provide further reassurance with respect to efficiency and safety. Viewpoints Citation: Chinnery PF, Craven L, Mitalipov S, Stewart JB, Herbert M, et al. (2014) The Challenges of Mitochondrial Replacement. PLoS Genet 10(4): e1004315. doi:10.1371/journal.pgen.1004315 Date deposited: Newcastle University ePrints - eprint.ncl.ac.uk Editor: David R. Thorburn, Royal Children’s Hospital, Australia Patrick F. Chinnery1,2, Lyndsey Craven1, Shoukhrat Mitalipov3, James B. Stewart4, Mary Herbert1,5, 1 Recently [10], it has been suggested that the possibility of a harmful interaction between the mitochon- Copyright: 2014 Chinnery 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: PFC, MH and DMT are all supported by The Wellcome Trust Centre for Mitochondrial Research [G906919]. PFC is a Wellcome Trust Senior Fellow in Clinical Science (084980/Z/08/Z), and a UK NIHR Senior Investigator and receives additional support from the Medical Research Council (UK) Centre for Translational Muscle Disease research, the Association Franc¸aise contre les Myopathies, and EU FP7 TIRCON, and the National Institute for Health Research (NIHR) Newcastle Biomedical Research Centre based at Newcastle upon Tyne Hospitals NHS Foundation Trust and Newcastle University. SM is supported by grants from the National Institutes of Health R01HD057121, R01HD059946, R01HD063276, R01EY021214 and 8P51OD01109. SM also receives support from the Leducq Foundation and other OHSU institutional funds. DMT is also supported by Newcastle University Centre for Brain Ageing and Vitality (supported by the BBSRC, EPSRC, ESRC and MRC) [G0700718]), MRC Centre for Neuromuscular Disease [G000608-1], The MRC Centre for Translational Research in Neuromuscular Disease Mitochondrial Disease Patient Cohort (UK) [G0800674], Lily Foundation, the UK NIHR Newcastle Biomedical Research Centre in Age and Age Related Diseases. The funders had no role in the preparation of the article. Competing Interests: The authors have declared that no competing interests exist. * E-mail: doug.turnbull@newcastle.ac.uk * E-mail: doug.turnbull@newcastle.ac.uk April 2014 \| Volume 10 \| Issue 4 \| e1004315 1 PLOS Genetics \| www.plosgenetics.org the world—and without any known adverse effects. and differentiation potential [7,8]. As suggested by the HFEA [9], it is possible to match mitochondrial haplotype be- tween the mother and the mitochondrial donor to avoid any concern, even though the evidence says it should not be needed. We do not believe this important devel- opment should be delayed—for families carrying mtDNA mutations, the clock is ticking, and the desire to have children free of mitochondrial DNA disease is entirely justified. Ultimately, we believe those that carry mitochondrial DNA mutations must be fully informed of the potential risks, and that they will decide which option to take. considerable mixing of the genome over recent generations. complete sequencing of asthenozoospermic males. Mol Biol Evol 24: 868–874. Patrick F. Chinnery1,2, Lyndsey Craven1, Shoukhrat Mitalipov3, James B. Stewart4, Mary Herbert1,5, 1 In these populations the mixing of alleles will inevitably dilute the effect of potentially harmful nuclear DNA-mitochondrial DNA interactions. There has never been any direct evidence of a ‘‘mismatch’’ between the two in humans—either on an evolutionary scale or in the context of disease. This is even the case for couples from divergent haplogroups, where potential nuclear- mitochondrial mismatches are at their most extreme. Thus, from the mitochon- drial DNA perspective, any mitochondrial transfer experiment is just recapitulating what is happening every day all around Whilst we accept that any new tech- nique is associated with risk, we think the lack of any reliable evidence of mitochondrial-nuclear interaction as a cause of disease in human outbred popu- lations provides the necessary reassurance to proceed. The recent studies in ma- caques after mitochondrial replacement are also supportive that the possible harmful interactions are unlikely to occur in man [6,7]. Human preimplantation embryos and embryonic stem cells gener- ated with ‘‘unmatched’’ mtDNA replace- ment demonstrated normal development p 16. Battersby BJ, Shoubridge EA (2001) Selection of a mtDNA sequence variant in hepatocytes of heteroplasmic mice is not due to differences in respiratory chain function or efficiency of repli- cation. Hum Mol Genet 10: 2469–2479. 15. Lee HS, Ma H, Juanes RC, Tachibana M, Sparman M, et al. (2012) Rapid mitochondrial DNA segregation in primate preimplantation embryos precedes somatic and germline bottle- neck. Cell Rep 1: 506–515. 9. HFEA (2011) Review of scientific methods to avoid mitochondrial disease 2011 (including 2013 update). Available: http://www.hfea.gov.uk/ 6372.html. Accessed 29 March 2014. 14. Mossman JA, Slate J, Birkhead TR, Moore HD, Pacey AA (2012) Mitochondrial haplotype does not influence sperm motility in a UK population of men. Hum Reprod 27: 641– 651. 7. Tachibana M, Amato P, Sparman M, Woodward J, Sanchis DM, et al. (2013) Towards germline gene therapy of inherited mitochondrial diseases. Nature 493: 627–631. References 7. Tachibana M, Amato P, Sparman M, Woodward J, Sanchis DM, et al. (2013) Towards germline gene therapy of inherited mitochondrial diseases. Nature 493: 627–631. 1. HFEA, Mitochondria public consultation (2012) Available: http://www.hfea.gov.uk/6896.html. Accessed 29 March 2014. 14. Mossman JA, Slate J, Birkhead TR, Moore HD, Pacey AA (2012) Mitochondrial haplotype does not influence sperm motility in a UK population of men. Hum Reprod 27: 641– 651. 2. Koopman WJ, Willems PH, Smeitink JA (2012) Monogenic mitochondrial disorders. N Engl J Med 366: 1132–1141. 8. Craven L, Tuppen HA, Greggains GD, Harbottle SJ, Murphy JL, et al. (2010) Pronuclear transfer in human embryos to prevent transmission of mitochondrial DNA disease. Nature 465: 82– 85. 3. Wellcome Trust Centre for Mitochondrial Re- search (2014) Patient Care Guidelines. Available: http://www.newcastle-mitochondria.com/ service/patient-care-guidelines/. Accessed 29 March 2014. 15. Lee HS, Ma H, Juanes RC, Tachibana M, Sparman M, et al. (2012) Rapid mitochondrial DNA segregation in primate preimplantation embryos precedes somatic and germline bottle- neck. Cell Rep 1: 506–515. 9. HFEA (2011) Review of scientific methods to avoid mitochondrial disease 2011 (including 2013 update). Available: http://www.hfea.gov.uk/ 6372.html. Accessed 29 March 2014. 4. Steffann J, Frydman N, Gigarel N, Burlet P, Ray PF, et al. (2006) Analysis of mtDNA variant segregation during early human embryonic de- velopment: a tool for successful NARP preim- plantation diagnosis. J Med Genet 43: 244–247. p 16. Battersby BJ, Shoubridge EA (2001) Selection of a mtDNA sequence variant in hepatocytes of heteroplasmic mice is not due to differences in respiratory chain function or efficiency of repli- cation. Hum Mol Genet 10: 2469–2479. 10. Reinhardt K, Dowling DK, Morrow EH (2013) Mitochondrial replacement, evolution, and the clinic. Science 341: 1345–1346. p g J 5. Steffann J, Gigarel N, Corcos J, Bonnie`re M, Encha-Razavi F, et al. (2007) Stability of the m.8993TRG mtDNA mutation load during human embryofetal development has implications for the feasibility of prenatal diagnosis in NARP syndrome. J Med Genet 44: 664–669. 11. Yu-Wai-Man P, Griffiths PG, Hudson G, Chin- nery PF (2009) Inherited mitochondrial optic neuropathies. J Med Genet 46: 145–158. 17. Cannon MV, Dunn DA, Irwin MH, Brooks AI, Bartol FF, et al. (2011) Xenomitochondrial mice: investigation into mitochondrial compensatory mechanisms. Mitochondrion 11: 33–39. 12. Giordano C, Montopoli M, Perli E, Orlandi M, Fantin M, et al. (2011) Oestrogens ameliorate mitochondrial dysfunction in Leber’s hereditary optic neuropathy. Brain 134: 220–234. 18. 18. Gregorova´ S, Divina P, Storchova R, Trachtulec Z, Fotopulosova V, et al. (2008) Mouse consomic strains: exploiting genetic divergence between Mus m. musculus and Mus m. domesticus subspecies. Genome Res 18: 509–515. 10. Reinhardt K, Dowling DK, Morrow EH (2013) Mitochondrial replacement, evolution, and the clinic. Science 341: 1345–1346. complete sequencing of asthenozoospermic males. Mol Biol Evol 24: 868–874. References Gregorova´ S, Divina P, Storchova R, Trachtulec Z, Fotopulosova V, et al. (2008) Mouse consomic strains: exploiting genetic divergence between Mus m. musculus and Mus m. domesticus subspecies. Genome Res 18: 509–515. 6. Tachibana M, Sparman M, Sritanaudomchai H, Ma H, Clepper L, et al. (2009) Mitochondrial gene replacement in primate offspring and embryonic stem cells. Nature 461: 367–372. 13. Pereira L, Gonc¸alves J, Franco-Duarte R, Silva J, Rocha T, et al. (2007) No evidence for an mtDNA role in sperm motility: data from April 2014 \| Volume 10 \| Issue 4 \| e1004315 PLOS Genetics \| www.plosgenetics.org 2
https://openalex.org/W2902630415	https://europepmc.org/articles/pmc6395889?pdf=render	English	null	Beliefs About Medication and Uptake of Preventive Therapy in Women at Increased Risk of Breast Cancer: Results From a Multicenter Prospective Study	Clinical breast cancer	2,019	cc-by	8,350	Original Study Original Study Original Study Clinical Breast Cancer, Vol. 19, No. 1, e116-26 ª 2018 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Keywords: BMQ, Breast cancer prevention, Chemoprevention, Decision-making, Medication beliefs Beliefs About Medication and Uptake of Preventive Therapy in Women at Increased Risk of Breast Cancer: Results From a Multicenter Prospective Study Rachael Jane Thorneloe,1 Rob Horne,2 Lucy Side,3 Michael Scott Wolf,4 Samuel George Smith,1 on behalf of the ENGAGE Investigators Rachael Jane Thorneloe,1 Rob Horne,2 Lucy Side,3 Michael Scott Wolf,4 Samuel George Smith,1 on behalf of the ENGAGE Investigators Abstract Preventive therapies, such as tamoxifen, are a risk reduction option for women at increased risk of breast cancer. Little is known about the psychological factors inﬂuencing the decision to use chemoprevention. Using latent proﬁle analysis, women who reported a low need for preventive therapy and strong medication concerns were less likely to initiate tamoxifen treatment. Medication beliefs are targets for supporting informed decision- making. Introduction: Uptake of preventive therapies for breast cancer is low. We examined whether women at increased risk of breast cancer can be categorized into groups with similar medication beliefs, and whether belief group membership was prospectively associated with uptake of preventive therapy. Patients and Methods: Women (n ¼ 732) attending an appointment to discuss breast cancer risk were approached; 408 (55.7%) completed the Beliefs About Medicines and the Perceived Sensitivity to Medicines questionnaires. Uptake of tamoxifen at 3 months was reported in 258 (63.2%). The optimal number of belief groups were identiﬁed using latent proﬁle analysis. Results: Uptake of tamoxifen was 14.7% (38/258). One in 5 women (19.4%; 78/402) reported a strong need for tamoxifen. The model ﬁt statistics supported a 2-group model. Both groups held weak beliefs about their need for tamoxifen for current and future health. Group 2 (38%; 154/406 of the sample) reported stronger concerns about tamoxifen and medicines in general, and stronger perceived sensitivity to the negative effects of medicines compared with group 1 (62%; 252/ 406). Women with low necessity and lower concerns (group 1) were more likely to initiate tamoxifen (18.3%; 33/180) than those with low necessity and higher concerns (group 2) (6.4%; 5/78). After adjusting for demographic and clinical factors, the odds ratio was 3.37 (95% conﬁdence interval, 1.08-10.51; P ¼ .036). Conclusion: Uptake of breast cancer preventive therapy was low. A subgroup of women reported low need for preventive therapy and strong medication concerns. These women were less likely to initiate tamoxifen. Medication beliefs are targets for supporting informed decision-making. 4Division of General Internal Medicine and Geriatrics, Northwestern University, Evanston, IL S.G.S. was supported by a Cancer Research UK postdoctoral fellowship (C42785/ A17965) during the collection of these data. He also acknowledges funding support from a Yorkshire Cancer Research University Academic Fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the report. 1526-8209/ª 2018 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1016/j.clbc.2018.10.008 1Leeds Institute of Health Sciences, University of Leeds, Leeds, United Kingdom 2Centre for Behavioural Medicine, School of Pharmacy, University College London, London, United Kingdom 3Wessex Clinical Genetics Service, University Hospitals Southampton, Southampton, United Kingdom 4Division of General Internal Medicine and Geriatrics, Northwestern University, Evanston, IL Introduction was calculated for each subscale, with scores ranging from 1 to 5. The proportion of women who agreed (¼ 4) or strongly agreed (¼ 5) with each item within the subscales were also examined. Breast cancer is the most common cancer in women worldwide.1 Preventive therapy is a risk reduction approach for women at increased risk of breast cancer. In a meta-analysis of 9 randomized trials, women at increased risk of breast cancer had at least a 30% lower risk of the disease if they used selective estrogen receptor modulators.2 The IBIS-I (International Breast Cancer Intervention Study) indicated the preventive effect of tamoxifen lasts for at least 20 years.3 The effectiveness of preventive therapy depends on adequate uptake but initiation rates remain low.4-7 The Perceived Sensitivity to Medicines (PSM) scale13 is used to assesses perceived sensitivity to potential adverse effects of medicines (5 items). Each item is scored on a 5-point scale (“strongly disagree” [¼1] to “strongly agree” [¼ 5]), with higher scores indicating higher perceived sensitivity to the negative effects of medicines. A mean score was calculated, with scores ranging from 1 to 5. The pro- portion of women who agreed (¼ 4) or strongly agreed (¼ 5) with each individual scale item was examined. Individual’s beliefs about medication are modiﬁable drivers of treatment decision-making.8 These beliefs include perceptions of personal need for medication (necessity beliefs) and concerns about its usage (concern beliefs), as well as more general concerns relating to the nature of medications and how they are used by doctors. The Beliefs About Medicines Questionnaire (BMQ) is the tool most commonly used to assess and quantify medication beliefs.8,9 Women’s concerns about side effects are a barrier to initiating preventive therapy.5,10 However, there can be heterogeneity in individual’s beliefs.11 Understanding subgroup differences in medication beliefs can sup- port the development of personalized interventions. The baseline survey obtained the following data: marital status; ethnicity; education level; employment status; nulliparity; and self-reported health. Age was calculated from date of birth provided from National Health Service records; women were coded as 35 years; 36 to 49 years and; 50 years for analysis. Abstract Submitted: Jul 31, 2018; Revised: Oct 12, 2018; Accepted: Oct 24, 2018; Epub: Dec 3, 2018 Address for correspondence: Samuel George Smith, BSc, MSc, PhD, University of Leeds, Leeds Institute of Health Sciences, Worsley Building, Clarendon Way, Leeds LS2 9NL, United Kingdom E il i h1@l d k 1Leeds Institute of Health Sciences, University of Leeds, Leeds, United Kingdom 2Centre for Behavioural Medicine, School of Pharmacy, University College London, London, United Kingdom 3 g E-mail contact: s.smith1@leeds.ac.uk g 3Wessex Clinical Genetics Service, University Hospitals Southampton, Southampton, United Kingdom 1526-8209/ª 2018 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1016/j.clbc.2018.10.008 1526-8209/ª 2018 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1016/j.clbc.2018.10.008 e116 Clinical Breast Cancer February 2019 Patients Women were approached after their appointment at 1 of the following clinic types; family history clinic (n ¼ 12), breast clinic (n ¼ 4), clinical genetic centers (n ¼ 3), and a family history clinic with genetics support (n ¼ 1). In the United Kingdom, women are referred to secondary care if their general practitioner (family doc- tor) believes they are likely to meet National Institute for Health and Care Excellence (NICE) criteria for breast cancer risk.12 Recruitment took place at 20 clinics in England between September 2015 and December 2016. Eligibility criteria included: women aged 18 years or older; English-speaking; had discussed preventive therapy with a health care professional; were classiﬁed as having a moderately high or high risk of breast cancer according to NICE guidelines12; and had no known contraindications for tamoxifen use. Women were excluded if they were unable to consent, read English, or had a previous diagnosis of breast cancer. Introduction Index of Multiple Deprivation scores were calculated from participant postcodes, and women were classiﬁed into tertiles of neighborhood deprivation.14 Breast cancer risk category (moderately high or high) as outlined in the NICE guidelines, was provided by clinic staff (with partici- pant consent).12 Uptake of tamoxifen was assessed in the 3-month follow-up questionnaire. Women were classiﬁed as initiating tamoxifen if they reported having a prescription for tamoxifen from their general practitioner or were currently taking tamoxifen. This is because some women might not have had the opportunity to collect their prescription and start treatment at the time of the 3-month follow-up period. The baseline survey obtained the following data: marital status; ethnicity; education level; employment status; nulliparity; and self-reported health. Age was calculated from date of birth provided from National Health Service records; women were coded as 35 years; 36 to 49 years and; 50 years for analysis. Index of Multiple Deprivation scores were calculated from participant postcodes, and women were classiﬁed into tertiles of neighborhood deprivation.14 The objectives of this study were to: (1) assess whether women at increased risk of breast cancer can be categorized into groups with similar medication beliefs; (2) determine whether sociodemographic and clinical variables are related to medication belief group mem- bership and; (3) examine whether medication belief groups are associated with tamoxifen uptake. Patients and Methods Patients The analysis was preregistered.15 The association between the BMQ subscales and the PSM scale were analyzed using Pearson correlation coefﬁcients. Differences in medication beliefs between those who completed the baseline survey and women who returned a baseline and follow-up survey were analyzed using t tests. Theory- driven latent proﬁle analysis (LPA) was used to investigate whether women could be categorized into medication belief groups. LPA is used to categorize individuals with similar proﬁles on a set of continuous variables (BMQ and PSM scales) into discrete groups represented by a categorical latent variable (medication belief groups). Participants’ mean scores for the BMQ subscales and the PSM scale were included in the LPA analysis. Two participants had missing data for all 5 variables and were excluded from analysis (n ¼ 406 included in baseline analysis). Model ﬁt statistics for LPA models with 1 through 5 class solutions were examined. These were the Akaike Information Criterion (AIC) and the Bayesian Infor- mation Criterion (BIC), where smaller values indicate a better ﬁt. The VuongeLoeMendelleRubin likelihood ratio test and the LoeMendelleRubin adjusted likelihood ratio test were used to compare the current model with a model with 1 less latent class. Entropy provides a measure of the classiﬁcation quality of the model, with values approaching 1 indicating a good separation of classes. Materials Women were invited to complete a baseline survey containing the following measures: the BMQ9 is used to assess perceptions about personal need for tamoxifen (3 items, speciﬁc necessity); concerns about negative effects from tamoxifen (6 items, speciﬁc concerns); beliefs relating to the nature of medication (4 items, general harmfulness); and beliefs about how they are used by doc- tors (4 items, general overuse). The BMQ was adapted for use in chemoprevention decision-making. Each item is scored on a 5-point scale (“strongly disagree: [¼ 1] to “strongly agree” [¼ 5]), with higher scores indicating stronger medication beliefs. A mean score Two planned sensitivity analyses were performed. The LPA model was run with and without the PSM scale. The LPA model was also run on individuals who provided baseline and 3-month follow-up data on tamoxifen uptake (n ¼ 258) to ensure that the reduction in sample size would not bias the results. e117 Clinical Breast Cancer February 2019 Beliefs About Medication and Perceived Sensitivity to its Effects ff Women reported low perceived need for tamoxifen; 19.4% (78/ 402) believed their current health depends on them taking tamox- ifen and 18.2% (73/401) believed they would become very ill without it (Table 2). Concerns about tamoxifen were common; 72.4% (291/402) worried about its long-term effects and 56.9% (230/404) believed tamoxifen use would result in unpleasant side effects. A signiﬁcant proportion of women reported poor under- standing about tamoxifen; 22.6% (91/402) believed tamoxifen was a “mystery” to them. Perceptions of perceived need for tamoxifen were unrelated to concerns about its usage (see Supplemental Table 2 in the online version). Data are presented as mean (SD) for continuous variables and n (%) for categorical variables. Abbreviation: SES ¼ socioeconomic status. have had reactions to medicines in the past, with 10.7% (43/403) believing that even very small amounts of medication can upset their body. A signiﬁcant proportion of women reported concerns about the nature of medicines and how they are used by doctors. This included the belief that doctors use too many medicines (28.9%; 117/405) and would prescribe fewer medicines if they had more time with patients (35.3%; 143/405). Some women also reported heightened sensitivity to the effects of medication; 22.8% (92/404) reported that they were particularly sensitive to medicines and they Ethical Approval Ethical approval was awarded by the National Research Ethics Service Committee North WestePreston (14/NW/1408). Informed consent was implied with the return of a questionnaire. Availability of Data and Material Participants did not provide explicit consent for their data to be shared in public repositories. Therefore, data may not be made publicly available because of ethical restrictions. We can share the anonymized version of the data with individual qualiﬁed researchers upon request. Data requests may be sent to the corresponding author of this report. Medication Beliefs and Uptake of Tamoxifen Table 1 Demographic, Clinical, and Psychological Variables at Baseline (n [ 408) Variable Value Demographic and Clinical Age 45.30 (7.82) Children Yes 314 (77.0) No 94 (23.0) Ethnic group White 384 (95.5) Other 18 (4.5) Education level Degree or above 176 (44.2) Below degree level 222 (55.8) Health status Poor 16 (4.0) Fair 78 (19.5) Good 240 (60.0) Excellent 66 (16.5) Risk level Moderate 243 (59.6) High 159 (39.0) Unclear 6 (1.4) SES Low (most deprived) 120 (29.9) Middle 131 (32.7) High (least deprived) 150 (37.4) Employment Full-time 348 (85.3) All other employment 60 (14.7) Marital status Married or cohabiting 298 (74.3) Unmarried 103 (25.7) Beliefs about Medicines Questionnaire Speciﬁc necessity 2.63 (0.77) Speciﬁc concerns 3.11 (0.60) General overuse 2.68 (0.73) General harmfulness 2.28 (0.61) Perceived Sensitivity to Medicines Score 2.34 (0.77) Data are presented as mean (SD) for continuous variables and n (%) for categorical variables. Abbreviation: SES ¼ socioeconomic status. A multivariable logistic regression model was used to examine the association between participant characteristics and medication belief group membership. Multivariable logistic regression was also used to examine the role of medication belief group membership on uptake. The analysis was done using Mplus 716 and SPSS version 24.0 (IBM Corp). Statistical signiﬁcance was set at a 2-sided P < .05. at Baseline (n [ 408) Variable Value Demographic and Clinical Age 45.30 (7.82) Children Yes 314 (77.0) No 94 (23.0) Ethnic group White 384 (95.5) Other 18 (4.5) Education level Degree or above 176 (44.2) Below degree level 222 (55.8) Health status Poor 16 (4.0) Fair 78 (19.5) Good 240 (60.0) Excellent 66 (16.5) Risk level Moderate 243 (59.6) High 159 (39.0) Unclear 6 (1.4) SES Low (most deprived) 120 (29.9) Middle 131 (32.7) High (least deprived) 150 (37.4) Employment Full-time 348 (85.3) All other employment 60 (14.7) Marital status Married or cohabiting 298 (74.3) Unmarried 103 (25.7) Beliefs about Medicines Questionnaire Speciﬁc necessity 2.63 (0.77) Speciﬁc concerns 3.11 (0.60) General overuse 2.68 (0.73) General harmfulness 2.28 (0.61) Perceived Sensitivity to Medicines Score 2.34 (0.77) Data are presented as mean (SD) for continuous variables and n (%) for categorical variables. Abbreviation: SES ¼ socioeconomic status. Results In total, 732 women were invited to complete a survey; 408 women (55.7%) returned the baseline survey (Table 1) and 258 (63.2%) women provided uptake data at least 3 months after their appointment (see Supplemental Figure 1 in the online version). Demographic and clinical differences between responders and nonresponders and between those who did and did not provide 3-month data are published elsewhere.6 There were no differences between responders and nonresponders with regard to clinical risk, socioeconomic status (SES), or age group. Women were more likely to provide follow-up data if they were from a higher SES group. There were no differences in medication beliefs between women who provided baseline data and those who provided baseline and 3-month data (see Supplemental Table 1 in the online version). Medication Belief Groups Model ﬁt statistics for 1 through 5 class solutions are presented in Supplemental Table 3 in the online version. Although the AIC, BIC, and entropy values supported a 3-class solution, the log ratio (LR) tests were nonsigniﬁcant, suggesting that extraction of 3 classes did not improve model ﬁt above a 2-class solution. Furthermore, the e118 Clinical Breast Cancer February 2019 Rachael Jane Thorneloe et al Rachael Jane Thorneloe et al Table 2 Beliefs About Medication and Perceived Sensitivity to its Effects for the Entire Sample and Medication Belief Groups (n [ 408) (n [ 408) Sample (n [ 408) Group 1 (Low Need, Lower Concerns) (62%; n [ 252) Group 2 (Low Need, Higher Concerns) (38%; n [ 154) BMQ Speciﬁc Necessity Beliefs 1. My current health depends on me taking tamoxifen 19.4 21.4 16.2 2. Without tamoxifen, I could become very ill 18.2 18.1 18.4 3. My future health depends on me taking tamoxifen 22.1 25.3 16.9 BMQ Speciﬁc Concern Beliefs 1. Taking tamoxifen would worry me 61.3 56.9 68.6 2. I worry about the long-term effects of tamoxifen 72.4 66.3 82.4 3. Tamoxifen is a mystery to me 22.6 17.7 30.5 4. Taking tamoxifen would disrupt my life 23.8 21.6 27.3 5. I worry I would become dependent on tamoxifen 9.2 6.0 14.5 6. Tamoxifen would give me unpleasant side effects 56.9 52.0 64.9 BMQ General Overuse Beliefs 1. Doctors use too many medicines 28.9 10.4 59.1 2. Natural remedies are safer than medicines 17.0 6.0 35.1 3. Doctors place too much trust in medicines 14.3 2.0 34.4 4. If doctors had more time with patients they would prescribe fewer medicines 35.3 16.7 66.0 BMQ General Harmfulness Beliefs 1. People who take medicines should stop for a while every now and again 23.7 10.8 44.8 2. Most medicines are addictive 13.3 3.2 29.9 3. Medicines do more harm than good 3.2 0.4 7.9 4. All medicines are poisons 5.9 1.2 13.6 PSM 1. My body is very sensitive to medicines 22.8 17.1 32.0 2. My body over-reacts to medicines 8.9 5.2 14.9 3. I usually have stronger reactions to medicines than most people 7.2 4.8 11.0 4. I have had a bad reaction to medicines in the past 24.2 21.0 29.4 5. Medication Belief Groups Even very small amounts of medicines can upset my body 10.7 8.0 15.0 Data are the percentage who agreed or strongly agreed with each statement; reference category: strongly disagree, disagree, and unsure. Abbreviations: BMQ ¼ Beliefs about Medicines Questionnaire; PSM ¼ Perceived Sensitivity to Medicines Scale. Predictors of Tamoxifen Uptake Uptake of chemoprevention was 14.7% (38/258); 31 women were currently taking tamoxifen and 7 women reported having a prescription. Uptake according to clinic setting is presented in Supplemental Table 7 in the online version. Women classiﬁed into group 1 (low necessity, lower concerns) were more likely to initiate tamoxifen (18.3%; 33/180) than those classiﬁed into group 2 (low necessity, higher concerns) (6.4%; 5/78). After adjusting for de- mographic and clinical factors, the OR was 3.37 (95% CI, 1.08- 10.51; P ¼ .036; Table 3). Factors Related to Medication Belief Group Membership Factors Related to Medication Belief Group Membership Women classiﬁed into group 2 (low need, higher concerns) were more likely to be: aged 50 years or older (vs. 36-49 years), from nonwhite ethnic groups (vs. white ethnic group), not working full-time (vs. full-time employment), and unmarried (vs. married or cohabiting; see Supplemental Table 6 in the online version). Only age (50 years or older vs. 36-49 years) remained signiﬁcantly associated with medication belief group membership in multivariable analyses (odds ratio [OR], 0.56; 95% CI, 0.34-0.93; P ¼ .024). It is important to determine whether preventive therapies can create or exacerbate existing inequalities in breast cancer out- comes.17 We have previously shown within this cohort that there are no sociodemographic differences in tamoxifen uptake.6 In this study, medication belief group membership was associated with key indicators of SES, which might help identify those who would most beneﬁt from additional decision-making support. Medication beliefs are key modiﬁable determinants of treatment decision-making.8 Beliefs about breast cancer risk and its treatment are complex and inﬂuenced by family experiences of cancer and medication use.6 We have illustrated the speciﬁc medication beliefs held by individuals at increased risk, with the identiﬁcation of subgroup differences having implications for supporting informed decision-making. Perceived need for tamoxifen was low, suggesting intervention strategies should focus on communicating the role tamoxifen could play in cancer prevention, while balancing this with information about harms and respecting women’s decision to decline. Although women who reported low need and lower con- cerns (group 1) were more likely to initiate tamoxifen, uptake was still low in this group. For those who initiate tamoxifen, continued uncertainty about personal need might result in lower adherence, which has been shown to be problematic in clinical trials.18-20 BMQ Speciﬁc Necessity Beliefs Data are the percentage who agreed or strongly agreed with each statement; reference category: strongly disagree, disagree, and unsure. Abbreviations: BMQ ¼ Beliefs about Medicines Questionnaire; PSM ¼ Perceived Sensitivity to Medicines Scale. and perceived sensitivity to medicines. Women classiﬁed into group 2 (38%; 154) reported the strongest concerns about tamoxifen and medicines in general, as well as stronger perceived sensitivity to the effects of medicines, compared with women classiﬁed into group 1 (62%; 252). The largest difference between the groups was for concerns about the overuse and harmfulness of medicines in general (Table 2). A higher proportion of women classiﬁed into group 2 (low necessity and higher concerns) believed that doctors use too many (59.1% [91/154] vs. 10.4% [26/251]) and place too much trust in medicines (34.4% [53/154] vs. 2.0% [5/251]), and would prescribe fewer medicines if they had more time with patients (66% [101/153] vs. 16.7% [42/252]). A higher proportion of women in second class was a small group (n ¼ 14). A 2-class solution was selected; both LR tests were signiﬁcant with good sample sizes within each latent class. Excluding the PSM scale did not improve model ﬁt (see Supplemental Table 4 in the online version). Rerunning the analysis using only participants who had completed baseline and had 3-month uptake data indicated a 2-class solution with similar medication belief proﬁles (see Supplemental Table 5 in the online version). Sample means (95% conﬁdence interval [CI]) of medication beliefs for the 2-class solution are presented in Figure 1. Both medication belief groups perceived a low need for tamoxifen (sub- scale: speciﬁc necessity), but differed in their medication concerns e119 Clinical Breast Cancer February 2019 Clinical Breast Cancer February 2019 Medication Beliefs and Uptake of Tamoxifen Medication Beliefs and Uptake of Tamoxifen Figure 1 Sample Means [95% CI] of Medication Beliefs for the 2-Class Solution (n [ 406). Chart Shows Differences in Medication Beliefs Between Group 1 (Low Need, Lower Concerns) and Group 2 (Low Need, Higher Concerns) Figure 1 Sample Means [95% CI] of Medication Beliefs for the 2-Class Solution (n [ 406). Chart Shows Differences in Medication Beliefs Between Group 1 (Low Need, Lower Concerns) and Group 2 (Low Need, Higher Concerns) Figure 1 Sample Means [95% CI] of Medication Beliefs for the 2-Class Solution (n [ 406). BMQ Speciﬁc Necessity Beliefs Chart Shows Differences in Medication Beliefs Between Group 1 (Low Need, Lower Concerns) and Group 2 (Low Need, Higher Concerns) Figure 1 Sample Means [95% CI] of Medication Beliefs for the 2-Class Solution (n [ 406). Chart Shows Differences in Medication Beliefs Between Group 1 (Low Need, Lower Concerns) and Group 2 (Low Need, Higher Concerns) group 2 also believed that medicines are poisons (13.6% [21/154] vs. 1.2% [3/252]), addictive (29.9% [46/154] vs. 3.2% [8/252]), and people who take medicines should stop for a while every now and again (44.8% [69/154] vs. 10.8% [27/251]). worries about its long-term effects and more than half reported concerns about potential unpleasant side effects. A subgroup of women, accounting for almost two-ﬁfths of the sample, reported the strongest medication concerns and perceived sensitivity to medi- cines. Women with low necessity and lower concerns were more likely to initiate tamoxifen than those with low necessity and higher concerns. Discussion In this United Kingdom multicenter study, only 1 in 5 women at increased risk of breast cancer reported a strong need for tamoxifen preventive therapy. More than 70% of women reported strong An important subgroup of women reported low need for tamoxifen and stronger medication concerns, and these beliefs e120 Clinical Breast Cancer February 2019 Rachael Jane Thorneloe et al Rachael Jane Thorneloe et al p g p g g (n [ 258) Uptake, n (%) Univariable Multivariable OR (95% CI) P OR (95% CI) P Age 35 Yearsa 1 (3.8)a e e 36-49 Years 29 (17.3) 1.46 (0.63-3.39) .378 1.19 (0.44-3.18) .731 50 Years 8 (12.5) Ref Ref Children Yes 36 (17.6) 5.43 (1.26-23.34) .023 3.66 (0.76-17.64) .106 No 2 (3.8) Ref Ref Ethnic Groupa White 37 (15) e e e e Other 1 (11.1) e e e e Education Level Degree or above 20 (17.2) 1.41 (0.71-2.82) .327 1.50 (0.66-3.42) .335 Below degree level 18 (12.9) Ref Ref Health Status Poora 0 e e Fair 5 (10.6) 0.68 (0.20-2.32) .538 0.53 (0.13-2.13) .372 Good 25 (16.6) 1.13 (0.46-2.82) .787 0.97 (0.37-2.60) .958 Excellent 7 (14.9) Ref Ref Risk Level Moderate 24 (15.1) 1.05 (0.52-2.15) .885 0.84 (0.38-1.82) .651 High 14 (14.4) Ref Ref Uncleara 0 e SES Low (most deprived) 7 (11.9) 0.78 (0.30-2.03) .613 1.23 (0.44-3.39) .695 Middle 14 (16.3) 1.13 (0.52-2.47) .759 1.38(0.57-3.33) .479 High (least deprived) 16 (14.7) Ref Ref Employment Full-time 32 (14.5) Ref Ref All other employment 6 (16.2) 1.14 (0.44-2.96) .783 1.82 (0.63-5.22) .269 Marital Status Married or cohabiting 33 (16.7) 2.16 (0.80-5.81) .127 1.47 (0.44-4.93) .534 Unmarried 5 (8.5) Ref Ref Medication Belief Group Group 1 (low need, lower concerns) 33 (18.3) 3.28 (1.23-8.75) .018 3.37 (1.08-10.51) .036 Group 2 (low need, higher concerns) 5 (6.4) Ref Ref Bold P values indicate statistical signiﬁcance P < .05. Abbreviations: OR ¼ odds ratio; Ref ¼ reference; SES ¼ socioeconomic status. aCategory not included in univariable and multivariable analyses because of insufﬁcient cases; the multivariable model included 213 respondents. Bold P values indicate statistical signiﬁcance P < .05. Abbreviations: OR ¼ odds ratio; Ref ¼ reference; SES ¼ socioeconomic status. aCategory not included in univariable and multivariable analyses because of insufﬁcient cases; the multivariable model included 213 respondents. Acknowledgments 12. National Institute for Health and Care Excellence (NICE). Familial breast cancer (CG164): Classiﬁcation, care and managing breast cancer and related risks in people with a family history of breast cancer. Issued: June. Available from: https:// www.nice.org.uk/guidance/cg164. Accessed June 1, 2018. The authors acknowledge the contribution of the ENGAGE col- laborators (in alphabetical order): Vanessa Adamson, Sarah Ains- worth, Malin Akerlund, Ivanna Baker, Julian Barwell, Jayne Beesley, Lisa Brock, Chrissie Butcher, Janice Carpenter, Martyn Clark, Shirley Cocks, Veronica Conteh, Martina Coulding, Sue Darby, Angela Duckworth, Gareth Evans, Catherine Fensom, Julie Fletcher, Kate Foster, Sara Grieg, Elaine Gullaksen, Jana Gurasashvili, Lisa Hard- staff, Rachel Hart, Kathryn Hoare, Jonathan Hoffman, Christopher Holcombe, Lynne Horton, Antony Howell, Farah Islam, Emma Jenkinson, Karen Jewers, Manisha Joshi, Amy Kirkby, Peter Knee- shaw, Natalie Knife, Jalal Kokan, Jin Li, Nicola Lunt, Douglas Macmillan, Karen Makinson, Evangelos Mallidis, Sarah Many- angadze, Charity Masvaure, Raksha Mistry, Alice Ngumo, Jane Ooi, Ashraf Patel, Vanessa Pope, Laura Price, Fiona Rabson, Lisa Richardson, Stephanie Ridgway, Karen Riley, Lorraine Roberts, Janet Ryan-Smith, Vian Salih, Nicky Scott, Mike Shere, Andrew Sloan, Nita Solanky, Amanda Taylor, Dinesh Thekkinkattil, Heather Thomas, Mangesh Thorat, Barbara Townley,Jayant S.Vaidya, Lynda Wagstaff, Shane Walsh, Lynsey Waring, Donna Watterson, Char- lotte Westley, Lesley Wilkinson, Nicola Willis, and Julia Wiseman. 13. Horne R, Faasse K, Cooper V, et al. The Perceived Sensitivity to Medicines (PSM) scale: an evaluation of validity and reliability. Br J Health Psychol 2013; 18:18-30. 14. McLennan D, Barnes H, Noble M, et al. The English indices of deprivation 2010. Available at: https://www.gov.uk/government/uploads/system/uploads/ attachment_data/ﬁle/6320/1870718.pdf. Accessed June 1, 2018. p J 15. Smith S, Thorneloe R. Decision-making in breast cancer prevention: the ENGAGE study. Available at: https://osf.io/bn6tw. Accessed January 24 2018. 16. Muthén LK, Muthén BO. Mplus User’s Guide. 7th ed. Los Angeles, CA: Muthén & Muthén; 1998-2017. 17. Cancer Research UK. Breast cancer statistics. Available at: http://www. cancerresearchuk.org/health-professional/cancer-statistics/statistics-by-cancer-type/ breast-cancer. Accessed June 1, 2018. 18. Smith SG, Sestak I, Howell A, et al. Participant-reported symptoms and their effect on long-term adherence in the International Breast Cancer Intervention Study I (IBIS I). J Clin Oncol 2017; 35:2666-73. 19. Land SR, Walcott FL, Liu Q, et al. Symptoms and QOL as predictors of che- moprevention adherence in NRG Oncology/NSABP Trial P-1. J Natl Cancer Inst 2016; 108:djv365. 20. Sestak I, Smith SG, Howell A, et al. Clinical Practice Points 3. Cuzick J, Sestak I, Cawthorn S, et al. Tamoxifen for prevention of breast cancer: extended long-term follow-up of the IBIS-I breast cancer prevention trial. Lancet Oncol 2015; 16:67-75. The effectiveness of preventive therapy for breast cancer depends on adequate uptake, but initiation rates remain low. Across many disease contexts, individuals’ beliefs about medication have been shown to inﬂuence treatment decision-making. Little is known about the psychological factors inﬂuencing the decision to use chemoprevention. 4. Smith SG, Sestak I, Forster A, et al. Factors affecting uptake and adherence to breast cancer chemoprevention: a systematic review and meta-analysis. Ann Oncol 2016; 27:575-90. 4. Smith SG, Sestak I, Forster A, et al. Factors affecting uptake and adherence to breast cancer chemoprevention: a systematic review and meta-analysis. Ann Oncol 2016; 27:575-90. 5. Donnelly LS, Evans DG, Wiseman J, et al. Uptake of tamoxifen in consecutive premenopausal women under surveillance in a high-risk breast cancer clinic. Br J Cancer 2014; 110:1681-7. 6. Hackett J, Thorneloe R, Side L, et al. Uptake of breast cancer preventive therapy in the UK: results from a multicentre prospective survey and qualitative interviews. Breast Cancer Res Treat 2018; 170:633-40. Our multicenter prospective study showed that uptake of breast cancer preventive therapy was low. Using LPA, we identiﬁed an important subgroup of women who reported low need for pre- ventive therapy and strong medication concerns. These women were less likely to initiate tamoxifen. 7. Curtis HJ, Walker AJ, Goldacre B. Impact of NICE guidance on tamoxifen prescribing in England 2011-2017: an interrupted time series analysis. Br J Cancer 2018; 118:1268-75. 8. Horne R, Chapman SC, Parham R, et al. Understanding patients’ adherence- related beliefs about medicines prescribed for long-term conditions: a meta- analytic review of the Necessity-Concerns Framework. PLoS One 2013; 8:e80633. 9. Horne R, Weinman J, Hankins M. The Beliefs About Medicines questionnaire: the development and evaluation of a new method for assessing the cognitive representation of medication. Psychol Health 1999; 14:1-24. This study identiﬁed why some women might decide to opt out of taking tamoxifen as a preventive measure. Identifying and addressing medication beliefs might help support informed decision-making. p y 10. Bober SL, HokeLA, Duda RB, et al.Decision-making about tamoxifen in women at high risk for breast cancer: clinical and psychological factors. J Clin Oncol 2004; 22:4951-7. Th l G fﬁh C l l l d l 11. Clinical Practice Points Thorneloe RJ, Grifﬁths CE, Emsley R, et al. Intentional and unintentional medication non-adherence in psoriasis: the role of patients’ medication beliefs and habit strength. J Invest Dermatol 2018; 138:785-94. Medication Beliefs and Uptake of Tamoxifen The authors also thank the women who participated in the study. however, some women might not have made their decision at the time of follow-up. These data are self-reported, and therefore uptake estimates might be biased. A number of sociodemographic, clinical, and psychological factors have been reported to be associated with uptake.4 We did not explore the quality of clinicianepatient communication, which might inﬂuence women’s knowledge, understanding, and beliefs about tamoxifen. However, our ﬁndings point to potentially modiﬁable targets to help women make an informed choice regarding preventive therapy. References 1. Jemal A, Bray F, Center MM, et al. Global cancer statistics. CA Cancer J Clin 2011; 61:69-90. 2. Cuzick J, Sestak I, Bonanni B, et al. Selective oestrogen receptor modulators in prevention of breast cancer: an updated meta-analysis of individual participant data. Lancet 2013; 381:1827-34. Disclosure R.J.T. has received honorarium from Novartis. S.G.S. has received consulting fees from Luto Research. The remaining authors have stated that they have no conﬂicts of interest. Conclusion In this multicenter study, the decision to initiate tamoxifen was predicted by women’s beliefs about tamoxifen and medicines in general, as well as perceived sensitivity to its negative effects. Elic- iting and addressing women’s beliefs might help support informed decision-making. Supplemental Data A supplemental tables and ﬁgure accompanying this article can be found in the online version at https://doi.org/10.1016/j.clbc.2018. 10.008. Strengths and Limitations inﬂuenced uptake decisions. This group might beneﬁt from additional support that focuses on eliciting and addressing unre- solved medication concerns.21 Treatment expectations have been shown to increase the risk of treatment-speciﬁc side effects and nonadherence in the context of secondary breast cancer preven- tion.22 Our study shows how previous treatment expectations can inﬂuence primary prevention decision-making and emphasizes the need for clinicians to address concerns and ensure realistic treat- ment expectations. inﬂuenced uptake decisions. This group might beneﬁt from additional support that focuses on eliciting and addressing unre- solved medication concerns.21 Treatment expectations have been shown to increase the risk of treatment-speciﬁc side effects and nonadherence in the context of secondary breast cancer preven- tion.22 Our study shows how previous treatment expectations can inﬂuence primary prevention decision-making and emphasizes the need for clinicians to address concerns and ensure realistic treat- ment expectations. The participation of more than 400 women from 20 centers across England reﬂects the experiences of treatment decision- making in clinical practice. The sample size was reduced for data on tamoxifen uptake, but sensitivity analyses did not indicate bias. Although the low level of uptake is comparable with other studies,4 it might have reduced statistical power. All women were given 3 months to decide whether they would like to initiate tamoxifen, Clinical Breast Cancer February 2019 e121 Clinical Breast Cancer February 2019 P value tests for signiﬁcant differences between baseline and baseline and 3-month cohorts using t tests. g g Abbreviations: BMQ ¼ Beliefs about Medicines Questionnaire; PSM ¼ Perceived Sensitivity to Medicines Scale. Acknowledgments Early participant-reported symptoms as predictors of adherence to anastrozole in the International Breast Cancer Inter- vention Studies II. Ann Oncol 2018; 29:504-9. 21. Phillips LA, Leventhal H, Leventhal EA. Physicians’ communication of the common-sense self-regulation model results in greater reported adherence than physicians’ use of interpersonal skills. Br J Health Psychol 2012; 17:244-57. 22. Nestoriuc Y, von Blanckenburg P, Schuricht F, et al. Is it best to expect the worst? Inﬂuence of patients’ side effect expectations on endocrine treatment outcome in a 2-year prospective clinical cohort study. Ann Oncol 2016; 27:1909-15. e122 Clinical Breast Cancer February 2019 Supplemental Figure 1 Recruitment Flow Diagram Did not consent and did not complete baseline assessment (n = 324) Consented and completed baseline assessment (n = 408) Shown invitaon and screened (n = 732) Did not complete 3-month assessment (n = 150) Completed 3-month assessment (n = 258) Supplemental Data Rachael Jane Thorneloe et al Supplemental Figure 1 Recruitment Flow Diagram Did not consent and did not complete baseline assessment (n = 324) Consented and completed baseline assessment (n = 408) Shown invitaon and screened (n = 732) Did not complete 3-month assessment (n = 150) Completed 3-month assessment (n = 258) Supplemental Data Rachael Jane Thorneloe et al Rachael Jane Thorneloe et al Did not consent and did not complete baseline assessment (n = 324) Consented and completed baseline assessment (n = 408) Did not complete 3-month assessment (n = 150) Supplemental Table 1 Univariable Comparison of Retention According to Medication Beliefs (n [ 408) Mean (SD) Baseline Only (n [ 150) Baseline and 3 Months (n [ 258) P BMQ Speciﬁc Necessity 2.66 (0.72) 2.61 (0.80) .549 BMQ Speciﬁc Concerns 3.07 (0.61) 3.14 (0.59) .297 BMQ General Overuse 2.71 (0.72) 2.67 (0.73) .611 BMQ General Harmfulness 2.30 (0.58) 2.27 (0.63) .629 PSM 2.32 (0.80) 2.34 (0.75) .798 P value tests for signiﬁcant differences between baseline and baseline and 3-month cohorts using t tests. Abbreviations: BMQ ¼ Beliefs about Medicines Questionnaire; PSM ¼ Perceived Sensitivity to Medicines Scale. Acknowledgments Medication Beliefs and Uptake of Tamoxifen Medication Beliefs and Uptake of Tamoxifen Supplemental Table 3 Model Fit Statistics for BMQ and PSM Variables (n [ 406) Class 1 2 3 4 5 Parameters 10 16 22 28 34 LL 2090.764 1997.354 1964.527 1942.876 1931.403 AIC 4201.528 4026.709 3973.054 3941.752 3930.807 BIC 4241.592 4090.810 4061.194 4053.930 4067.023 Entropy e 0.666 0.759 0.777 0.730 Sample Size per Class, % (n) e Class 1 ¼ 62 (252) Class 2 ¼ 38 (154) Class 1 ¼ 49.3 (200) Class 2 ¼ 3.4 (14) Class 3 ¼ 47.3 (192) Class 1 ¼ 9.9 (40) Class 2 ¼ 33 (134) Class 3 ¼ 2.7 (11) Class 4 ¼ 54.4 (221) Class 1 ¼ 17 (69) Class 2 ¼ 3.0 (12) Class 3 ¼ 32.2 (131) Class 4 ¼ 43.1 (175) Class 5 ¼ 4.7 (19) VLMR-LRT P Value e .0001 .2951 .1590 .7835 LMR-LRT P Value e .0001 .3023 .1652 .7856 Two participants had missing data for all 5 variables and were excluded from the analysis. Abbreviations: AIC ¼ Akaike Information Criterion; BIC ¼ Bayesian Information Criterion; BMQ ¼ Beliefs about Medicines Questionnaire; LL ¼ log-likelihood; LMR-LRT ¼ LoeMendelleRubin adjusted likelihood ratio test; PSM ¼ Perceived Sensitivity to Medicines Scale; VLMR-LRT ¼ VuongeLoeMendelleRubin likelihood ratio test. Supplemental Table 3 Model Fit Statistics for BMQ and PSM Variables (n [ 406) Class 1 2 3 4 5 Parameters 10 16 22 28 34 LL 2090.764 1997.354 1964.527 1942.876 1931.403 AIC 4201.528 4026.709 3973.054 3941.752 3930.807 BIC 4241.592 4090.810 4061.194 4053.930 4067.023 Entropy e 0.666 0.759 0.777 0.730 Sample Size per Class, % (n) e Class 1 ¼ 62 (252) Class 2 ¼ 38 (154) Class 1 ¼ 49.3 (200) Class 2 ¼ 3.4 (14) Class 3 ¼ 47.3 (192) Class 1 ¼ 9.9 (40) Class 2 ¼ 33 (134) Class 3 ¼ 2.7 (11) Class 4 ¼ 54.4 (221) Class 1 ¼ 17 (69) Class 2 ¼ 3.0 (12) Class 3 ¼ 32.2 (131) Class 4 ¼ 43.1 (175) Class 5 ¼ 4.7 (19) VLMR-LRT P Value e .0001 .2951 .1590 .7835 LMR-LRT P Value e .0001 .3023 .1652 .7856 Two participants had missing data for all 5 variables and were excluded from the analysis. Acknowledgments Clinical Breast Cancer February 2019 - e123 Supplemental Table 1 Univariable Comparison of Retention According to Medication Beliefs (n [ 408) Mean (SD) Baseline Only (n [ 150) Baseline and 3 Months (n [ 258) P BMQ Speciﬁc Necessity 2.66 (0.72) 2.61 (0.80) .549 BMQ Speciﬁc Concerns 3.07 (0.61) 3.14 (0.59) .297 BMQ General Overuse 2.71 (0.72) 2.67 (0.73) .611 BMQ General Harmfulness 2.30 (0.58) 2.27 (0.63) .629 PSM 2.32 (0.80) 2.34 (0.75) .798 P value tests for signiﬁcant differences between baseline and baseline and 3-month cohorts using t tests. Abbreviations: BMQ ¼ Beliefs about Medicines Questionnaire; PSM ¼ Perceived Sensitivity to Medicines Scale. e123 Supplemental Table 2 Correlations Between Medication Belief Variables (n [ 408) 1 2 3 4 Speciﬁc Necessity e e e e Speciﬁc Concerns .040 e e e General Overuse .099a .293b e e General Harmfulness .060 .294b .623b e Perceived Sensitivity to Medicines .032 .252b .193b .174b Data presented are Pearson correlation coefﬁcients. aCorrelation is signiﬁcant at the .05 level. bCorrelation is signiﬁcant at the .01 level. Medication Beliefs and Uptake of Tamoxifen Supplemental Table 2 Correlations Between Medication Belief Variables (n [ 408) 1 2 3 4 Speciﬁc Necessity e e e e Speciﬁc Concerns .040 e e e General Overuse .099a .293b e e General Harmfulness .060 .294b .623b e Perceived Sensitivity to Medicines .032 .252b .193b .174b Data presented are Pearson correlation coefﬁcients. aCorrelation is signiﬁcant at the .05 level. bCorrelation is signiﬁcant at the .01 level. Two participants had missing data for all 5 variables and were excluded from the analysis. Abbreviations: AIC ¼ Akaike Information Criterion; BIC ¼ Bayesian Information Criterion; BMQ ¼ Beliefs about Medicines Questionnaire; LL ¼ log-likelihood; LMR-LRT ¼ LoeMendelleRubin adjusted likelihood ratio test; PSM ¼ Perceived Sensitivity to Medicines Scale; VLMR-LRT ¼ VuongeLoeMendelleRubin likelihood ratio test. Acknowledgments Abbreviations: AIC ¼ Akaike Information Criterion; BIC ¼ Bayesian Information Criterion; BMQ ¼ Beliefs about Medicines Questionnaire; LL ¼ log-likelihood; LMR-LRT ¼ LoeMendelleRubin adjusted likelihood ratio test; VLMR-LRT ¼ VuongeLoeMendelleRubin likelihood ratio test. Supplemental Table 4 Model Fit Statistics for BMQ Variables Only (n [ 406) Class 1 2 3 4 5 Parameters 8 13 18 23 28 LL 1632.263 1546.829 1520.696 1498.641 1492.457 AIC 3280.526 3119.659 3077.391 3043.283 3040.914 BIC 3312.576 3171.741 3149.505 3135.429 3153.092 Entropy e 0.661 0.706 0.815 0.790 Sample Size per Class, % (n) e Class 1 ¼ 61.3 (249) Class 2 ¼ 38.7 (157) Class 1 ¼ 12 (49) Class 2 ¼ 62.6 (254) Class 3 ¼ 25.4 (103) Class 1 ¼ 60.1 (244) Class 2 ¼ 11.1 (45) Class 3 ¼ 1 (4) Class 4 ¼ 27.8 (113) Class 1 ¼ 26.4 (107) Class 2 ¼ 11.1 (45) Class 3 ¼ 53 (215) Class 4 ¼ 9 (37) Class 5 ¼ 0.5 (2) VLMR-LRT P Value e .0000 .1910 .0638 .4124 LMR-LRT P Value e .0001 .1990 .0673 .4194 Two participants had missing data for all 4 variables and were excluded from the analysis. Abbreviations: AIC ¼ Akaike Information Criterion; BIC ¼ Bayesian Information Criterion; BMQ ¼ Beliefs about Medicines Questionnaire; LL ¼ log-likelihood; LMR-LRT ¼ LoeMendelleRubin adjusted likelihood ratio test; VLMR-LRT ¼ VuongeLoeMendelleRubin likelihood ratio test. Supplemental Table 4 Model Fit Statistics for BMQ Variables Only (n [ 406) Two participants had missing data for all 4 variables and were excluded from the analysis. Abbreviations: AIC ¼ Akaike Information Criterion; BIC ¼ Bayesian Information Criterion; BMQ ¼ Beliefs about Medicines Questionnaire; LL ¼ log-likelihood; LMR-LRT ¼ LoeMendelleRubin adjusted likelihood ratio test; VLMR-LRT ¼ VuongeLoeMendelleRubin likelihood ratio test. Acknowledgments Abbreviations: AIC ¼ Akaike Information Criterion; BIC ¼ Bayesian Information Criterion; BMQ ¼ Beliefs about Medicines Questionnaire; LL ¼ log-likelihood; LMR-LRT ¼ LoeMendelleRubin adjusted likelihood ratio test; PSM ¼ Perceived Sensitivity to Medicines Scale; VLMR-LRT ¼ VuongeLoeMendelleRubin likelihood ratio test. Two participants had missing data for all 5 variables and were excluded from the analysis. Abbreviations: AIC ¼ Akaike Information Criterion; BIC ¼ Bayesian Information Criterion; BMQ ¼ Beliefs about Medicines Questionnaire; LL ¼ log-likelihood; LMR-LRT ¼ LoeMendelleRubin adjusted likelihood ratio test; PSM ¼ Perceived Sensitivity to Medicines Scale; VLMR-LRT ¼ VuongeLoeMendelleRubin likelihood ratio test. Two participants had missing data for all 5 variables and were excluded from the analysis. Two participants had missing data for all 5 variables and were excluded from the analysis. Abbreviations: AIC ¼ Akaike Information Criterion; BIC ¼ Bayesian Information Criterion; BMQ ¼ Beliefs about Medicines Questionnaire; LL ¼ log-likelihood; LMR-LRT ¼ LoeMendelleRubin adjusted likelihood ratio test; PSM ¼ Perceived Sensitivity to Medicines Scale; VLMR-LRT ¼ VuongeLoeMendelleRubin likelihood ratio test. Two participants had missing data for all 5 variables and were excluded from the analysis. Abbreviations: AIC ¼ Akaike Information Criterion; BIC ¼ Bayesian Information Criterion; BMQ ¼ Beliefs about Medicines Questionnaire; LL ¼ log-lik adjusted likelihood ratio test; PSM ¼ Perceived Sensitivity to Medicines Scale; VLMR-LRT ¼ VuongeLoeMendelleRubin likelihood ratio test. e124 Clinical Breast Cancer February 2019 Clinical Breast Cancer February 2019 Rachael Jane Thorneloe et al Supplemental Table 4 Model Fit Statistics for BMQ Variables Only (n [ 406) Class 1 2 3 4 5 Parameters 8 13 18 23 28 LL 1632.263 1546.829 1520.696 1498.641 1492.457 AIC 3280.526 3119.659 3077.391 3043.283 3040.914 BIC 3312.576 3171.741 3149.505 3135.429 3153.092 Entropy e 0.661 0.706 0.815 0.790 Sample Size per Class, % (n) e Class 1 ¼ 61.3 (249) Class 2 ¼ 38.7 (157) Class 1 ¼ 12 (49) Class 2 ¼ 62.6 (254) Class 3 ¼ 25.4 (103) Class 1 ¼ 60.1 (244) Class 2 ¼ 11.1 (45) Class 3 ¼ 1 (4) Class 4 ¼ 27.8 (113) Class 1 ¼ 26.4 (107) Class 2 ¼ 11.1 (45) Class 3 ¼ 53 (215) Class 4 ¼ 9 (37) Class 5 ¼ 0.5 (2) VLMR-LRT P Value e .0000 .1910 .0638 .4124 LMR-LRT P Value e .0001 .1990 .0673 .4194 Two participants had missing data for all 4 variables and were excluded from the analysis. Two participants had missing data for all 4 variables and were excluded from the analysis. Abbreviations: AIC ¼ Akaike Information Criterion; BIC ¼ Bayesian Information Criterion; BMQ ¼ Beliefs about Medicines Questionnaire; LL ¼ log-likelihood; LMR-LRT ¼ LoeMendelleRubin adjusted likelihood ratio test; VLMR-LRT ¼ VuongeLoeMendelleRubin likelihood ratio test. Acknowledgments e125 Clinical Breast Cancer February 2019 Supplemental Table 6 Medication Belief Group Membership According to Participant Characteristics and Univariable and Multi- variable Logistic Regression Model (n [ 406) Group 2: Low Need, Higher Concerns, n (%) Univariable Multivariable OR (95% CI) P OR (95% CI) P Age 35 years 16 (39) 0.70 (0.34-1.46) .346 0.65 (0.28-1.56) .337 36-49 years 87 (33.7) 0.56 (0.35-0.88) .013 0.56 (0.34-0.93) .024 50 years 51 (47.7) Ref Ref Children Yes 115 (36.6) 0.79 (0.49-1.26) .317 0.88 (0.49-1.57) .653 No 39 (42.4) Ref Ref Ethnic Group White 140 (36.5) Ref Ref Other 11 (61.1) 2.74 (1.04-7.23) .042 2.40 (0.81-7.14) .117 Education Level Degree or above 58 (33) 0.74 (0.49-1.11) .143 0.71 (0.44-1.13) .148 Below degree level 89 (40.1) Ref Ref Health Status Poor 8 (50) 1.64 (0.55-4.92) .378 1.50 (0.43-5.25) .526 Fair 38 (48.7) 1.56 (0.80-3.04) .192 1.32 (0.64-2.72) .457 Good 80 (33.3) 0.82 (0.47-1.44) .491 0.69 (0.38-1.27) .234 Excellent 25 (37.9) Ref Ref Risk Level Moderate 96 (39.7) 1.20 (0.79-1.81) .395 1.41 (0.89-2.23) .144 High 56 (35.4) Ref Ref Uncleara 2 (33.3) e e SES Low (most deprived) 56 (47.1) 1.63 (0.99-2.66) .052 1.20(0.69-2.08) .525 Middle 43 (32.8) 0.89 (0.55-1.47) .658 0.77 (0.45-1.33) .352 High (least deprived) 53 (35.3) Ref Ref Employment Full-time 125 (35.9) Ref Ref All other employment 29 (50) 1.78 (1.02-3.12) .043 1.39 (0.74-2.62) .313 Marital Status Married or cohabiting 100 (33.6) Ref Ref Unmarried 51 (49.5) 1.94 (1.23-3.06) .004 1.63(0.96-2.76) .071 Bold P values indicate statistical signiﬁcance P < .05. Abbreviation: OR ¼ odds ratio; Ref ¼ reference; SES ¼ socioeconomic status. aCategory not included in univariable and multivariable analyses because of insufﬁcient cases. The multivariable model included 379 respondents. Acknowledgments Supplemental Table 5 Model Fit Statistics for BMQ and PSM Variables, for Baseline and 3 Months (n [ 258) Class 1 2 3 4 5 Parameters 10 16 22 28 34 LL 1343.119 1281.764 1257.355 1232.145 1222.712 AIC 2706.238 2595.527 2558.710 2520.291 2513.423 BIC 2741.767 2652.374 2636.875 2619.774 2634.224 Entropy e 0.719 0.758 0.852 0.862 Sample size per class (%; n) e Class 1 ¼ 70 (180) Class 2 ¼ 30 (78) Class 1 ¼ 50 (129) Class 2 ¼ 46.1 (119) Class 3 ¼ 3.9 (10) Class 1 ¼ 11.6 (30) Class 2 ¼ 26 (67) Class 3 ¼ 60.9 (157) Class 4 ¼ 1.5 (4) Class 1 ¼ 11.6 (30) Class 2 ¼ 1.6 (4) Class 3 ¼ 58.5 (151) Class 4 ¼ 26.4 (68) Class 5 ¼ 1.9 (5) VLMR-LRT P Value e .0019 .2136 .1444 .7560 LMR-LRT P Value e .0022 .2215 .1500 .7598 Two participants had missing data for all 4 variables and were excluded from the analysis. Abbreviations: AIC ¼ Akaike Information Criterion; BIC ¼ Bayesian Information Criterion; BMQ ¼ Beliefs about Medicines Questionnaire; LL ¼ log-likelihood; LMR-LRT ¼ LoeMendelleRubin adjusted likelihood ratio test; PSM ¼ Perceived Sensitivity to Medicines Scale; VLMR-LRT ¼ VuongeLoeMendelleRubin likelihood ratio test. Two participants had missing data for all 4 variables and were excluded from the analysis. Abbreviations: AIC ¼ Akaike Information Criterion; BIC ¼ Bayesian Information Criterion; BMQ ¼ Beliefs about Medicines Questionnaire; LL ¼ log-likelihood; LMR-LRT ¼ LoeMendelleRubin adjusted likelihood ratio test; PSM ¼ Perceived Sensitivity to Medicines Scale; VLMR-LRT ¼ VuongeLoeMendelleRubin likelihood ratio test. Two participants had missing data for all 4 variables and were excluded from the analysis. Abbreviations: AIC ¼ Akaike Information Criterion; BIC ¼ Bayesian Information Criterion; BMQ ¼ Beliefs about Medicines Questionnaire; LL ¼ log-likelihood; LMR-LRT ¼ LoeMendelleRubin adjusted likelihood ratio test; PSM ¼ Perceived Sensitivity to Medicines Scale; VLMR-LRT ¼ VuongeLoeMendelleRubin likelihood ratio test. g Abbreviation: OR ¼ odds ratio; Ref ¼ reference; SES ¼ socioeconomic status. aCategory not included in univariable and multivariable analyses because of insufﬁcient cases. The multivariable model included 379 respondents. Acknowledgments Medication Beliefs and Uptake of Tamoxifen Medication Beliefs and Uptake of Tamoxifen Supplemental Table 6 Medication Belief Group Membership According to Participant Characteristics and Univariable and Multi- variable Logistic Regression Model (n [ 406) Group 2: Low Need, Higher Concerns, n (%) Univariable Multivariable OR (95% CI) P OR (95% CI) P Age 35 years 16 (39) 0.70 (0.34-1.46) .346 0.65 (0.28-1.56) .337 36-49 years 87 (33.7) 0.56 (0.35-0.88) .013 0.56 (0.34-0.93) .024 50 years 51 (47.7) Ref Ref Children Yes 115 (36.6) 0.79 (0.49-1.26) .317 0.88 (0.49-1.57) .653 No 39 (42.4) Ref Ref Ethnic Group White 140 (36.5) Ref Ref Other 11 (61.1) 2.74 (1.04-7.23) .042 2.40 (0.81-7.14) .117 Education Level Degree or above 58 (33) 0.74 (0.49-1.11) .143 0.71 (0.44-1.13) .148 Below degree level 89 (40.1) Ref Ref Health Status Poor 8 (50) 1.64 (0.55-4.92) .378 1.50 (0.43-5.25) .526 Fair 38 (48.7) 1.56 (0.80-3.04) .192 1.32 (0.64-2.72) .457 Good 80 (33.3) 0.82 (0.47-1.44) .491 0.69 (0.38-1.27) .234 Excellent 25 (37.9) Ref Ref Risk Level Moderate 96 (39.7) 1.20 (0.79-1.81) .395 1.41 (0.89-2.23) .144 High 56 (35.4) Ref Ref Uncleara 2 (33.3) e e SES Low (most deprived) 56 (47.1) 1.63 (0.99-2.66) .052 1.20(0.69-2.08) .525 Middle 43 (32.8) 0.89 (0.55-1.47) .658 0.77 (0.45-1.33) .352 High (least deprived) 53 (35.3) Ref Ref Employment Full-time 125 (35.9) Ref Ref All other employment 29 (50) 1.78 (1.02-3.12) .043 1.39 (0.74-2.62) .313 Marital Status Married or cohabiting 100 (33.6) Ref Ref Unmarried 51 (49.5) 1.94 (1.23-3.06) .004 1.63(0.96-2.76) .071 Bold P values indicate statistical signiﬁcance P < .05. Abbreviation: OR ¼ odds ratio; Ref ¼ reference; SES ¼ socioeconomic status. aCategory not included in univariable and multivariable analyses because of insufﬁcient cases. The multivariable model included 379 respondents. Medication Beliefs and Uptake of Tamoxifen Bold P values indicate statistical signiﬁcance P < .05. Abbreviation: OR ¼ odds ratio; Ref ¼ reference; SES ¼ socioeconomic status. aCategory not included in univariable and multivariable analyses because of insufﬁcient cases. The multivariable model included 379 respondents. Supplemental Table 7 Uptake of Tamoxifen According to Clinic Setting (n [ 258) Clinic Setting Uptake of Tamoxifen, % (n) Genetics 6.7 (1/15) Breast Clinic 6.9 (2/29) Family History 15.5 (28/181) Family History Clinic and Genetics 21.2 (7/33) e126 - Clinical Breast Cancer February 2019 e126
https://openalex.org/W4207069177	https://dugi-doc.udg.edu/bitstream/10256/21291/1/biomedicines-10-00106.pdf	English	null	Clinical Genetics of Inherited Arrhythmogenic Disease in the Pediatric Population	Biomedicines	2,022	cc-by	21,075	Citation: Martínez-Barrios, E.; Cesar, S.; Cruzalegui, J.; Hernandez, C.; Arbelo, E.; Fiol, V.; Brugada, J.; Brugada, R.; Campuzano, O.; Sarquella-Brugada, G. Clinical Genetics of Inherited Arrhythmogenic Disease in the Pediatric Population. Biomedicines 2022, 10, 106. https://doi.org/ 10.3390/biomedicines10010106 Keywords: Brugada syndrome; catecholaminergic polymorphic ventricular tachycardia; chan- nelopathies; long QT syndrome; short QT syndrome 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. biomedicines biomedicines biomedicines biomedicines Review Estefanía Martínez-Barrios 1, Sergi Cesar 1, José Cruzalegui 1, Clara Hernandez 1, Elena Arbelo 2,3, Victoria Fiol 1, Josep Brugada 1,2,3, Ramon Brugada 2,4,5,6, Oscar Campuzano 2,4,5,,† and Georgia Sarquella-Brugada 1,4,,† 1 Arrhythmias Unit, Hospital Sant Joan de Déu, University of Barcelona, 08007 Barcelona, Spain; estefania.martinez@sjd.es (E.M.-B.); sergio.cesar@sjd.es (S.C.); josecarlos.cruzalegui@sjd.es (J.C.); clara.hernandez@sjd.es (C.H.); victoria.ﬁol@sjd.es (V.F.); jbrugada@clinic.cat (J.B.) 1 Arrhythmias Unit, Hospital Sant Joan de Déu, University of Barcelona, 08007 Barcelona, Spain; estefania.martinez@sjd.es (E.M.-B.); sergio.cesar@sjd.es (S.C.); josecarlos.cruzalegui@sjd.es (J.C.); clara.hernandez@sjd.es (C.H.); victoria.ﬁol@sjd.es (V.F.); jbrugada@clinic.cat (J.B.) j j j g 2 Centro de Investigación Biomédica en Red, Enfermedades Cardiovasculares (CIBERCV), 28029 M EARBELO@clinic.cat (E.A.); rbrugada@idibgi.org (R.B.) 3 Arrhythmias Unit, Hospital Clinic, University of Barcelona-IDIBAPS, 08036 Barcelona, Spain 4 Medical Science Department, School of Medicine, University of Girona, 17004 Girona, Spain 5 Cardiovascular Genetics Center, University of Girona-IDIBGI, 17190 Girona, Spain 6 Cardiology Service, Hospital Josep Trueta, University of Girona, 17007 Girona, Spain * Correspondence: oscar@brugada.org (O.C.); georgia@brugada.org (G.S.-B.) p g g g g † These authors contributed equally to this work. p g g g g g g † These authors contributed equally to this work. Abstract: Sudden death is a rare event in the pediatric population but with a social shock due to its presentation as the ﬁrst symptom in previously healthy children. Comprehensive autopsy in pediatric cases identify an inconclusive cause in 40–50% of cases. In such cases, a diagnosis of sudden arrhythmic death syndrome is suggested as the main potential cause of death. Molecular autopsy identiﬁes nearly 30% of cases under 16 years of age carrying a pathogenic/potentially pathogenic alteration in genes associated with any inherited arrhythmogenic disease. In the last few years, despite the increasing rate of post-mortem genetic diagnosis, many families still remain without a conclusive genetic cause of the unexpected death. Current challenges in genetic diagnosis are the establishment of a correct genotype–phenotype association between genes and inherited arrhythmogenic disease, as well as the classiﬁcation of variants of uncertain signiﬁcance. In this review, we provide an update on the state of the art in the genetic diagnosis of inherited arrhythmogenic disease in the pediatric population. We focus on emerging publications on gene curation for genotype–phenotype associations, cases of genetic overlap and advances in the classiﬁcation of variants of uncertain signiﬁcance. Our goal is to facilitate the translation of genetic diagnosis to the clinical area, helping risk stratiﬁcation, treatment and the genetic counselling of families. Citation: Martínez-Barrios, E.; Cesar, S.; Cruzalegui, J.; Hernandez, C.; Arbelo, E.; Fiol, V.; Brugada, J.; Brugada, R.; Campuzano, O.; Sarquella-Brugada, G. Clinical Genetics of Inherited Arrhythmogenic Disease in the Pediatric Population. Biomedicines 2022, 10, 106. https://doi.org/ 10.3390/biomedicines10010106 1. Introduction While sudden cardiac death (SCD) is a rare event in pediatrics, it has a signiﬁcant social impact, since it often presents as the ﬁrst symptom in previously healthy children. The reported incidence rate of SCD in children and young adults is estimated to be between 1.3 and 1.7 per 100,000 persons-year [1,2], with twice as many cases in males than females. SCD is almost 10 times higher in young adults aged 31–35 years, while very low in children aged 6–10 years of age [3]. In addition to age and sex, other demographic factors such as ethnicity, exercise habits and geographic location impact on the incidence and survival rate of SCD, but the causes remain to be established [4,5]. During the ﬁrst year of life, unexpected deaths remain unexplained after a comprehensive autopsy accounts for the highest infant mortality rate and is usually labelled as sudden infant death syndrome (SIDS) [6]. 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/biomedicines Biomedicines 2022, 10, 106. https://doi.org/10.3390/biomedicines10010106 Biomedicines 2022, 10, 106 2 of 28 The most prevalent etiologies identiﬁed by medico-legal autopsy in children and young adults are cardiomyopathies, accounting for about 30% of SCD [7]. Nevertheless, a negative autopsy result is the most common ﬁnding in pediatrics, accounting for 40–50% of cases in the population under 16 years old. This outcome suggests a diagnosis of sudden arrhythmic death syndrome (SADS), the leading cause of SCD in children [8]. In SADS, the regular heart rhythm is abruptly replaced by a lethal ventricular arrhythmia, in the context of a structurally normal heart [9], the so-called inherited arrhythmia syndromes (IASs) or cardiac channelopathies. p Thanks to recent technological advances in the ﬁeld of genetics, a considerable number of potentially IASs-causing genes have been identiﬁed although not comprehensively characterized. Current panels used in genetic testing of IASs range from the most common genes recommended by the clinical guidelines to the less common genes described in a few families, in large part not deﬁnitively associated with any IASs. Those that can be tested in a fast and cost-effective approach in clinical practice remain a current matter of argument. Therefore, studying a larger number of genes represents new challenges, such as limitations in establishing valid associations between genes and phenotypes. 1. Introduction To address this problem, in 2013 the National Institute of Health (NIH) encouraged the development of ClinGen (Clinical Genome Resource https://clinicalgenome.org/ accessed on 3 December 2021), an international consortium of geneticists, genomic scientists and experts in the clinical ﬁeld, which has established an evidence-based gene curation approach to establish gene– disease associations. Recently, evaluations of IAS-associated genes according to ClinGen’s approach have been published [10–13]. A further major challenge is due to the large number of variants of uncertain signiﬁcance (VUS) yielded in next-generation sequencing (NGS) studies. The American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines are the current gold standard for classifying genetic variants. However, these guidelines were designed mainly for the classiﬁcation of genetic variants in recessive or dominant pathologies with complete penetrance. IASs are characterized as diseases with an autosomal dominant inheritance pattern (AD), incomplete penetrance and variable expressivity, thus some criteria of the ACMG/AMP guidelines present limitations for their classiﬁcation. Together with its strict criteria, this leads to high rates of VUS in the genetic diagnosis of IASs [14–17], making it necessary to work with adjusted ACMG/AMP criteria for each pathology and gene. Some researchers in the ﬁeld of primary arrhythmias have aimed to perform a quantitative implementation of the ACMG/AMP guidelines for IAS’s genetic testing, with success in reducing the VUS rate [18]. We provide a state of the art overview of the genetic diagnosis of IASs in the pediatric population and its translation into clinical practice, based on international expert guidelines, recent advances in evidence-based genetic curation, and IAS-focused variant classiﬁcation. We will emphasize genotype–phenotype and variant–phenotype correlation. Our goal is to facilitate translation of the genetic diagnosis to the clinical area, helping in risk stratiﬁcation, treatment and the genetic counseling of families. 2. Inherited Arrhythmia Syndromes Cardiac channelopathies are caused by defects in the genes encoding sodium (Na+), potassium (K+) and calcium (Ca2+) ion channels or their associated proteins. Defects in these proteins impair the generation and transmission of the action potential (AP) predispos- ing the patient to fatal arrhythmias [19]. The arrhythmias are usually of the type ventricular tachycardia (VT) or rapid ventricular ﬁbrillation (VF), and generally polymorphic [20]. De- spite the fact that each channelopathy usually has a characteristic electrocardiogram (ECG) proﬁle, an associated clinical phenotype and speciﬁc genes involved, these factors can often be shared by two or more syndromes, leading to difﬁculties in establishing a differential diagnosis. In addition, some of these syndromes are not associated with baseline ECG abnormalities, which make them difﬁcult to diagnose, with the added distress that SCD or resuscitated cardiac arrest is the initial symptom in most cases [21]. Therefore, an accurate Biomedicines 2022, 10, 106 3 of 28 3 of 28 genetic diagnosis can help to predict the prognosis and management of patients and their families. IASs are predominantly monogenic syndromes with an AD inheritance pattern, characterized by incomplete penetrance and variable expressivity [22]. Phenotypes with an autosomal recessive (AR) and X-linked inheritance pattern occur, but they are a minor- ity [23]. Four major IASs can be typically observed in pediatrics: long QT syndrome (LQTS), short QT syndrome (SQTS), Brugada syndrome (BrS) and catecholaminergic polymorphic ventricular tachycardia (CPVT) [24]. 3. Long QT Syndrome LQTS is the most common of the IASs and the main contributor for SCD in young people under 20 years of age. LQTS affects approximately 1 per 2000–2500 persons-year [25] with a slight predominance of females [26]. Moreover, it is the major arrhythmogenic syndrome, responsible for infants’ deaths, especially in the ﬁrst days of life [27]. It is characterized by QT interval prolongation and T-wave abnormalities in ECG, polymorphic ventricular tachycardia (PVT) in torsade de pointes (TdP), VF and syncope or SCD [28]. LQTS can be hereditary, congenital or acquired, usually associated with drugs and electrolyte imbalance (hypokalemia) (http://www.torsades.org accessed on 3 December 2021). 3.1. Genetics Originally characterized as an AD inheritance syndrome, called Romano–Ward syn- drome (LQT1–6 and LQT9–13), today 17 LQTS-associated genes are known (AKAP9, ANK2, CACNA1C, CALM1, CALM2, CAV3, KCNE1, KCNE2, KCNH2, KCNJ2, KCNJ5, KCNQ1, SCN1B, SCN4B, SCN5A, SNTA1 and TRDN) (Table 1) [29,30]. Most manifest with an AD inheritance pattern, except for the Jervell and Lange–Nielsen syndromes (JLNS) [31] and the recently characterized triadin knock out syndrome (LQT17) [32], which are inherited in an AR manner. In the most recent evaluation by an expert consensus, only three of these genes were considered to have adequate evidence to be classiﬁed as deﬁnitive for typical LQTS (KCNQ1, KCNH2, SCN5A) and four genes for LQTS with atypical features (CALM1, CALM2, CALM3, TRDN). The remaining 10 genes, despite being associated with LQTS, were considered to require further evidence for their classiﬁcation as causals [10]. 3.2. Deﬁnitive Genes for LQTS Cardiac Phenotype Inheritance Model Frequency Gene Curation Genes Main Type of Mutations Current Affected Non-Cardiac Phenotype Phenotypic Overlap (Both LOF/GOF Variants) Ref. LQTS LQT1 AD 30–35% Deﬁnitive genes KCNQ1 LOF IKs SNHL (AR), seizures JLNS (AR), SQTS, AF [10,33–38] LQT2 AD 25–30% KCNH2 LOF IKr Seizures SQTS, BrS, AF [10,33,34,39] LQT3 AD 5–10% SCN5A GOF INa Multiple (including seizures) BrS, SQTS, CPVT, ERS, AF, AFL, ARVC/D, HCM, DCM, LVNC, SVT, AVB, SND, PCCD, WPW [10,33,34,40–42] LQT14–16 AD <1% Deﬁnitive genes with atypical characteristics CALM1–3 GOF ICa Seizures, DD CPVT [10,33,43–45] LQT17 (TKOS) AR <1% TRDN LOF ICa Muscle weakness CPVT [10,33,46,47] LQT5 AD <1% Genes with moderate or limited evidence, associated with multiorgan syndromes KCNE1 LOF IKs SNHL (AR) JLNS (AR) [10,33,34,48] LQT8 AD <1% CACNA1C GOF ICa Dysmorphic and neurodevelop- mental features TS, SQTS, BrS, ERS, HCM, AF, SND, CND [10,33,49–53] LQT7 AD <1% KCNJ2 LOF IK1 Muscle weakness, dysmorphic features, DD, seizures ATS, SQTS, AF, CPVT, DCM [10,33,37,42,54–58] LQTS AD <1% each Other genes with limited evidence ANK2 KCNJ5 KCNE2 AKAP9 SCN4B CAV3 SNTA1 LOF LOF LOF LOF GOF GOF GOF Many IK-Ach IKr IKs INa INa INa Seizures CPVT, BrS, CND AF, SND AF [10,33,34,42,48,59–63] Table 1. Genotype-phenotype IASs correlation. Based on ClinGen [13]. Adapted from Nakajima, et al. [33]. ACM: Arrhythmogenic cardiomyopathy; AE3: Anion exchanger; AF: Atrial ﬁbrillation; AFL: Atrial ﬂutter; AR: autosomal recessive; ATS: Andersen-Tawil syndrome; AVB: atrioventricular block; BrS: Brugada syndrome; CDSP: Systemic primary carnitine deﬁciency; CPVT: catecholaminergic polymorphic ventricular tachycardia; CND: complex neurodevelopmental disorder; CRDS: Ca2+-release deﬁciency syndrome; DCM: Dilated cardiomyopathy; DD: developmental delay; DM: diabetes mellitus; ERS: Early repolarization syndrome; HCM: cardiomyopathy; Hypertrophic GOF: gain-of-function; ICa: Voltage-gated calcium currents; Ih: Hyperpolarization-activated non-selective cation channels/currents; IK: Delayed rectiﬁer potassium currents; IK-Ach: Acetylcholine-activated inward rectiﬁer potassium currents; IK-ATP: ATP-sensitive inward rectiﬁer potassium currents; IKr: Rapidly activating IK; IKs: Slowly activating IK; IK1: Inward rectiﬁer potassium currents; INa: Voltage-gated sodium currents; Ito: Transient outward potassium currents; TKOS: triadin knockout syndrome, TS: Timothy syndrome; JLNS: Jervell and Lange–Nielsen syndrome; LOF: loss-of-function; LQTS: Long QT syndrome; LVNC: Left ventricular non-compaction; PCCD: progressive cardiac conduction disease; SCA: Spinocerebellar ataxia; SND: Sinus node dysfunction; SNHL: Sensorineural hearing loss; SQTS: Short QT syndrome; SVT: Supraventricular tachyarrhythmia; WPW: Wolff–Parkinson–White.; XD: X-linked dominant. Cardiac Phenotype Inheritance Model Frequency Gene Curation Genes Main Type of Mutations Current Affected Non-Cardiac Phenotype Phenotypic Overlap (Both LOF/GOF Variants) Ref. 3.2. Deﬁnitive Genes for LQTS Pathogenic variants in 3 genes (KCNQ1, KCNH2 and SCN5A) are responsible for approximately 90% of all diagnosed cases of LQTS [98]. With LQT1 (KCNQ1) account- ing for 30–35% of cases, LQT2 (KCNH2) between 25–30% and LQT3 (SCN5A) around 5–10% [99,100]. The age of onset of clinical manifestations in each type is variable. LQT1 affect predominantly the pediatric population between 5–15 years of age, whereas LQT2 and LQT3 occur during puberty or later [34,101]. Even though severe causes of neonatal or even fetal manifestation have been reported, a similar mechanism for arrhythmogenesis is found in types 1 and 2. Both syndromes are caused by loss-of-function pathogenic variants in genes encoding for K+ channels. Disruption of these channels causes a repolarization delay with an increase in the AP (phase 3), leading to the QT interval prolongation. In comparison, in LQT3, this phenotype is caused by gain-of-function pathogenic variants in the SCN5A gene, which codes for a Na+ channel [102]. Adrenergic stimuli have an important role as a trigger of symptoms in LQTS types 1 and 2. Physical exercise is the main trigger of SCD in LQT1. A strong sudden startle, loud noise or emotional stress are triggers of LQT2. Moreover, LQT3 presents with malignant arrhythmias at rest or during sleep [34]. Biomedicines 2022, 10, 106 4 of 28 4 of 28 Table 1. Genotype-phenotype IASs correlation. Based on ClinGen [13]. Adapted from Nakajima, et al. [33]. ACM: Arrhythmogenic cardiomyopathy; AE3: Anion exchanger; AF: Atrial ﬁbrillation; AFL: Atrial ﬂutter; AR: autosomal recessive; ATS: Andersen-Tawil syndrome; AVB: atrioventricular block; BrS: Brugada syndrome; CDSP: Systemic primary carnitine deﬁciency; CPVT: catecholaminergic polymorphic ventricular tachycardia; CND: complex neurodevelopmental disorder; CRDS: Ca2+-release deﬁciency syndrome; DCM: Dilated cardiomyopathy; DD: developmental delay; DM: diabetes mellitus; ERS: Early repolarization syndrome; HCM: cardiomyopathy; Hypertrophic GOF: gain-of-function; ICa: Voltage-gated calcium currents; Ih: Hyperpolarization-activated non-selective cation channels/currents; IK: Delayed rectiﬁer potassium currents; IK-Ach: Acetylcholine-activated inward rectiﬁer potassium currents; IK-ATP: ATP-sensitive inward rectiﬁer potassium currents; IKr: Rapidly activating IK; IKs: Slowly activating IK; IK1: Inward rectiﬁer potassium currents; INa: Voltage-gated sodium currents; Ito: Transient outward potassium currents; TKOS: triadin knockout syndrome, TS: Timothy syndrome; JLNS: Jervell and Lange–Nielsen syndrome; LOF: loss-of-function; LQTS: Long QT syndrome; LVNC: Left ventricular non-compaction; PCCD: progressive cardiac conduction disease; SCA: Spinocerebellar ataxia; SND: Sinus node dysfunction; SNHL: Sensorineural hearing loss; SQTS: Short QT syndrome; SVT: Supraventricular tachyarrhythmia; WPW: Wolff–Parkinson–White.; XD: X-linked dominant. 3.2. Deﬁnitive Genes for LQTS LQTS LQT1 AD 30–35% Deﬁnitive genes KCNQ1 LOF IKs SNHL (AR), seizures JLNS (AR), SQTS, AF [10,33–38] LQT2 AD 25–30% KCNH2 LOF IKr Seizures SQTS, BrS, AF [10,33,34,39] LQT3 AD 5–10% SCN5A GOF INa Multiple (including seizures) BrS, SQTS, CPVT, ERS, AF, AFL, ARVC/D, HCM, DCM, LVNC, SVT, AVB, SND, PCCD, WPW [10,33,34,40–42] LQT14–16 AD <1% Deﬁnitive genes with atypical characteristics CALM1–3 GOF ICa Seizures, DD CPVT [10,33,43–45] LQT17 (TKOS) AR <1% TRDN LOF ICa Muscle weakness CPVT [10,33,46,47] LQT5 AD <1% Genes with moderate or limited evidence, associated with multiorgan syndromes KCNE1 LOF IKs SNHL (AR) JLNS (AR) [10,33,34,48] LQT8 AD <1% CACNA1C GOF ICa Dysmorphic and neurodevelop- mental features TS, SQTS, BrS, ERS, HCM, AF, SND, CND [10,33,49–53] LQT7 AD <1% KCNJ2 LOF IK1 Muscle weakness, dysmorphic features, DD, seizures ATS, SQTS, AF, CPVT, DCM [10,33,37,42,54–58] LQTS AD <1% each Other genes with limited evidence ANK2 KCNJ5 KCNE2 AKAP9 SCN4B CAV3 SNTA1 LOF LOF LOF LOF GOF GOF GOF Many IK-Ach IKr IKs INa INa INa Seizures CPVT, BrS, CND AF, SND AF [10,33,34,42,48,59–63] 5 of 28 Biomedicines 2022, 10, 106 Table 1. Cont. Cardiac Phenotype Inheritance Model Frequency Gene Curation Genes Main Type of Mutations Current Affected Non-Cardiac Phenotype Phenotypic Overlap (Both LOF/GOF Variants) Ref. 3.4. Genes with Moderate or Limited Evidence for LQTS According to the recent evaluation by Adler et al., there is insufﬁcient evidence to classify 10 of the 17 LQTS-related genes (CACNA1C, AKAP9, ANK2, CAV3, KCNE1, KCNE2, KCNJ2, KCNJ5, SCN4 and SNTA1) as LQTS-causing genes [31983240]. Each of these types represents less than 1% of LQTS. Some of them are associated with phenotypic syndromes and speciﬁc ECG features (Table 1). These include Andersen–Tawil syndrome (LQT7, KCNJ2) in which skeletal developmental abnormalities are observed [54]; Timothy syn- drome (LQT8, CACNA1C) with characteristic neurological, facial and limb features [106] and JLNS syndrome, associated with sensorineural deafness (KCNQ1-KCNE1 genes) [107]. Although the association of pathogenic variants in these genes with multiorganic syn- dromes is clear, the level of evidence for the speciﬁc cardiac phenotype is not so clear, and further studies are needed [10]. 3.6. Diagnosis According to the 2015 ESC Guideline, LQTS is diagnosed when the patient presents one of the following criteria: a corrected QT (QTc) ≥480 ms (repeated 12-lead ECGs), a LQTS risk score >3, or when a pathogenic alteration in one of the LQTS-causing genes is identiﬁed [116]. The LQTS risk score combines the altered ECG parameters with the patient’s clinical and family history and is based on the clinical score proposed by Schwartz in 1993 [117]. Regardless, in the presence of unexplained syncope a QTc ≥460 ms is sufﬁcient to diagnose LQTS [116] [26318695]. LQTS presents cardiac and extracardiac phenotypic features, as well as ECG characteristics that allow its classiﬁcation, although 25% of individuals with positive genetics show a normal baseline ECG [118]. 3.3. Deﬁnitive Genes for LQTS with Atypical Characteristics 3.3. Deﬁnitive Genes for LQTS with Atypical Characteristics Loss-of-function pathogenic variants in the CALM1, CALM2, CALM3 and TRDN genes cause LQTS types 14 to 17, respectively. The CALM, CALM2 and CALM3 genes code for calmodulin, and the TRDN gene for triadin, proteins involved in calcium-dependent processes and ion channel regulation [43,46]. LQT14–16 present atypical features, including seizures and neurodevelopmental delay [103] with symptoms manifesting in infants and young children between the ages of 0–5 years old and a high mortality rate [104]. LQT17 or triadin knockout syndrome (TKOS) also has poor prognosis, and exercise-induced SCD occurs in children aged 0–3 years old. Almost all patients are symptomatic by the age of 10 years old. The observation of negative T waves in the precordial leads is characteristic of LQT17 [47,105]. 3.5. Genetic Modiﬁers and Acquired LQTS GWAS studies have identiﬁed polymorphisms associated with increased risk of trig- gering LQTS [108,109]. Common variants in the NOS1AP (Nitric Oxide Synthase 1 Adaptor Protein) gene confer an increased risk of SCD in patients with LQT1 [110]. Likewise, some variants have a protective effect, as is the case of p.H558R (SCN5A), which reduces the pathogenic effect of other pathogenic variants, producing a less severe phenotype [22]. The common variants p.D85N (KCNE1) and T8A-MiRP1 (KCNE2) in Caucasians [111,112] and p.S1103Y (SCN5A) in African Americans [113], confer risk in the presence of other triggers such as drugs, a phenotype known as acquired LQTS (aLQTS). These variants are insufﬁcient by themselves to cause LQTS in the absence of other interval-prolonging factors [10,114]. However, they are of major importance due to the high frequency of aLQTS [115]. 3.2. Deﬁnitive Genes for LQTS BrS BrS1 AD 20–30% Deﬁnitive gene SCN5A LOF INa Multiple (including seizures) BrS, SQTS, CPVT, ERS, AF, AFL, ARVC, HCM, DCM, LVNC, SVT, AVB, SND, PCCD, WPW [12,64–76] BrS AD <5% Genes with moderate or limited evidence ABCC9 ANK2 KCNH2 KCNJ8 KCND3 KCNE3 CACNA1C CACNB2 CACNA2D1 HCN4 PKP2 GPD1-L TRPM4 SCN1B–3B SCN10A SLMAP RANGRF GOF GOF GOF GOF GOF GOF LOF LOF LOF LOF LOF LOF LOF LOF LOF LOF LOF IK-ATP Many IKr IK-ATP Ito Ito ICa ICa ICa Ih INa INa INa INa INa INa INa Seizures Seizures Seizures, SCA (See LQT8) ERS, AF, DCM LQTS, CPVT, CND LQTS, SQTS, AF ERS, AF ERS, AF, CND AF Multiple (see LQT8) SQTS, ERS, CND SQTS, ERS, CND AF, SND ARVC, DCM, ACM, CPVT DM LQTS AF AF [12,33,37,64,77–81] XD KCNE5 LOF Ito AF CPVT CPVT1 AD 55–60% Deﬁnitive genes RYR2 GOF ICa LQTS, HCM, LVNC, CRDS [11,48,82–87] CPVT2 AR 3–5% CASQ2 LOF ICa [11,48,88] CPVT3 AR 1–2% TECRL LOF ICa [11,48,89,90] CPVT4 AD <1% CALM1–3 LOF ICa Seizures, DD LQTS [11,43–45,48] CPVT5 AR 1–2% TRDN LOF ICa Muscle weakness LQTS [11,47,48,91] CPVT AD <1% Genes with moderate or limited evidence SCN5A PKP2 ANK2 KCNJ2 LOF LOF LOF LOF INa INa Many IK1 Multiple Seizures Seizures (See LQT7) Multiple (see BrS1) ARVC, DCM, ACM, CPVT LQTS, BrS, CND Multiple (see LQT7) [11,48,92] SQTS SQT1 AD 15% Deﬁnitive gene KCNH2 GOF IKr Seizures LQTS, AF, BrS [11,33,34,93] SQT2–3 AD <5% each Genes with strong-moderate evidence KCNQ1 KCNJ2 GOF GOF IKs IK1 (See LQT1) (See LQT7) JLNS (AR), SQTS, AF Multiple (see LQT7) [10,11,33–38] SQTS AD <1% Gene with moderate evidence SLC4A3 LOF AE3 [11,94] Biomedicines 2022, 10, 106 6 of 28 Table 1. Cont. Cardiac Phenotype Inheritance Model Frequency Gene Curation Genes Main Type of Mutations Current Affected Non-Cardiac Phenotype Phenotypic Overlap (Both LOF/GOF Variants) Ref. SQTS AD AR <1% each Genes with limited evidence CACNA1C CACNB2 CACNA2D1 SCN5A SLC22A5 LOF LOF LOF LOF LOF ICa ICa ICa INa INa (See LQT8) Multiple Metabolic decompensation, skeletal myopathy Multiple (see LQT8) SQTS, ERS, CND SQTS, ERS, CND Multiple (see BrS1) CDSP [11,33,65,95–97] 7 of 28 7 of 28 Biomedicines 2022, 10, 106 3.8. Genetic Counselling LQTS has incomplete penetrance and variable expressivity, even in the same family. The penetrance is estimated to be about 40%, a range that can vary depending on genotype, pathogenic variants type and location, age and sex, among other things [22,128]. The identiﬁcation of a pathogenic variant in one of the LQTS-associated genes (Table 1) is important to establish a differential diagnosis of patients and their relatives. In LQTS, treatment and risk stratiﬁcation differ depending on the gene causing the disease, and can be variant-speciﬁc [129]. According to guideline recommendations, only genes with deﬁnitive evidence for LQTS (KCNQ1, KCNH2 and SCN5A) should be routinely used in the evaluation of patients and their families. In patients with clinical ﬁndings consistent with the phenotypic expression demonstrated in LQTS with atypical features, related genes (CALM1, CALM2, CALM3 and TRDN) should also be tested [10]. Genetic testing can identify an LQTS-causing alteration in 70–80% of cases [99]. The proportion of LQTS caused by de novo pathogenic variants is difﬁcult to estimate, but is expected to be low [130]. Between 5–9% of familial cases of LQTS have two or more pathogenic variants (biallelic or digenic), which have been associated with a more severe phenotype [131,132]. The coexistence of two or more pathogenic variants could explain the variable expressivity observed in some families [133]. The copy number variants (CNV) detection rate among LQTS families is not very clear, but is estimated to be between 2–11% [134–138]. Population screening by ECG has been promoted to identify individuals at risk of LQTS, which has been successful in reducing SCD rates among patient family members [139] neonates [27,140] and athletes [141,142]. 3.7. Risk Stratiﬁcation Multiple factors are known to raise the likelihood of SCD in patients with LQTS. The presence of a QTc >500 ms is the strongest of these predictors [119,120]. In children, this parameter can be modiﬁed by the patient’s age, sex and genotype, with a critical transition period between 12–14 years of age [121]. Patients who have suffered syncope during Biomedicines 2022, 10, 106 8 of 28 8 of 28 childhood have an increased risk of recurrent episodes, which can be reduced through the use of beta-blockers (BB) and/or implantable cardiac deﬁbrillators (ICD) [122,123]. Among the types, LQT3 has a worse prognosis and the ﬁrst presentation is usually SCD [119,124]. Likewise, LQT14–16 types have been associated with a very severe phenotype in infants and poor response to available therapies [103]. Women with LQT2 have an increased risk of SCD in the ﬁrst 6 months postpartum, suggesting a potential hormonal effect [125]. Pathogenic variants type and location, as well as other additive genetic factors may increase the risk of SCD. For example, female adults with LQT2 run a higher risk of SCD than males. However, when missense pathogenic variants in the KCNH2 gene are in the pore loop regions, males are at a higher risk of SCD than their female counterparts [126]. Indeed, pathogenic variants in these regions have been found to be associated with QT interval prolongation and the development of TdP during fever, suggesting that fever may be a potential trigger of arrhythmias in patients with LQT2 [127]. 4.1. Genetics Genetically described as a Mendelian syndrome with an autosomal dominant inheri- tance pattern and incomplete penetrance [155], recent evidence suggests that BrS may be an oligogenic disease, involving several genetic factors [156]. However, the lack of conclusive data on these genetic alterations leads it to remain classiﬁed as a monogenic syndrome [157]. To date, more than 20 genes have been associated with BrS (ABCC9, ANK2, CACNA1C, CACNA2D1, CACNB2, GPD1-L, HCN4, KCND3, KCNE3, KCNE5, KCNH2, KCNJ8, PKP2, RANGRF, SCN10A, SCN1B, SCN5A, SCN2B, SLMAP and TRPM4), reappraisal of these genes by Hosseini et al., established that only SCN5A had deﬁnitive evidence of being a causal gene [12] and its genetic analysis is the only one recommended by current guide- lines [23,158]. 3.9. Management and Treatment In children with LQTS the ﬁrst approach is to avoid genotype-speciﬁc triggers, such as competitive sports, especially swimming in LQT1, and exposure to loud noise in children with LQT2 as well as the avoidance of the QT interval prolonging drugs in all carriers of LQTS-associated variants (http://crediblemeds.org/ accessed on 3 December 2021) [116]. Long-acting BB (nadolol) are recommended in all types, including asymptomatic genetic carriers [143,144], as their use decreases the risk of SCD [145]. Speciﬁc treatment of LQT3 with mexiletine and/or ﬂecainide has proven to be highly effective. In LQT1 the use of BB is very effective, and some authors suggest that it is not necessary to place an ICD in patients at low risk of SCD (asymptomatic prepubertal girls and adults >20 years with normal ECG) [146]. Left-cardiac sympathetic denervation is indicated in patients with LQT1 or when BB therapy is contraindicated or badly tolerated [147]. ICDs are used in patients at high risk of SCD despite previous therapies, in those who have previously presented syncope while taking BB, and effective LCSD (left cardiac sympathetic denervation) has been performed [148]. Despite its efﬁcacy, ICD has a high economic cost and can present numerous complications. Its use in the pediatric population should be assessed on a case-by-case basis by specialists [149]. Biomedicines 2022, 10, 106 9 of 28 4.3. BrS2–12 and Other Susceptibility Genes with Limited Evidence Pathogenic variants associated with BrS2–12, genes (GPD1-L, CACNA1C, CACNB2, SCN1B, KCNE3, SCN3B, HCN4, KCND3, KCNJ8, CACNA2D1 and MOG1) together represent less than 5% of all diagnosed cases [159]. Over the past few years, other genes have been suggested as possible causes of BrS (ABCC9, ANK2, FGF12, HEY2, KCND2, KCNH2, KCNE5, LRRC10, SEMA3A, PKP2, RANGRF, SCN10A, SCN2B, SLMAP and TRPM4), but no comprehensive clinical and cellular studies have conﬁrmed this association [157]. All of them follow an AD inheritance pattern, except for the KCNE5 gene, which follows an X-linked dominant pattern [160,161]. 4. Brugada Syndrome BrS in children and young adults is rare, and its incidence rate and clinical implications remain unclear. In the general population, its prevalence is estimated to be between 1 in 2000–5000 person-years [150]. The syndrome has a higher prevalence in Southeast Asian countries [151], and is more frequent among males than females [152]. It is characterized by a right bundle branch block, a very sharp T wave and spontaneous or drug-induced ST- segment elevation (J point) in the right precordial leads (V1–V3), known as ‘type-1’ BrS ECG pattern [64]. Clinical manifestations may appear between the ages of 2 months and 77 years old, but the mean age of presentation is 40 years old [153]. Symptoms present at rest, during sleep or febrile episodes, including nocturnal agonal respirations, palpitations, seizures, and polymorphic ventricular tachycardia (PVT) or VF. Most individuals remain asymptomatic, although SCD occurs in 17–42% of the cases and can be the initial presentation [154]. 4.2. Deﬁnitive Gene for BrS Loss-of-function pathogenic variants in the SCN5A gene account for approximately 30% of genetically positive BrS cases. This gene encodes the alpha subunit of the cardiac sodium channel Nav1.5, responsible for phase 0 of the AP. Inactivation of the channel leads to a delay in ventricular polarization, resulting in the development of ventricular tachycardia and ﬁbrillation (VT/VF) [150]. 4.3. BrS2–12 and Other Susceptibility Genes with Limited Evidence 4.4. Diagnosis According to the 2015 ESC guidelines, BrS is diagnosed in patients with a ‘type 1’ ECG pattern, ST-segment elevation ≥2 mm (J-point) in one or more of the right precordial leads (V1–V3) [116]. This ECG pattern may occur spontaneously or be unmasked by a provocation test with a class Ic drug (sodium channel blockers such as ajmaline, ﬂecainide, procainamide, or pilsicainide), in this case additional clinical criteria are required for diagnosis [64]. Fever is a trigger for ventricular arrhythmias in patients with BrS and may unmask the characteristic ECG pattern, especially in children under 5 years of age [162]. A 12-lead ECG is recommended during febrile episodes in children with a family history of BrS and in all children with febrile seizures [163]. Initially presumed not to have any structural abnormalities, postmortem histological studies and endomyocardial biopsies have shown changes at the tissular and molecular level in patients with BrS. These changes Biomedicines 2022, 10, 106 10 of 28 10 of 28 include localized electroanatomical and structural abnormalities in the right ventricular outﬂow tract (RVOT), ﬁbrosis, fatty inﬁltration, increased epicardial collagen, and decreased expression of Connexin 43 at right ventricular gap junctions [164–166]. 4.5. Risk Stratiﬁcation Despite advances in risk stratiﬁcation of IAS, in BrS it remains challenging. The most important risk marker is the presentation of a previous arrhythmogenic event (AE, AF, syncope, or SCD), which increases the likelihood of SCD in both young and adults [167,168]. The age of onset is a notable prognostic marker. For instance, although BrS is uncommon in children, they present with a more severe form of the disease [169]. Gender is an important risk factor, with males being up to 5–8 times more affected than females, presenting a more severe phenotype, earlier symptom debut and a higher number of events [152,170]. These gender differences have not been observed in children under 12 years old [167]. Family history of SCD and the presence of pathogenic variants in SCN5A could also be predictors of high risk in adults and adolescents, [167] even though their role in risk stratiﬁcation is still controversial [171,172]. Other risk factors observed are spontaneous variation of the ‘type 1’ ECG pattern and fragmentation of the QRS complex [171,173], nevertheless further studies are required to conﬁrm this association. 4.7. Genetic Counseling BrS penetrance is highly variable in the different published studies. It is estimated to range from 12.5% to 50% [22,179]. About 70–80% of families with BrS do not have a genetic diagnosis. However, although the results of genetic screening do not currently inﬂuence prognosis or treatment, genetic testing should be performed in all ﬁrst-degree relatives if the index case tested positive [99,116]. Moreover, the ECG pattern of BrS ‘type 1’ is uncommon in children and genetic testing may help with their diagnosis [21]. In BrS families counseling should also include an ECG, because negative-genotype positive- phenotype cases are not uncommon [180]. The proportion of cases caused by de novo pathogenic variants is estimated at 1% and the number of cases with a CNV variant is approximately 1.3–2.9% [138,181]. ECG screening using a provocation test for BrS detection is controversial [182,183], but should be performed when an abnormal ECG is found. ECG screening has beneﬁts in the prevention of SCD in neonates [140] and the young population [184]. 4.6. Management and Treatment In children with BrS or a family history, BrS-inducing drugs should be avoided (http://www.brugadadrugs.org accessed on 3 December 2021). Appropriate treatment of any fever with antipyretic drugs should be provided [167]. ICD implantation is the only treatment that reduces the risk of SCD in BrS. It is indicated in all patients resuscitated from arrhythmic syncope, those with documented VT or VF or with a spontaneous ‘type 1’ ECG pattern [116,149]. In patients with electrical storms, administration of isoproterenol is recommended [174,175]. Quinidine is recommended for patients who refuse ICD im- plantation or in those who, despite having an ICD, still have a high risk of SCD [176,177]. Catheter ablation is useful in high-risk patients with a history of electrical storms or re- peated appropriate ICD shocks [178]. ICD implantation in asymptomatic individuals with a ‘type 1’ ECG pattern for primary prevention, including children, remains controversial and is a challenge for specialists who have to handle it on a case-by-case basis [149,177]. 5.5. Risk Stratiﬁcation On account of the limited number of patients with SQTS and the phenotypic variability of the syndrome, risk stratiﬁcation currently represents a challenge. To date, the only predictor of SCD found in patients with SQTS is a history of cardiac arrest [93]. Genotype- phenotype correlation studies have found that SQTS1 manifests at an older age and patients have a shorter QTc than other patients with SQTS. Nevertheless, no association of this reduction with an increased risk of SCD has been found [191]. 5.3. Genes with Strong or Moderate Evidence for SQTS SQT2 and SQT3 are driven by gain-of-function pathogenic variants in genes encoding for K+ channels (KCNQ1 and KCNJ2, respectively). The mechanism of arrhythmogenicity is similar to that presented by KCNH2. The SLC4A3 gene, recently associated with SQTS, presents an uncommon mechanism for the development of malignant arrhythmia. SLC4A3 encodes the plasma membrane anion exchange protein 3 (AE3) and acts by mediating part of the Cl−/HCO3−exchange in cardiac myocytes. Loss-of-function pathogenic variants in the SLC4A3 gene would cause an increase in pHi and a decrease in [Cl−]i, shortening the duration of the AP [94]. 5.1. Genetics At present, nine genes have been associated with SQTS (CACNA1C, CACNA2D1, CACNB2, KCNH2, KCNJ2 and KCNQ1, SLC22A5, SLC4A3 and SCN5A) [190]. Evaluation of these genes, by Walsh et al., showed that only the KCNH2 gene had deﬁnitive evidence for SQTS causality. Three other genes (KCNQ1, KCNJ2, SLC4A3) presented strong to moderate evidence. Causality of the other SQTS-associated genes remains still in dispute [11]. These data are consistent with the ﬁndings published by Campuzano et al., who found that all variants with a conclusive pathogenic role in SQTS clustered in three genes (KCNQ1, KCNH2 and KCNJ2). In that study, the SLC4A3 gene was excluded, since carriers were in a gray zone of SQTS diagnosis (with a QTc ≤370 ms) [190]. 5.4. Diagnosis According to the 2015 ESC guidelines, SQTS is diagnosed by the presence of a QTc ≤340 ms, or ≤360 ms when one the following clinical criteria occur: the detection of a known pathogenic alteration, a family history of SQTS, a family history of SCD before the age of 40 years, or reanimated cardiac arrest with a structurally normal heart [116]. 5. Short QT Syndrome SQTS is an extremely rare inherited disease associated with SCD. To date, less than 200 cases have been reported worldwide [93]. The estimated prevalence varies between 0.18–2.9%, with a higher incidence in males than females [25]. The incidence rate can even be lower (0.02–0.10%) if more restrictive values are considered for its diagnosis [185,186]. While prevalence of the syndrome in children and adolescents is low (about 0.05%), early 11 of 28 Biomedicines 2022, 10, 106 11 of 28 detection is important, as it is potentially lethal for all age groups. It is characterized by a short QT interval on the ECG (<330 ms), with an asymmetric and peaked T wave. Symptoms occur mostly in men between the ages of 14 and 40 and may be favored by hormonal causes [187]. Cardiac events usually occur in adrenergic situations (noise or exercise), although it can also occur at rest. Clinical presentation includes ventricular repolarization abnormalities (AF and VT) and syncope. The probability of presenting SCD as the ﬁrst symptom increases with age, reaching 41% at the age of 40 [188]. Currently, approximately 40% of cases remain asymptomatic [189]. 5.2. SQT1 Deﬁnitive Gene: KCNH2 The p.T618I and p.N588K pathogenic variants in the KCNH2 gene are the most frequent associated with SQTS, accounting for 85% of SQT1 and 55% of all genetically identiﬁed cases of SQTS [189]. Gain-of-function pathogenic variants in KCNH2 lead to prolonged K+ channel activation and accelerated cardiac repolarization with shorter refractory periods, potentially triggering life-threatening supraventricular and ventricular arrhythmias [189]. 5.6. Management and Treatment ICD implantation is recommended for all patients with SQTS, especially for patients who have survived an aborted cardiac arrest or have presented spontaneous sustained Biomedicines 2022, 10, 106 12 of 28 12 of 28 VT [186,192]. QT interval prolonging drugs (quinidine and sotalol) should be considered for all patients at risk of SQTS in both, asymptomatic and symptomatic patients who don’t have an ICD, especially young children [186]. 6.1. Genetics According to the recent evaluation by Walsh et al., seven genes were classiﬁed as causing of CPVT with deﬁnite to moderate evidence. Four of them present an AD inheri- tance pattern (RYR2, CALM1, CALM2, CALM3) and three AR inheritance (CASQ2, TRDN, TECRL). Three genes (KCNJ2, PKP2, SCN5A) were reported for phenotypes that were not representative of CPVT, while the reported variants in the ANK2 gene were considered too common in the population to be disease-causing (Table 1) [11]. 6. Catecholaminergic Polymorphic Ventricular Tachycardia The prevalence of CPVT is estimated to be 1 per 10,000 population. However, the real prevalence is uncertain, as it might be underestimated due to its high lethality at a young age and difﬁculty in diagnosis [25,200]. CPVT is characterized by a bidirectional polymorphic VT, triggered by an adrenergic stimulus mainly during exertion, extreme stress or emotion that can lead to syncope and SCD. Syncopal episodes are increasingly reported during “awake rest” possibly due to anxiety, stress, or other psychological stimuli unrelated to exertion [201]. The age of onset can range from infancy to the age of 30, although it is more common in children aged 7–10 years old [202]. By the age of 10, about 35% of patients are symptomatic, increasing to 72% by the age of 21 [203]. A younger age of debut is often accompanied by more severe phenotypes and increased risk of SCD [42]. 5.7. Genetic Counselling Due to the low number of cases, the penetrance of SQTS is difﬁcult to estimate. However, pathogenic variants with a penetrance of 100% have been reported [189]. The diagnostic yield of genetic testing in SQTS is low (<25%) [186]. Current guidelines recom- mend analysis of ﬁve genes: KCNH2, KCNQ1, KCNJ2, KCNJ2, CACNA1C and CACNB2 in the diagnosis of SQTS [116], with the KCNH2 gene as the most cost-effective option [193]. Familial genetic analysis is recommended, both to clarify the pathogenic role of newly identiﬁed variants and to identify family members at risk for SCD. To date, there is no published data on CNV analysis on patients with SQTS. De novo variants in the KCNQ1 gene have been associated with a particular in utero phenotype with clinical diagnosis of AF with concomitant bradycardia and short QT interval [194,195]. Some researchers support the screening of SQTS in the pediatric population, given its high lethality and the beneﬁts of early diagnosis in the prevention of SCD. These studies have shown that the diagnostic criteria for QTc should be adjusted in each population based on factors including sex and age, to avoid false positives [196–199]. 6.5. Management and Treatment In children with CPVT, avoidance of phenotype triggers such as competitive sports, strenuous exercise (especially swimming), and stressful environments is recommended. BB are the ﬁrst-line of treatment, their use is recommended in all patients, even in genetically identiﬁed asymptomatic patients [116]. About 25% of children experience syncope or cardiac arrest despite treatment with BB [215]. In these patients, it is advisable to include ﬂecainide therapy and/or left cardiac sympathetic denervation (LCSD). LCSD has been proven to reduce the rate of arrhythmic events in patients with LQTS and CPVT [200,216]. It should be noted that ICD therapy may be counterproductive in CPVT, because the discharge may activate adrenergic production and exacerbate the VT storm, so its implantation should be assessed by the specialist [217]. 6.4. Risk Stratiﬁcation Between 30–50% of patients with CPVT will experience SCD before the age of 30 [210,211]. The event rate in untreated children under 8 years old has been estimated at 58% that can be reduced to 27% with adherence to BB treatments [203]. RYR2 is one of the most prevalent genes in cohorts of patients with unexplained SCD, occurring in 5–10% of cases [212]. While the variable expressivity of the CPVT phenotype could be explained by the inﬂuence of other genetic and non-genetic factors, no genetic modiﬁers have been identiﬁed in CPVT to date [108,213]. Patients with the recessive form of CPVT and a younger age at diagnosis have a more severe phenotype [48,203]. The location of the rare variant may be a possible disease modiﬁer [201]. No gender- or age-dependent differences in arrhythmic risk in children have been found to date [214]. However, further studies in CPVT risk stratiﬁcation are needed to draw deﬁnitive conclusions. 6.3. Diagnosis CPVT is diagnosed when exercise- or emotion-induced bidirectional or polymorphic VT is detected, in the presence of a structurally normal heart or in patients carrying pathogenic alterations in the deﬁnitive genes for CPVT [200]. Since the resting ECG is usually normal, ECG during exercise and Holter monitoring play a relevant role in the diagnosis. The disease can be easily missed or misdiagnosed; for instance, many children are initially diagnosed with epilepsy, as syncope may be associated with seizure movements. 6.2. Deﬁnitive Genes for CPVT CPVT1 is the most prevalent variant, accounting for more than 60% of all genetically diagnosed cases of CPVT [204]. CPVT1 with a high incidence in children around 10 years of age [205]. Thus, it is caused by pathogenic variants in the RYR2 gene, which encodes for ryanodine receptor 2, responsible for calcium regulation in the cardiomyocyte. A majority of pathogenic variants in the RYR2 gene are gain-of-function, which promote an increased Ca2+ release from the sarcoplasmic reticulum of cardiomyocytes into the cytoplasm leading to late after-depolarizations [48]. The remaining types of CPVT represent about 10% of cases. Most are caused by loss-of-function pathogenic variants in genes encoding proteins involved in the storage and release of Ca2+ in the sarcoplasmic reticulum [206]. CPVT2 is caused by homozygous or compound heterozygous pathogenic variants in the CASQ2 gene, following an AR inheritance model. CPVT2 accounts for about 3–5% of cases [203], affecting mainly children around the age of 7 years [205]. Pathogenic variants in TRDN, CALM1 and TECRL are responsible for CPVT3, CPVT4, and CPVT5, respectively. Each Biomedicines 2022, 10, 106 13 of 28 13 of 28 represents about 1–2% of the cases. CPVT3 and CPVT5 follow an AR inheritance pattern, even though AD inheritance has also been observed in some families [42,91]. In contrast, CPVT5 is presented with an AD inheritance pattern, a younger age of onset (approximately 2.3 years) and more severe phenotypes, being highly lethal in children [205,207]. Along with TRDN and CALM1, gain-of-function pathogenic variants in the CALM2 and CALM3 genes have been associated with the development of atypical CPVT, presenting more complex and variable associated phenotypes than classic [208,209]. 7. Genetic Overlap Several causative genes for IASs are common to two or more syndromes (LQTS, SQTS, BrS, and CPVT) (Figure 1) and have been found to overlap with other cardiac and extracardiac clinical phenotypes (Table 1). This genetic overlap could be explained since pathogenic variants can alter different properties of the channels or proteins, affecting ion exchange, interaction with auxiliary proteins, as well as gene expression. The ﬁnal phenotype would depend not only on which property is affected, but also how it is affected. Notably, some genes that cause LQTS are the same genes that also cause SQTS (KCNQ1, KCNH2, KCNJ2, CACNA1C, CACNB2, and CACNA2D1), CPVT (CALM1, CALM2, CALM3, TRDN, KCNJ2, SCN5A, ANK2 and TECRL) or BrS (SCN5A) [42,89]. However, the pathogenic variants usually cause an opposite effect on the channel. EVIEW 15 of 30 Figure 1. Diagram of the overlap between the genes IASs-associated genes: Brugada syndrome (BrS); short QT syndrome (SQTS); long short QT syndrome (LQTS) and catecholaminergic polymor- phic ventricular tachycardia (CPVT) Figure 1. Diagram of the overlap between the genes IASs-associated genes: Brugada syndrome (BrS); short QT syndrome (SQTS); long short QT syndrome (LQTS) and catecholaminergic polymorphic ventricular tachycardia (CPVT). Figure 1. Diagram of the overlap between the genes IASs-associated genes: Brugada syndrome (BrS); short QT syndrome (SQTS); long short QT syndrome (LQTS) and catecholaminergic polymor- phic ventricular tachycardia (CPVT). Figure 1. Diagram of the overlap between the genes IASs-associated genes: Brugada syndrome (BrS); short QT syndrome (SQTS); long short QT syndrome (LQTS) and catecholaminergic polymorphic ventricular tachycardia (CPVT). Some pathogenic variants have been found to lead to complex overlapping forms of two or more syndromes. Several publications support the fact that pathogenic variants in KCNQ1, ANK2, KCNE1, KCNE2, KCNH2, KCNJ2 and SCN5A can lead to a complex over- lapping phenotype of LQTS, CPVT and ventricular ectopy [42,48,92]. Hirose et al., recently described in a cohort of children (<16 years old) the association of loss-of-function patho- genic variants in RYR2 with various types of arrhythmia, including LQTS, VF and scTdP, depending on the alteration of channel activity [83,84]. The pathogenic variants p.I4855M and deletion of exon 3 in the RYR2 gene have been associated with the rare syndrome of left ventricular non-compaction (LVNC) overlap and CPVT, presenting a high lethality [85]. 6.6. Genetic Counseling CPVT penetrance can vary between 63–78% [22,213,218]. The diagnostic yield is high, with a positive genetic result in 60–65% of the studied cases [25]. Genetic testing is recommended by expert consensus, with RYR2 and CASQ2 genes as the most cost-effective options [23]. Identiﬁcation of family members at risk is critical to avoid SCD, which is the ﬁrst manifestation in up to 30–50% of cases [210]. CPVT has a high incidence of de novo variants, which are found in approximately 50% of genetically diagnosed CPVT patients [25]. CNV in RYR2 have been associated with CPVT [138,219,220], while the other CPVT-related genes have not been examined so far. 14 of 28 14 of 28 Biomedicines 2022, 10, 106 7. Genetic Overlap Pathogenic variants in the TRPM4 gene, both gain and loss of function, have been identified in patients with different forms of cardiac disorder including conduction de- fects, BrS and LQTS [77]. The PKP2 gene, the main gene mutated in arrhythmogenic car- diomyopathy (ACM), has been recently associated with BrS and CPVT. The p.S183N path- ogenic variant has been reported in both a patient with BrS and a patient with a definite diagnosis of ACM [221]. However, further studies are needed to establish the pathophys- i l i l h i f th l ti Some pathogenic variants have been found to lead to complex overlapping forms of two or more syndromes. Several publications support the fact that pathogenic variants in KCNQ1, ANK2, KCNE1, KCNE2, KCNH2, KCNJ2 and SCN5A can lead to a complex overlapping phenotype of LQTS, CPVT and ventricular ectopy [42,48,92]. Hirose et al., recently described in a cohort of children (<16 years old) the association of loss-of-function pathogenic variants in RYR2 with various types of arrhythmia, including LQTS, VF and scTdP, depending on the alteration of channel activity [83,84]. The pathogenic variants p.I4855M and deletion of exon 3 in the RYR2 gene have been associated with the rare syndrome of left ventricular non-compaction (LVNC) overlap and CPVT, presenting a high lethality [85]. Pathogenic variants in the TRPM4 gene, both gain and loss of function, have been identiﬁed in patients with different forms of cardiac disorder including conduction defects, BrS and LQTS [77]. The PKP2 gene, the main gene mutated in arrhythmogenic cardiomyopathy (ACM), has been recently associated with BrS and CPVT. The p.S183N pathogenic variant has been reported in both a patient with BrS and a patient with a deﬁnite diagnosis of ACM [221]. However, further studies are needed to establish the pathophysiological mechanisms of these correlations. 15 of 28 15 of 28 Biomedicines 2022, 10, 106 7.3. Non-Genetic Phenotype Overlapping Multiple phenotypic overlaps between IASs have been described, in which no un- derlying genetic cause has been observed ERS and BrS [66,236], BrS and CA [237], ACM and BrS [238]. The overlap of ACM and BrS is controversial, since their diagnostic criteria exclude the coexistence of both syndromes in the same individual. This overlap would not be fully explained by a genetic overlap. The ECG pattern of BrS (drug-induced) was observed both in patients with ACM and pathogenic variants in the SCN5A gene [239] as well as in patients without SCN5A pathogenic variants [240,241]. Therefore, it is possible that genetic (coding or non-coding variants) and non-genetic (demographic variables or exogenous factors) modiﬁers could be involved in this variability, all of them contributing additively to the expression of the phenotype. An alternative explanation for phenotypic overlap could be misdiagnosis due to the use of drugs in ECG testing. Notably, it has been shown that ﬂecainide can induce ST-segment elevation in ECG in patients with LQTS3, and lead to misdiagnosis in some cases [242]. 7.2. Genetic Overlap of Arrhythmogenic Phenotypes and Epilepsy 7.2. Genetic Overlap of Arrhythmogenic Phenotypes and Epilepsy Recently, evidence for genetic overlap between IASs and epilepsy has been reported. Sudden unexpected death in epilepsy (SUDEP) share many features with SADS in the young and may have a similar genetic contribution [233,234]. In a systematic review, Anwar and Chahal, et al., found that 11% of the most frequent pathogenic variants identiﬁed by molecular autopsy in SUDEP were found in genes related to cardiac channelopathies [235]. 7.1. SCN5A Clinical Overlap Pathogenic variants affecting SCN5A have been found in all major IAS, as well as in other associated cardiac phenotypes (Figure 2). Gain-of-function pathogenic variants in SCN5A (LQT3), have been associated with other arrhythmias including multifocal ectopic Purkinje-related premature contractions [222,223] and atypical CPVT-like phe- notype [224]. For instance, p.T1857I and p.I141V variants, have been associated with tachyarrhythmias and exercise-induced polymorphic ventricular arrhythmia [224,225]. However, the correlation between SCN5A pathogenic variants and CPVT remains de- bated. Meanwhile, loss-of-function pathogenic variants in SCN5A (usually associated with BrS) have also been implicated in certain phenotypes including isolated cardiac conduc- tion defect and sick sinus syndrome (SSS) [226,227]. In addition, both loss-of-function and gain-of-function pathogenic variants can cause dilated cardiomyopathy (DCM), AF, and overlap syndromes [226,228]. The founder pathogenic variants p.E1784K [229,230], p.F1617del [231], p.1795insD [232], among others, can be manifested as a mixed clinical phenotype of LQTS and/or BrS, even between affected individuals in the same family. Recently, Sasaki, et al. have described the p.A735E pathogenic variant, which could be associated with the overlap of multiple phenotypes in eight carrier individuals, although further studies are needed to ratify this hypothesis [76]. Figure 2. Examples of clinical overlap due to pathogenic variants in the SCN5A gene. I. Phenotype caused by gain-of-function variants in the SCN5A gene: (a) LQTS (Long QT syndrome type 3); II. Phenotype caused by both gain-of-function and loss-of-function variants in the SCN5A gene: (b) Atrial ﬂutter; III Phenotypes caused by loss-of-function variants in the SCN5A gene: (c) Hisian tachycardia + sinus rhythm + I◦AV block; (d) I◦AV block + complete RBBB (right bundle branch block); (e) Complete RBBB + pathologic elevation of ST; (f) BrS (Brugada syndrome) ‘type 1’ ECG; and (g) SNS (sinus node syndrome), atrial standstill. Figure 2. Examples of clinical overlap due to pathogenic variants in the SCN5A gene. I. Phenotype caused by gain-of-function variants in the SCN5A gene: (a) LQTS (Long QT syndrome type 3); II. Phenotype caused by both gain-of-function and loss-of-function variants in the SCN5A gene: (b) Atrial ﬂutter; III Phenotypes caused by loss-of-function variants in the SCN5A gene: (c) Hisian tachycardia + sinus rhythm + I◦AV block; (d) I◦AV block + complete RBBB (right bundle branch block); (e) Complete RBBB + pathologic elevation of ST; (f) BrS (Brugada syndrome) ‘type 1’ ECG; and (g) SNS (sinus node syndrome), atrial standstill. 16 of 28 Biomedicines 2022, 10, 106 16 of 28 8. How to Deal with the Variants of Uncertain Signiﬁcance in Inherited Arrhythmia Syndromes (IASs) The classiﬁcation of VUS remains a current challenge in genetic ﬁeld. When functional and segregation studies are not feasible due to the rarity and exclusivity of some variants, population allele frequency (https://gnomad.broadinstitute.org/ accessed on 3 Decem- ber 2021) and familial segregation are a fundamental tool for variant classiﬁcation [132]. Continued reclassiﬁcation of rare variants based on the ACMG-AMP criteria has led to an increasing understanding of the potential impact of a variant on a disease; for example, it has been shown that rare variants previously described as pathogenic may have too high a population frequency to be responsible for IASs, and that a large number of rare variants may be benign. On the other hand, as mentioned, the ACMG/AMP guidelines present limitations for their application in the classiﬁcation of rare variants in IASs and their criteria need to be adjusted. To date, few validations have been performed with adjusted criteria. The reasons are probably due to the difﬁculty of establishing valid and homoge- neous criteria that can be generally applied to such heterogeneous diseases such as IAS. Assigning erroneous classiﬁcations to variants carries great danger, both for false positives (assigning pathogenic causality to variants that are not) that can have severe consequences, for example leading to the implantation of an unnecessary ICD or, on the contrary, leaving as VUS variants those that are truly causative of the disease. Nevertheless, working with criteria adjusted to each disease could be a useful approach to achieve greater success in the classiﬁcation of genetic variants in IASs and decrease the number of cases without a conclusive genetic diagnosis. In the recent validation of variants in LQTS and BrS by Walsh et al. they developed a quantitative implementation (disease-speciﬁc) of the ACMG-AMP guidelines following the ClinGen recommendations [243]. These reﬁnements consisted of deﬁning population frequency thresholds for rare variants taking into account both disease prevalence and estimated penetrance, together with the maximum allelic contribu- tion. They further used data from case-control studies to identify genetic regions highly enriched in rare variants in IASs cohorts compared to the control population, together with the information derived from functional studies. These data proved to be effective, and implementation of these criteria led to a signiﬁcant reduction in the proportion of cases with VUS in both syndromes [18]. 17 of 28 Biomedicines 2022, 10, 106 17 of 28 9. Conclusions 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: Not applicable. Data Availability Statement: Not applicable. Data Availability Statement: Not applicable. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. 9. Conclusions Nowadays, the diagnosis, management and risk stratiﬁcation of SADS in the pediatric population is still a challenge for clinicians. Genetic diagnosis plays an important role in the differential diagnosis of SADS. In addition to directing the clinical management, treatment and risk stratiﬁcation of patients in most cases, it allows risk stratiﬁcation and prevention of their relatives, who may remain asymptomatic. International guidelines recommend genetic analysis in families with IASs, testing only the main causal genes associated with each syndrome [244], always in the context of pretest and posttest genetic counseling. For a genetic result of VUS, it is important to emphasize that it does not necessarily imply lower or higher risk for any carrier patient. It means that there is currently insufﬁcient evidence to support or rule out the pathogenic role of the variant in the phenotype. Therefore, clinical translation of VUS should be undertaken with caution and should not be excluded or used in clinical decision-making until follow-up testing is completed, and its clinical role clariﬁed [15]. Continuous reﬁnements in clinical and genetic tools have improved diagnosis in families. Nevertheless, more than 50% of families remain without a conclusive genetic result, with the concern that unexpected death is often the ﬁrst manifestation of the disease. Owing to recent evaluations of IASs-associated genes according to the evidence-based approach proposed by ClinGen, we now have a better genotype–phenotype correlation of the main causative genes. However, it is necessary to continue this curation process to clarify the association of minority genes with these diseases. Additionally, adjustment of the ACMG-AMP criteria considering the inheritance model, population frequency, prevalence and penetrance of the IASs, among other factors, will allow a more accurate classiﬁcation of these rare variants before applying knowledge to clinical practice in a personalized approach. Author Contributions: O.C., G.S.-B., E.A., J.B. and R.B. developed the concept. E.M.-B., S.C., J.C., C.H., E.A. and V.F. acquired, pre-processed, and analyzed the data. O.C., E.M.-B. and G.S.-B. prepared the manuscript. O.C., G.S.-B., J.B. and R.B. supervised the study. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by Obra Social “La Caixa Foundation” (LCF/PR/GN16/50290001 and LCF/PR/GN19/50320002). CIBERCV is an initiative of the ISCIII, Spanish Ministry of Econ- omy and Competitiveness. Funders had no role in study design, data collection, data analysis, interpretation, or writing of the report. 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https://openalex.org/W1925501079	https://www.frontiersin.org/articles/10.3389/fphar.2015.00199/pdf	English	null	Systems pharmacology of adiposity reveals inhibition of EP300 as a common therapeutic mechanism of caloric restriction and resveratrol for obesity	Frontiers in pharmacology	2,015	cc-by	10,425	Abbreviations: AcCoA, acetyl coenzyme A; BMI, body mass index; CR, caloric restriction; DIO, diet-induced obese; dpf, days post-fertilization; FXR, farnesoid X receptor; GEO, Gene Expression Omnibus; mpf, months post-fertilization; OF, overfeeding; RSV, resveratrol; TF, transcription factors; TGs, triglycerides. Systems pharmacology of adiposity reveals inhibition of EP300 as a common therapeutic mechanism of caloric restriction and resveratrol for obesity Yuhei Nishimura1,2,3,4,5, Shota Sasagawa1, Michiko Ariyoshi1, Sayuri Ichikawa1, Yasuhito Shimada1, Koki Kawaguchi1, Reiko Kawase1, Reiko Yamamoto6, Takuma Uehara6, Takaaki Yanai6, Ryoji Takata6 and Toshio Tanaka1,2,3,4,5* 1 Department of Molecular and Cellular Pharmacology, Pharmacogenomics and Pharmacoinformatics, Mie University Graduate School of Medicine, Tsu, Japan, 2 Mie University Medical Zebraﬁsh Research Center, Tsu, Japan, 3 Department of Systems Pharmacology, Mie University Graduate School of Medicine, Tsu, Japan, 4 Department of Omics Medicine, Mie University Industrial Technology Innovation Institute, Tsu, Japan, 5 Department of Bioinformatics, Mie University Life Science Research Center, Tsu, Japan, 6 Product Development Research Institute, Mercian Corporation, Fujisawa, Japan ORIGINAL RESEARCH published: 15 September 2015 doi: 10.3389/fphar.2015.00199 Edited by: Chiranjib Chakraborty, Galgotias University, India Both caloric restriction (CR) and resveratrol (RSV) have beneﬁcial effects on obesity. However, the biochemical pathways that mediate these beneﬁcial effects might be complex and interconnected and have not been fully elucidated. To reveal the common therapeutic mechanism of CR and RSV, we performed a comparative transcriptome analysis of adipose tissues from diet-induced obese (DIO) zebraﬁsh and obese humans. We identiﬁed nine genes in DIO zebraﬁsh and seven genes in obese humans whose expressions were regulated by CR and RSV. Although the gene lists did not overlap except for one gene, the gene ontologies enriched in the gene lists were highly overlapped, and included genes involved in adipocyte differentiation, lipid storage and lipid metabolism. Bioinformatic analysis of cis-regulatory sequences of these genes revealed that their transcriptional regulators also overlapped, including EP300, HDAC2, CEBPB, CEBPD, FOXA1, and FOXA2. We also identiﬁed 15 and 46 genes that were dysregulated in the adipose tissue of DIO zebraﬁsh and obese humans, respectively. Bioinformatics analysis identiﬁed EP300, HDAC2, and CEBPB as common transcriptional regulators for these genes. EP300 is a histone and lysyl acetyltransferase that modulates the function of histone and various proteins including CEBPB, CEBPD, FOXA1, and FOXA2. We demonstrated that adiposity in larval zebraﬁsh was signiﬁcantly reduced by C646, an inhibitor of EP300 that antagonizes acetyl-CoA. The reduction of adiposity by C646 was not signiﬁcantly different from that induced by RSV or co- treatment of C646 and RSV. These results indicate that the inhibition of EP300 might be a common therapeutic mechanism between CR and RSV in adipose tissues of obese individuals. Reviewed by: Partha Krishnamurthy, University of Kansas Medical Center, USA Reviewed by: Partha Krishnamurthy, University of Kansas Medical Center, USA Luca Vanella, University of Catania, Italy Correspondence: Toshio Tanaka, Department of Molecular and Cellular Pharmacology, Pharmacogenomics and Pharmacoinformatics, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507, Japan tanaka@doc.medic.mie-u.ac.jp Specialty section: This article was submitted to Experimental Pharmacology and Drug Discovery, a section of the journal Frontiers in Pharmacology Received: 06 August 2015 Accepted: 31 August 2015 Published: 15 September 2015 Keywords: resveratrol, caloric restriction, obesity, adipose tissue, ep300, zebraﬁsh, comparative transcriptome, systems pharmacology Edited by: Chiranjib Chakraborty, Galgotias University, India Reviewed by: Partha Krishnamurthy, University of Kansas Medical Center, USA Luca Vanella, University of Catania, Italy Correspondence: Toshio Tanaka, Department of Molecular and Cellular Pharmacology, Pharmacogenomics and Pharmacoinformatics, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507, Japan tanaka@doc.medic.mie-u.ac.jp Edited by: Chiranjib Chakraborty, Galgotias University, India Edited by: Chiranjib Chakraborty, Galgotias University, India Introduction According to the World Health Organization, an estimated 310 million people worldwide are obese (Bessesen, 2008). Such estimates are particularly alarming given the strong association between obesity and various adverse health consequences, including atherosclerosis, hypertension, type 2 diabetes and certain types of cancer (Bessesen, 2008). CR can alleviate these deleterious conditions in obesity (Guarente, 2013). However, eating less for the sake of creating a desirable metabolic proﬁle is unlikely to gain widespread compliance (Timmers et al., 2011). Accordingly, there has been an increasing interest in identifying compounds that elicit the beneﬁcial eﬀects of CR without requiring reduced calorie intake. Citation: Nishimura Y, Sasagawa S, Ariyoshi M, Ichikawa S, Shimada Y, Kawaguchi K, Kawase R, Yamamoto R, Uehara T, Yanai T, Takata R and Tanaka T (2015) Systems pharmacology of adiposity reveals inhibition of EP300 as a common therapeutic mechanism of caloric restriction and resveratrol for obesity. Front. Pharmacol. 6:199. doi: 10.3389/fphar.2015.00199 September 2015 \| Volume 6 \| Article 199 Frontiers in Pharmacology \| www.frontiersin.org 1 CR and RSV inhibit EP300 Nishimura et al. Compounds and Reagents Resveratrol and C646 were purchased from Sigma (St. Louis, MO, USA). Nile Red was purchased from Tokyo Kasei (Tokyo, Japan); 2-phenoxyethanol and TG L-type assay were purchased from Wako (Tokyo, Japan). Resveratrol is thought to mimic the eﬀects of CR in obesity (Guarente, 2013). In mice on a high-fat diet, RSV diminished total body fat content and decreased depots of epididymal, inguinal and retroperitoneal white adipose tissue (Lagouge et al., 2006). In obese Zucker rats, the administration of RSV resulted in a signiﬁcant reduction in plasma TGs, free fatty acids, cholesterol, and liver TGs when compared with untreated obese Zucker rats (Rivera et al., 2009). Ethics Statement Mie University Institutional Animal Care and Use Committee guidelines state that no approval is required for experiments using zebraﬁsh. However, animal experiments described in this manuscript conform to the ethical guidelines established by the Institutional Animal Care and Use Committee at Mie University. Zebraﬁsh Husbandry Zebraﬁsh were maintained according to the methods described by Westerﬁeld (2007) with some modiﬁcation. Brieﬂy, zebraﬁsh were raised at 28.5 ± 0.5◦C with a 14 h/10 h light/dark cycle. Embryos were obtained via natural mating and cultured in 0.3 × Danieau solution [19.3 mM NaCl, 0.23 mM KCl, 0.13 mM MgSO4, 0.2 mM Ca(NO3)2, 1.7 mM HEPES, pH 7.2]. To induce the adult DIO model, we used a transparent zebraﬁsh mutant line MieKomachi 001 (MK001) that was created by crossing nacre and rose. For the experiments using larva, we used an albino zebraﬁsh line that was obtained from the Max Planck Institute for Developmental Biology (Tübingen, Germany). The mechanism of how RSV exerts these favorable eﬀects was proposed to be related to the induction of genes encoding oxidative phosphorylation and mitochondrial biogenesis molecules (Chung et al., 2012; de Ligt et al., 2015). Numerous data indicate that the activation of NAD+-dependent protein deacetylase, SIRT1, is pivotal for the beneﬁcial eﬀect of RSV (Chung et al., 2012; de Ligt et al., 2015). SIRT1 catalyzes, among others, deacetylation and the activation of peroxisome proliferator gamma coactivator-1α, a cofactor in mitochondrial biogenesis (Chung et al., 2012; de Ligt et al., 2015). However, the biochemical pathways proposed to mediate the beneﬁcial eﬀects of RSV are highly interconnected (Chung et al., 2012), suggesting that systems pharmacology might be required to fully elucidate the therapeutic mechanism of RSV in obesity. Adult DIO Zebraﬁsh Model Female zebraﬁsh at 4 mpf were assigned into three dietary groups: OF, OF + RSV, and control, with ﬁve ﬁsh per 2-L tank. Zebraﬁsh in the OF and OF + RSV groups were fed three times per day with freshly hatched Artemia (corresponding to 60 mg cysts/ﬁsh/day). Zebraﬁsh in the control group were fed freshly hatched Artemia (corresponding to 5 mg cysts/ﬁsh/day) once per day. Zebraﬁsh in the OF + RSV groups were fed with gluten containing RSV (corresponding to 20 μg /ﬁsh/day) at 20 min before feeding Artemia in the morning. Zebraﬁsh in the OF and control groups were fed with gluten without RSV at 20 min before feeding Artemia in the morning. Gluten with or without RSV were prepared as previously described (Zang et al., 2011). The body weight and length of zebraﬁsh were measured weekly throughout the study. Zebraﬁsh length was measured from the head to the end of the body. Blood collection and TG measurement in the plasma were performed as described previously (Oka et al., 2010). Staining visceral adipose tissues was performed as previously described (Oka et al., 2010) except for the concentration of Nile Red (1 μg /ml) and the staining time (30 min). After staining, the visceral adipose tissues were observed with a ﬂuorescence microscope (MZ 16F, Leica, Tokyo, Japan) using a GFP2 ﬁlter (Leica). The ﬂuorescent intensity of Nile Red was calculated using Volocity 3D Image Analysis Software (Perkin-Elmer, Cambridge, MA, USA). It was demonstrated that CR increases the expression of SIRT1 protein (Cohen et al., 2004) and that the beneﬁcial eﬀects of CR are mediated via SIRT1 (Picard et al., 2004). However, the general up-regulation of Sirt1 expression was challenged by a study showing CR-regulated Sirt1 expression was tissue speciﬁc in mice (Chen et al., 2008). Moreover, SIRT1-independent eﬀects of CR were also reported (Kaeberlein and Powers, 2007). These ﬁndings suggest there common therapeutic mechanisms between CR and RSV might be both dependent and independent of SIRT1. To identify the common therapeutic mechanism between CR and RSV, we performed comparative transcriptome analysis of adipose tissues from DIO zebraﬁsh and obese humans with or without CR or RSV. Combined with bioinformatics analysis, we identiﬁed EP300, a histone and lysyl acetyltransferase, as a master regulator for genes dysregulated in obesity and normalized by CR and RSV. Frontiers in Pharmacology \| www.frontiersin.org Identiﬁcation of Transcriptional Regulators using iRegulon iRegulon (Janky et al., 2014) exploits the fact that genes that are co-regulated by the same TF commonly share binding sites for the TF and uses gene sets derived from ENCODE ChIP-seq data. We used iRegulon as an application in Cytoscape (Shannon et al., 2003). The lists of diﬀerentially expressed genes shown in Supplementary Table S1 were subjected to iRegulon and used to predict their transcriptional regulators using the default setting. The predicted transcriptional regulators with normalized enrichment scores (NES) >3 are shown in Supplementary Table S4 (Sheets 1–4) and used for further analysis. Adult DIO Zebraﬁsh Model We also demonstrated that the inhibition of EP300 by a competitive inhibitor of acetyl-CoA reduced the adiposity of larval zebraﬁsh. These results suggest that the inhibition of EP300 might be a common therapeutic mechanism of CR and RSV on adiposity in obesity. Assessment of Adiposity in Larval Zebraﬁsh Zebraﬁsh at three dpf were treated with 2 μM C646, 200 μM RSV or 2 μM C646 and 200 μM RSV for 48 h. After the treatment, September 2015 \| Volume 6 \| Article 199 Frontiers in Pharmacology \| www.frontiersin.org 2 CR and RSV inhibit EP300 Nishimura et al. 2015) and the probes that passed four criteria (gIsSaturated, gIsFeatNonUnifOL, gIsPosAndSignif, gIsWellAboveBG) across the dataset were used for further analysis. For the aﬀymetrix array, the transcriptome data were normalized using “aﬀy” (Bolstad et al., 2003) for GSE35710 or “oligo” (Carvalho et al., 2007) for GSE29718 and GSE42432. Data normalized by “aﬀy” were ﬁltered based on the presence-absence call and probes that were present or marginal across the dataset were used for further analysis. Data normalized by “oligo” were ﬁltered based on the normalized signal and probes with a normalized signal >3 across the dataset were used for further analysis. RankProd analysis (Hong et al., 2006) was performed to identify diﬀerentially expressed genes between two groups by calculating the FDR. FDR30% was used as the threshold. For transcriptome analysis of zebraﬁsh, diﬀerentially expressed genes were converted to human orthologs using the Life Science Knowledge Bank (World Fusion, Tokyo, Japan). zebraﬁsh were stained with Nile Red (5 ng/ml) for 30 min. After staining, zebraﬁsh were rinsed in 0.3 × Danieau solution and anesthetized with 2-phenoxyethanol (500 ppm). In vivo imaging of the zebraﬁsh was performed using a ﬂuorescence microscope (SMZ25, Nikon, Tokyo, Japan) with a GFP-L ﬁlter (Nikon). The area stained with Nile Red in the abdomen was measured using Volocity (Perkin-Elmer). Transcriptome Analysis of Visceral Adipose Tissues of Female DIO Zebraﬁsh The visceral adipose tissue of female DIO zebraﬁsh overfed Artemia for 1 week (OF 1 w) or 5 weeks (OF 5 w), or overfed Artemia for 5 weeks with RSV (OF + RSV 5 w), were stained with Nile Red and collected by surgical extraction under a ﬂuorescence microscope. The visceral adipose tissue was stored in RNA-later (Applied Biosystems, Foster City, CA, USA). Total RNA was then extracted using an RNeasy Plus Micro Kit (Qiagen, Valencia, CA, USA), qualiﬁed by an Agilent Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA) and quantiﬁed using a spectrophotometer (NanoDrop ND-100, Wilmington, DE, USA). Fifty nanograms of total RNA from each visceral adipose tissue depot were converted into labeled cRNA using the Low RNA Input Fluorescent Linear Ampliﬁcation Kit (Agilent). Cy3-labeled cRNA (860 ng) was hybridized to Agilent Zebraﬁsh Whole Genome Oligo Microarrays (G2519F) according to the manufacturer’s protocol. The hybridized microarrays were scanned (Agilent G2565BA) and analyzed using Feature Extraction software (Agilent). The data were normalized using Limma (Ritchie et al., 2015), a package in Bioconductor. Probes that passed four criteria (gIsSaturated, gIsFeatNonUnifOL, gIsPosAndSignif, gIsWellAboveBG) across the dataset were used for further analysis. RankProd analysis (Hong et al., 2006) was performed to identify diﬀerentially expressed genes between two groups by calculating the false discovery rate (FDR). Diﬀerentially expressed genes (FDR < 30%) were then converted to human orthologs using the Life Science Knowledge Bank (World Fusion, Tokyo, Japan). The gene symbols of human orthologs were used for functional analysis. The gene symbols of human orthologs were used for functional analysis. The microarray data has been deposited to GEO as GSE70281. Identiﬁcation of Transcriptional Regulators using Pathway Studio Pathway Studio (Nikitin et al., 2003) uses gene sets derived from natural language processing based text mining of published literature, including a gene set for transcriptional regulators, composed of genes whose promoters the transcriptional regulator bound to and genes whose expression were regulated by the transcriptional regulator. The lists of diﬀerentially expressed genes shown in Supplementary Table S1 were subjected to Pathway Studio and used to predict their transcriptional regulators using the subnetwork enrichment analysis for “expression target”. The predicted transcriptional regulators with p < 5.0 × 10−3 are shown in Supplementary Table S5 (Sheets 1–4) and were used for further analysis. anscriptome Analysis of Adipose Tissue in ale DIO Zebraﬁsh and Obese Humans We downloaded transcriptome data from the GEO analyzing visceral adipose tissues of male DIO zebraﬁsh with and without CR (GSE18566; Oka et al., 2010), subcutaneous adipose tissues of obese humans with and without CR (GSE35710; Nookaew et al., 2013), subcutaneous adipose tissues of obese human males with and without RSV (GSE42432; Konings et al., 2014), subcutaneous adipose tissue of obese human females (GSE44000; Deng et al., 2013), and subcutaneous adipose tissue of obese human males (GSE29718; Tam et al., 2011). Identiﬁcation of Cell Processes Enriched in a Gene List using Pathway Studio The lists of diﬀerentially expressed genes were subjected to Pathway Studio and used to predict cell processes related to the lists using the subnetwork enrichment analysis with p < 5.0 × 10−3 as the threshold. Identiﬁcation of Cell Processes Enriched in a Gene List using Pathway Studio The lists of diﬀerentially expressed genes were subjected to Pathway Studio and used to predict cell processes related to the lists using the subnetwork enrichment analysis with p < 5.0 × 10−3 as the threshold. K-Means Clustering Identiﬁcation of Transcriptional Regulators for Genes Commonly Regulated by RSV and CR in Obese Humans We then applied this analysis to determine the common mechanism of action of RSV and CR in adipose tissues of obese humans. We analyzed the transcriptome data of subcutaneous adipose tissues from obese humans with and without RSV (Konings et al., 2014) or CR (Nookaew et al., 2013) deposited to GEO as GSE42432 and GSE35710, respectively. GSE42432 contained data from males only, whereas GSE35710 contained data from both females and males. The correlation of gene expression between RSV and CR was low (Figure 2A, left and middle), which is consistent with the result in DIO zebraﬁsh (Figure 1A), while the correlation between female and male CR was high (r = 0.87, p < 1 × 10−4, Figure 2A, right). We identiﬁed seven genes in common (Figure 2B, Supplementary Table S1, Sheet 2) among 597 genes with altered expression by RSV (Supplementary Table S2, Sheet 3), and 391 and 1,017 genes with altered expression by CR in females and males, respectively (Supplementary Table S2, Sheets 4 and 5). Gene ontology analysis revealed that 177 cell processes, including lipid metabolism, lipid degradation, and mitochondrial depolarization were enriched in the seven genes (Supplementary Table S3, Sheet 2). Although only one gene (transferrin) was overlapped between the nine and seven common genes in zebraﬁsh and humans, respectively (Supplementary Table S1, Sheets 1 and 2), 36 gene ontologies were overlapped between the 56 and 177 gene ontologies in zebraﬁsh and humans, respectively (Supplementary Table S3, Sheets 1 and 2). iRegulon identiﬁed 19 transcriptional regulators for the seven genes (Supplementary Table S4, Sheet 2). The network between these transcriptional regulator and their targets is shown in Figure 2C. Pathway Studio identiﬁed 34 regulators for the seven common genes (Supplementary Table S5, Sheet 2). Three regulators were overlapped between data from the iRegulon and Pathway Studio analyses. The network between the three regulators Results Resveratrol Reduces Plasma Triglyceride and Visceral Fat in Diet-Induced Obese Zebraﬁsh We previously demonstrated that CR after OF signiﬁcantly lowered both plasma TG and visceral adiposity in zebraﬁsh (Oka et al., 2010). In this study, we ﬁrst examined whether RSV could also exert these eﬀects in the DIO zebraﬁsh model. Adult female zebraﬁsh were overfed with freshly hatched nauplii of Artemia that have a relatively high fat content compared with ﬂake foods (Oka et al., 2010) with and without RSV treatment (20 μg/day, 40 mg/kg body weight/day in 0.5 g zebraﬁsh). The BMI of OF zebraﬁsh was signiﬁcantly increased compared with the BMI of control zebraﬁsh after 1 week (Supplementary Figure S1A). The BMI of zebraﬁsh overfed with Artemia and treated with RSV (OF + RSV) was also signiﬁcantly increased compared with the BMI of control zebraﬁsh. However, the BMI of OF + RSV zebraﬁsh was not signiﬁcantly diﬀerent from that of OF zebraﬁsh. We then examined the eﬀects of RSV on plasma TG. As shown in Supplementary Figure S1B, plasma TG in OF zebraﬁsh was signiﬁcantly increased compared with that of control zebraﬁsh. In contrast, plasma TG levels in OF + RSV zebraﬁsh were signiﬁcantly lower than in OF zebraﬁsh. We also examined the eﬀects of RSV on visceral adiposity in DIO zebraﬁsh, which can be visualized by Nile Red staining (Oka et al., 2010). As shown in Supplementary Figure S1C, the ﬂuorescent intensity of Nile Red in OF zebraﬁsh was signiﬁcantly higher than in control zebraﬁsh. The ﬂuorescent intensity of Nile Red in OF + RSV zebraﬁsh was signiﬁcantly lower than in OF zebraﬁsh. These results suggest that RSV can reduce plasma TG and adiposity in DIO zebraﬁsh, consistent with previous reports in mammalian obesity (Rocha et al., 2009; Timmers et al., 2011). Statistical Analysis (Janky et al., 2014) to identify master regulators of the co- expressed genes based on the genome-wide ENCODE ChIP- seq data. iRegulon identiﬁed 20 transcriptional regulators for the nine genes with a NES >3 (Supplementary Table S4, Sheet 1). The network between these transcriptional regulators and their target genes is shown in Figure 1C. To validate the network identiﬁed by iRegulon, we used Pathway Studio (Nikitin et al., 2003) to identify the master regulators of co- expressed genes using natural language processing based text mining. Pathway Studio identiﬁed 50 regulators for the nine common genes (Supplementary Table S5, Sheet 1). Seven regulators were overlapped between iRegulon and Pathway Studio data. The network between the seven regulators and their target genes identiﬁed by Pathway Studio is shown in Figure 1D. The overlapping network correlated well with the data identiﬁed by iRegulon. For example, Pathway Studio identiﬁed FOXA1/FOXA2 as positive regulators of IGFBP1, TF, and UCP3. iRegulon also identiﬁed these three genes as targets of FOXA1/FOXA2 Supplementary Table S4, Sheet 1). Statistical analysis (analysis of variance followed by Tukey’s multiple comparisons test) was performed using Prism 6.0 (GraphPad Software, San Diego, CA, USA). K-Means Clustering For the Agilent array (GSE18566 and GSE44000), transcriptome data were normalized using Limma (Ritchie et al., K-means clustering was performed using MultiExperiment Viewer v4.8 (Howe et al., 2011). Frontiers in Pharmacology \| www.frontiersin.org September 2015 \| Volume 6 \| Article 199 3 CR and RSV inhibit EP300 Nishimura et al. Identiﬁcation of Transcriptional Regulators for Genes Commonly Regulated by RSV and CR in DIO Zebraﬁsh To identify common mechanisms of action between RSV and CR, we compared transcriptome data of visceral adipose tissue in DIO zebraﬁsh with RSV or CR (Oka et al., 2010). Although the correlation of change in gene expression between RSV and CR was low (Figure 1A), we identiﬁed nine genes in common (Figure 1B and Supplementary Table S1, Sheet 1) from 27 and 528 genes whose expression were changed by RSV (Supplementary Table S2, Sheet 1) and CR (Supplementary Table S2, Sheet 2), respectively. Gene ontology analysis revealed that 56 cell processes, including lipid peroxidation, lipid storage, and energy homeostasis, were enriched in the nine identiﬁed genes (Supplementary Table S3, Sheet 1). To reveal the molecular mechanism involved in the regulation of the expression of the nine common genes, we used iRegulon September 2015 \| Volume 6 \| Article 199 Frontiers in Pharmacology \| www.frontiersin.org 4 CR and RSV inhibit EP300 Nishimura et al. FIGURE 1 \| Identiﬁcation of transcriptional regulators for genes with changed expression levels common to RSV and CR in DIO zebraﬁsh. (A) Correlation of gene expression changes induced by RSV and CR in DIO zebraﬁsh. (B) Venn diagram of the numbers of DEG by RSV and CR in DIO zebraﬁsh. (C) A regulatory network identiﬁed by iRegulon for the nine genes common between the RSV and CR groups. Genes shown in blue and red are decreased and increased, respectively, by RSV or CR. (D) A regulatory network identiﬁed by Pathway Studio for seven regulators and their six target genes. Genes shown in blue and red indicate decreased and increased expression, respectively, by RSV or CR. pression levels common to RSV and CR in DIO zebraﬁsh. FIGURE 1 \| Identiﬁcation of transcriptional regulators for genes with changed expression levels common to RSV and CR in DIO zebraﬁsh. (A) Correlation of gene expression changes induced by RSV and CR in DIO zebraﬁsh. (B) Venn diagram of the numbers of DEG by RSV and CR in DIO zebraﬁsh. (C) A regulatory network identiﬁed by iRegulon for the nine genes common between the RSV and CR groups. Genes shown in blue and red are decreased and increased, respectively, by RSV or CR. (D) A regulatory network identiﬁed by Pathway Studio for seven regulators and their six target genes. Genes shown in blue and red indicate decreased and increased expression, respectively, by RSV or CR. Identiﬁcation of Transcriptional Regulators for Genes Commonly Regulated by RSV and CR in DIO Zebraﬁsh and 473 genes dysregulated in female and male DIO zebraﬁsh, respectively (Supplementary Table S2, Sheets 6 and 7). Gene ontology analysis revealed that 111 cell processes, including cell damage, superoxide anion generation and blood clotting, were enriched in the 15 genes identiﬁed (Supplementary Table S3, Sheet 3). iRegulon identiﬁed 14 transcriptional regulators for the 15 genes (Supplementary Table S4, Sheet 3). The network between these transcriptional regulators and their target genes is shown in Figure 3C. Pathway Studio identiﬁed 61 regulators for the 15 common genes (Supplementary Table S5, Sheet 3). Four regulators were overlapped between the iRegulon and Pathway Studio data. The network between the four regulators and their targets is shown in Figure 3D. The overlapping network correlated well with the data identiﬁed by iRegulon. Pathway Studio identiﬁed CEBPB as a positive regulator of SERPINA1, and their target genes identiﬁed by Pathway Studio is shown in Figure 2D. The overlapping network correlated well with the data identiﬁed by iRegulon. For example, Pathway Studio identiﬁed EP300 as a positive regulator of LEP, TF, and NQO1. iRegulon also identiﬁed these three genes as targets of EP300 (Supplementary Table S4, Sheet 2). Identiﬁcation of Transcriptional Regulators for Genes Dysregulated in DIO Zebraﬁsh We then analyzed transcriptome data of visceral adipose tissues from female and male (Oka et al., 2010) DIO zebraﬁsh to identify the network dysregulated in obesity. Although the correlation of gene expression between female and male DIO zebraﬁsh was not high (Figure 3A), we identiﬁed 15 genes in common (Figure 3B, Supplementary Table S1, Sheet 3) between 81 September 2015 \| Volume 6 \| Article 199 Frontiers in Pharmacology \| www.frontiersin.org 5 CR and RSV inhibit EP300 Nishimura et al. GURE 2 \| Identiﬁcation of the regulatory network of genes with changed expression levels common to RSV and CR in obese humans. (A) Correlation gene expression changes by RSV in obese males and CR in obese females (left) or males (middle) and CR in obese females and males (right). (B) Venn diagram of numbers of DEG by RSV in obese males, or CR in obese females or males. (C) A regulatory network identiﬁed by iRegulon for the seven common genes among V in obese males, and CR in obese females and males. Genes shown in blue and red indicate decreased and increased expression, respectively, by RSV or CR. A regulatory network identiﬁed by Pathway Studio between three regulators and their ﬁve target genes. Genes shown in blue and red indicate decreased and reased expression, respectively, by RSV or CR. FIGURE 2 \| Identiﬁcation of the regulatory network of genes with changed expression levels common to RSV and CR in obese humans. (A) Correlation of gene expression changes by RSV in obese males and CR in obese females (left) or males (middle) and CR in obese females and males (right). (B) Venn diagram of the numbers of DEG by RSV in obese males, or CR in obese females or males. (C) A regulatory network identiﬁed by iRegulon for the seven common genes among RSV in obese males, and CR in obese females and males. Genes shown in blue and red indicate decreased and increased expression, respectively, by RSV or CR. (D) A regulatory network identiﬁed by Pathway Studio between three regulators and their ﬁve target genes. Genes shown in blue and red indicate decreased and increased expression, respectively, by RSV or CR. CP, IGFBP1, and FGB, all of which were also identiﬁed as targets of CEBPB by iRegulon (Supplementary Table S4, Sheet 3). Identiﬁcation of Transcriptional Regulators for Genes Dysregulated in DIO Zebraﬁsh correlation of gene expression between obese human females and males was relatively high (r = 0.37, p < 1 × 10−4). We identiﬁed 46 genes in common (Figure 4B; Supplementary Table S1, Sheet 4) between 1,736 and 124 genes dysregulated in obese human females and males, respectively (Supplementary Table S2, Sheets 8 and 9). Gene ontology analysis revealed that 350 cell processes, including lipid peroxidation, oxidative stress, and inﬂammatory response were enriched in the 46 genes (Supplementary Table S3, Sheet 4). Although there was no overlap between the 15 and 46 common genes in zebraﬁsh and humans, respectively (Supplementary Table S1, Sheets Identiﬁcation of Transcriptional Regulators for Genes Dysregulated in Obese Humans To identify regulatory network for genes dysregulated in obese humans, we analyzed the transcriptome data of adipose tissues from obese human females (Deng et al., 2013) and males (Tam et al., 2011) deposited to GEO as GSE44000 and GSE29718, respectively. As shown in Figure 4A, the September 2015 \| Volume 6 \| Article 199 Frontiers in Pharmacology \| www.frontiersin.org 6 CR and RSV inhibit EP300 Nishimura et al. FIGURE 3 \| Identiﬁcation of the regulatory network for genes dysregulated in DIO zebraﬁsh. (A) Correlation of the change in gene expression in DIO female and male zebraﬁsh. (B) Venn diagram of the numbers of DEG in DIO female and male zebraﬁsh. (C) A regulatory network identiﬁed by iRegulon for the 15 common genes between female and male DIO zebraﬁsh. Genes shown in blue and red indicate decreased and increased expression, respectively, by obesity. (D) A regulatory network identiﬁed by Pathway Studio for four regulators and their eight target genes. Genes shown in blue and red indicate decreased and increased expression, respectively, by obesity. FIGURE 3 \| Identiﬁcation of the regulatory network for genes dysregulated in DIO zebraﬁsh. (A) Correlation of the change in gene expression in DIO female and male zebraﬁsh. (B) Venn diagram of the numbers of DEG in DIO female and male zebraﬁsh. (C) A regulatory network identiﬁed by iRegulon for the 15 common genes between female and male DIO zebraﬁsh. Genes shown in blue and red indicate decreased and increased expression, respectively, by obesity. (D) A regulatory network identiﬁed by Pathway Studio for four regulators and their eight target genes. Genes shown in blue and red indicate decreased and increased expression, respectively, by obesity. ontology analysis revealed that 161 cell processes were enriched in the eight transcriptional regulators in Clusters 5, 6, and 7 (Supplementary Table S3, Sheet 5). Five cell processes (adipocyte diﬀerentiation, aging, energy homeostasis, lipid metabolism, and lipid storage) were overlapped with those enriched in genes dysregulated in obesity and normalized by RSV and CR in both DIO zebraﬁsh and obese humans (Supplementary Table S3, Sheet 6). The network between these transcriptional regulators and cell processes identiﬁed by Pathway Studio is shown in Figure 5B. It was demonstrated that EP300 activates CEBPB, CEBPB, FOXA1, and FOXA2 by acetylation (Wang et al., 2006, 2013b; Cesena et al., 2007; Kohler and Cirillo, 2010). Identiﬁcation of Transcriptional Regulators for Genes Dysregulated in Obese Humans These ﬁndings suggest that EP300 might be a key transcriptional regulator involved in the common therapeutic mechanism of RSV and CR in adipose tissues of obese individuals. 3 and 4), 73 gene ontologies were overlapped between the respective 111 and 350 gene ontologies in zebraﬁsh and humans (Supplementary Table S3, Sheets 3 and 4). iRegulon identiﬁed 10 transcriptional regulators for the 46 common genes (Supplementary Table S4, Sheet 4). The network between these regulator and their targets is shown in Figure 4C. Pathway Studio identiﬁed 428 regulators for the 46 common genes (Supplementary Table S5, Sheet 4). Five regulators were overlapped between the iRegulon and Pathway Studio data. The regulatory network between the ﬁve regulators and their 18 target genes is shown in Figure 4D. Identiﬁcation of Common Transcriptional Regulators Targeted by RSV and CR To reveal which transcriptional regulators might be involved in the common mechanism of RSV and CR, we performed K-Means clustering (Soukas et al., 2000) of transcriptional regulators identiﬁed by iRegulon based on their NES (Supplementary Table S4). As shown in Figure 5A, the K-Means clustering classiﬁed these transcriptional regulators into seven clusters. The clustering revealed that HDAC2, CEBPB, and EP300 (Cluster 7) were transcriptional regulators for genes dysregulated in obesity and normalized by RSV and CR in both DIO zebraﬁsh and obese human. NR2F2, POLR2A, CEBPD (Cluster 5), and FOXA1, FOXA2 (Cluster 6) were transcriptional regulators for genes dysregulated in DIO zebraﬁsh and normalized by RSV and CR in both DIO zebraﬁsh and obese humans. Gene Inhibition of EP300 Reduces Adiposity in Larval Zebraﬁsh The network between EP300 and the target genes identiﬁed by iRegulon is shown in Figure 6A. It was demonstrated that EP300 increased the expression of TF (Chaudhary and Skinner, 2001), IGFBP1 (Nasrin et al., 2000), NQO1 (Macoch et al., 2015), and LEP genes (Cascio et al., 2008). The expression of these genes increased in obesity (Supplementary Figure S2; Supplementary Table S1, Sheets 3 and 4) and was normalized by RSV and CR (Supplementary Figure S2; Supplementary Table S1, Sheets 1 and 2) in adipose tissues of zebraﬁsh and humans. These results suggest that EP300 might be activated in obesity and September 2015 \| Volume 6 \| Article 199 Frontiers in Pharmacology \| www.frontiersin.org 7 CR and RSV inhibit EP300 Nishimura et al. FIGURE 4 \| Identiﬁcation of the regulatory network for genes dysregulated in obese humans. (A) Correlation of genes dysregulated in adipose tissues from female and male obese humans. (B) Venn diagram of the numbers of DEG in obese females and males. (C) A regulatory network identiﬁed by iRegulon for the 46 common genes between obese females and males. Genes shown in blue and red indicate decreased and increased expression, respectively, by obesity. (D) A regulatory network identiﬁed by Pathway Studio for ﬁve regulators and their 18 target genes. Genes shown in blue and red indicate decreased and increased expression, respectively, by obesity. FIGURE 4 \| Identiﬁcation of the regulatory network for genes dysregulated in obese humans. (A) Correlation of genes dysregulated in adipose tissues from female and male obese humans. (B) Venn diagram of the numbers of DEG in obese females and males. (C) A regulatory network identiﬁed by iRegulon for the 46 common genes between obese females and males. Genes shown in blue and red indicate decreased and increased expression, respectively, by obesity. (D) A regulatory network identiﬁed by Pathway Studio for ﬁve regulators and their 18 target genes. Genes shown in blue and red indicate decreased and increased expression, respectively, by obesity. the acetylation of FXR was constitutively activated in obesity (Kemper et al., 2009). EP300 also activated the transcription of genes encoding TG synthetic enzymes (Bricambert et al., 2010). Disrupting the function of EP300 in mice resulted in the reduction of white adipose tissue and plasma TG (Bedford et al., 2011). These ﬁndings suggest that EP300 might be activated in obesity and that the inhibition of EP300 would be a potential therapeutic strategy in obesity. Inhibition of EP300 Reduces Adiposity in Larval Zebraﬁsh that both RSV and CR may suppress the activity of EP300 in adipose tissues. To examine the eﬀect of EP300 on adiposity, we used a selective EP300 inhibitor C646 that competes for AcCoA binding to EP300 (Bowers et al., 2010). As shown in Figure 6B, the abdominal area of larval zebraﬁsh stained by Nile Red was signiﬁcantly decreased by 2 μM C646, 200 μM RSV, or co-treatment of 2 μM C646 and 200 μM RSV. However, the reduction of adiposity was not signiﬁcantly diﬀerent among the three treatment groups. These results suggest that the inhibition of EP300 might be a major therapeutic mechanism of RSV. It was shown that CR decreased intracellular AcCoA (Marino et al., 2014) suggesting that CR might inhibit EP300 through reducing AcCoA. p gy y It was shown that RSV inhibited EP300 through the activation of SIRT1 (Shakibaei et al., 2011). When activated, SIRT1 promoted the deacetylation of lysine residues on EP300, resulting in the inhibition of acetyltransferase activity of EP300 (Kume et al., 2007). Because SIRT1 is also activated by CR (Guarente, 2013), CR might inhibit EP300 through the activation of SIRT1. However, CR also decreases intracellular AcCoA through SIRT3 (Hebert et al., 2013). Furthermore, CR inhibited EP300 through the depletion of AcCoA in the heart and skeletal muscle of mice, leading to the deacetylation of cellular proteins and activation of AMP-dependent protein kinase (Marino et al., 2014). These ﬁndings suggest that EP300 might be inhibited by RSV and CR in adipose tissues either dependently or independently of SIRT1. Discussion In this study, we demonstrated that the inhibition of EP300 might be common therapeutic mechanism of CR and RSV in adipose tissues of obese individuals. EP300 is a transcriptional coactivator with various functions (Wang et al., 2013a), including linking DNA-bound TF to basal transcription machinery, relaxing chromatin structure through its histone acetyltransferase activity, and modulating the function of various proteins through the lysyl acetyltransferase activity (Wang et al., 2013a). In this study, we performed comparative transcriptome analysis of adipose tissues from DIO zebraﬁsh and obese humans to identify a common therapeutic mechanism of RSV and CR. There are multiple methods to identify common pathways using comparative transcriptome analysis. The It was demonstrated that EP300 acetylated FXR, a master regulator of lipid homeostasis (Modica et al., 2010) and that September 2015 \| Volume 6 \| Article 199 Frontiers in Pharmacology \| www.frontiersin.org 8 CR and RSV inhibit EP300 Nishimura et al. FIGURE 5 \| Identiﬁcation of common transcriptional regulators targeted by RSV and CR. (A) K-Means clustering of regulators identiﬁed by iRegulon based on their NES in each analysis. (B) Network between transcriptional regulators in Clusters 5, 6, and 7 and cellular functions enriched in these transcriptional regulators and the genes dysregulated in obesity and normalized by RSV or CR. FIGURE 5 \| Identiﬁcation of common transcriptional regulators targeted by RSV and CR. (A) K-Means clustering of regulators identiﬁed by iRegulon based on their NES in each analysis. (B) Network between transcriptional regulators in Clusters 5, 6, and 7 and cellular functions enriched in these transcriptional regulators and the genes dysregulated in obesity and normalized by RSV or CR. simplest way is to identify DEG that overlap among diﬀerent transcriptome data. For example, we identiﬁed 46 DEG that overlapped between obese human females and males. Among the DEG, several genes were also identiﬁed as DEG in other transcriptome analyses not included in this study. These genes include carboxyl esterase 1 (CES1; Jernas et al., 2009), NAD(P)H dehydrogenase, quinone 1 (NQO1; Kim et al., 2011), cathepsin S (CTSS; Lee et al., 2005), matrix metallopeptidase 9 (MMP9; Lee et al., 2005). The expression of these genes was linked to adiposity (Taleb et al., 2005; Palming et al., 2007; Jernas et al., 2009), suggesting that the DEG replicated in multiple transcriptome data is a good and robust marker for obesity. Frontiers in Pharmacology \| www.frontiersin.org Discussion overlapped DEG by comparative transcriptome analysis might be too stringent, resulting in overlooking important genes and pathways. To circumvent this, group testing is widely used. For example, the list of DEG from each transcriptome analysis can be compared with predeﬁned gene sets related to speciﬁc functions calculating which gene sets are represented in the list of DEG at a level greater than that expected by chance alone (Manoli et al., 2006). By comparing the gene sets signiﬁcantly enriched in each list of DEG, common pathways can be identiﬁed among multiple transcriptome data. Using this group analysis, several pathways have been successfully identiﬁed as common targets of CR and RSV, including “chromatin assembly or disassembly” (Barger et al., 2008). Functions related to chromatin were also identiﬁed as enriched categories in CR by another comparative transcriptome analysis (Wuttke et al., 2012). In this study, we performed cis-regulatory sequence analysis as a group analysis and identiﬁed EP300 as a common therapeutic target of CR and However, the lists of DEG do not always overlap between the same intervention in diﬀerent strains/species or between related interventions in the same strains/species (Allison et al., 2006; Antosh et al., 2011). Therefore, the identiﬁcation of September 2015 \| Volume 6 \| Article 199 Frontiers in Pharmacology \| www.frontiersin.org 9 CR and RSV inhibit EP300 Nishimura et al. FIGURE 6 \| Inhibition of EP300 reduces the adiposity in larval zebraﬁsh. (A) Network between EP300 and the target genes identiﬁed iRegulon in adipose tissues of DIO zebraﬁsh and obese humans with and without RES and CR. (B) Changes in the visceral adiposity of zebraﬁsh treated with RES and/or C646. The area stained with Nile Red is shown compared with that of control (arbitrarily deﬁned as 100%). Values are the means ± SEM. N = 10–12/group. #p < 0.05 vs control. FIGURE 6 \| Inhibition of EP300 reduces the adiposity in larval zebraﬁsh. (A) Network between EP300 and the target genes identiﬁed iRegulon in adipose tissues of DIO zebraﬁsh and obese humans with and without RES and CR. (B) Changes in the visceral adiposity of zebraﬁsh treated with RES and/or C646. The area stained with Nile Red is shown compared with that of control (arbitrarily deﬁned as 100%). Values are the means ± SEM. N = 10–12/group. #p < 0.05 vs control. FIGURE 6 \| Inhibition of EP300 reduces the adiposity in larval zebraﬁsh. Discussion (A) Network between EP300 and the target genes identiﬁed iRegulon in adipose tissues of DIO zebraﬁsh and obese humans with and without RES and CR. (B) Changes in the visceral adiposity of zebraﬁsh treated with RES and/or C646. The area stained with Nile Red is shown compared with that of control (arbitrarily deﬁned as 100%). Values are the means ± SEM. N = 10–12/group. #p < 0.05 vs control. obesity and normalized by CR and RSV. A previous study reported that mitogen-activated protein kinase phosphatase 3 deﬁcient mice were protected from DIO and that the activation of HDAC2 by increased phosphorylation of Ser394 had a major role in the protection (Feng et al., 2014). Further studies are required to examine whether HDAC2 is involved in the therapeutic mechanism of CR and RSV. RSV. Taken together, these ﬁndings suggest that both CR and RSV may normalize the dysregulation of chromatin functions by EP300. We also identiﬁed that the transcriptional regulators CEBPB and HDAC2 might be common therapeutic targets of RSV and CR. EP300 interacts with CEBPB at the promoter of targeted genes (Rastogi et al., 2013) and HDAC2 and EP300 compete for binding to promoters of their target genes (Meng et al., 2009). Indeed, the binding regions of EP300, CEBPB, and HDAC2 in the promoters of transferrin (TF; Supplementary Figure S2), insulin-like growth factor binding protein 1 (IGFBP1; Supplementary Figure S3) and NQO1 (Supplementary Figure S4), whose expression was increased in obesity and was normalized by CR or RSV (Figure 6A), overlap. This might explain why iRegulon identiﬁed CEBPB and HDAC2 as transcriptional regulators. However, we cannot exclude the possibility that the activity of HDAC2 might be inhibited in Supplementary Material The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fphar. 2015.00199 Table S4 \| Transcriptional regulators identiﬁed by iRegulon. Sheet 1: Transcriptional regulators identiﬁed by iRegulon for the common genes between RSV and CR in visceral adipose tissues of DIO zebraﬁsh. Sheet 2: Transcriptional regulators identiﬁed by iRegulon for the common genes between RSV and CR in subcutaneous adipose tissues of obese humans. Sheet 3: Transcriptional regulators identiﬁed by iRegulon for genes dysregulated in common between female and male DIO zebraﬁsh. Sheet 4: Transcriptional regulators identiﬁed by iRegulon for genes dysregulated in common between obese human females and males. FIGURE S1 \| Resveratrol reduces plasma triglyceride and visceral fat in DIO zebraﬁsh. (A) Changes in BMI (g/cm2) of zebraﬁsh in the control, OF and OF + RSV groups. Values are means ± SEM, N = 6/group. (B) Changes in plasma TG levels of zebraﬁsh in the control, OF and OF + RSV groups. Values are means ± SEM. Control: N = 19, OF: N = 14, OF + RSV: N = 15. (C) Changes in visceral adiposity of zebraﬁsh in the control, OF and OF + RSV groups. Values are means ± SEM. N = 9/group. #p < 0.05 vs. control, ∗p < 0.05 vs. OF. FIGURE S1 \| Resveratrol reduces plasma triglyceride and visceral fat in DIO zebraﬁsh. (A) Changes in BMI (g/cm2) of zebraﬁsh in the control, OF and OF + RSV groups. Values are means ± SEM, N = 6/group. (B) Changes in plasma TG levels of zebraﬁsh in the control, OF and OF + RSV groups. Values are means ± SEM. Control: N = 19, OF: N = 14, OF + RSV: N = 15. (C) Changes in visceral adiposity of zebraﬁsh in the control, OF and OF + RSV groups. Values are means ± SEM. N = 9/group. #p < 0.05 vs. control, ∗p < 0.05 vs. OF. FIGURE S2 \| Binding of transcriptional regulators to the human TF gene. These data were based on the UCSC Genome Browser. The binding of EP300, HDAC2, and CEBPB are shown as red, blue, or green arrows. Table S5 \| Transcriptional regulators identiﬁed by Pathway Studio. Sheet 1: Transcriptional regulators identiﬁed by Pathway Studio for the common genes between RSV and CR in visceral adipose tissues of DIO zebraﬁsh. Table S2 \| Differentially expressed genes in each transcriptome dataset. Table S2 \| Differentially expressed genes in each transcriptome dataset. Sheet 1: Genes regulated by RSV in visceral adipose tissues of female DIO zebraﬁsh. Sheet 2: Genes regulated by CR in visceral adipose tissues of male DIO zebraﬁsh. Sheet 3: Genes regulated by RSV in subcutaneous adipose tissues from obese human males treated with RSV. Sheet 4: Genes regulated by CR in subcutaneous adipose tissues from obese human females. Sheet 5: Genes regulated by CR in subcutaneous adipose tissues from obese human males. Sheet 6: Genes dysregulated in visceral adipose tissues of female DIO zebraﬁsh. Sheet 7: Genes dysregulated in visceral adipose tissues of male DIO zebraﬁsh. Sheet 8: Genes dysregulated in subcutaneous adipose tissues of obese human females. Sheet 9: Genes dysregulated in subcutaneous adipose tissues of obese human males. Table S2 \| Differentially expressed genes in each transcriptome dataset. Sheet 1: Genes regulated by RSV in visceral adipose tissues of female DIO zebraﬁsh. Sheet 2: Genes regulated by CR in visceral adipose tissues of male DIO zebraﬁsh. Sheet 3: Genes regulated by RSV in subcutaneous adipose tissues from obese human males treated with RSV. Sheet 4: Genes regulated by CR in subcutaneous adipose tissues from obese human females. Sheet 5: Genes regulated by CR in subcutaneous adipose tissues from obese human males. Sheet 6: Genes dysregulated in visceral adipose tissues of female DIO zebraﬁsh. Sheet 7: Genes dysregulated in visceral adipose tissues of male DIO zebraﬁsh. Sheet 8: Genes dysregulated in subcutaneous adipose tissues of obese human females. Sheet 9: Genes dysregulated in subcutaneous adipose tissues of obese human males. Conclusion We demonstrated that the inhibition of EP300 might be a common therapeutic mechanism of CR and RSV. To our knowledge, this is the ﬁrst study to indicate EP300 might be a potential therapeutic target of CR and RSV in adipose tissues. Chemicals and natural products that inhibit EP300 might function as CR mimetics to reduce adiposity in obesity. September 2015 \| Volume 6 \| Article 199 Frontiers in Pharmacology \| www.frontiersin.org 10 CR and RSV inhibit EP300 Nishimura et al. Acknowledgments This work was supported, in part, by, JSPS KAKENHI (25670127, 15K15051, 24510069), JST A-step (AS262Z00004Q) and Long- range Research Initiative of the Japan Chemical Industrial Association (13_PT01-01). We are grateful to Drs. Hirofumi Furuita and Toshiyuki Yamada (National Research Institute of Aquaculture) for helpful discussion about the DIO zebraﬁsh. We would also like to thank Junko Koiwa, Hiroko Nakayama, Yuka Hayakawa, Yuka Takahashi and Chizuru Hirota for assistance with the experiments, and Rie Ikeyama and Yuka Mizutani for secretarial support. Table S3 \| Cell processes enriched in the gene list. Sheet 1: Cell processes enriched in the common genes between RSV and CR in visceral adipose tissues of DIO zebraﬁsh. Sheet 2: Cell processes enriched in the common genes between RSV and CR in subcutaneous adipose tissues of obese humans. Sheet 3: Cell processes enriched in the common genes dysregulated in visceral adipose tissues of female and male DIO zebraﬁsh. Sheet 4: Cell processes enriched in the common genes dysregulated in subcutaneous adipose tissues of obese human females and males. Sheet 5: Cell processes enriched in HDAC2, EP300, CEBPB, CEBPD, FOXA1, FOXA2, NR2F2, and POLR2A. Sheet 6: Common cell processes enriched in genes between RSV and CR, in genes between obese females and males, and in eight regulators. Supplementary Material Sheet 2: Transcriptional regulators identiﬁed by Pathway Studio for the common genes between RSV and CR in subcutaneous adipose tissues of obese humans. Sheet 3: Transcriptional regulators identiﬁed by Pathway Studio for the common genes between female and male DIO zebraﬁsh. Sheet 4: Transcriptional regulators identiﬁed by Pathway Studio for the common genes between female and male DIO zebraﬁsh. FIGURE S3 \| Binding of transcriptional regulators to the human IGFBP1 gene. These data were based on the UCSC Genome Browser. The binding of EP300, HDAC2, and CEBPB are shown as red, blue, or green arrows. FIGURE S4 \| Binding of transcriptional regulators to the human NQO1 gene. These data were based on the UCSC Genome Browser. The binding of Author Contributions EP300, HDAC2, and CEBPB are shown as red, blue, or green arrows. EP300, HDAC2, and CEBPB are shown as red, blue, or green arrows. YN conceived the study, analyzed the data and wrote the manuscript. SS performed experiments using larval zebraﬁsh. MA, SI, and YS performed experiments using adult DIO zebraﬁsh. KK and RK helped to perform the experiments. RY, TU, TY, and RT conceived the study and measured RSV contents in food. TT conceived the study and wrote the manuscript. Table S1 \| Genes with changes in expression are common between multiple datasets. Sheet 1: Genes with changes in expression are common between RSV and CR in visceral adipose tissue in DIO zebraﬁsh. Sheet 2: Genes with changes in expression are between RSV and CR in subcutaneous adipose tissues of obese humans. 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https://openalex.org/W2744407719	https://biblio.ugent.be/publication/8531955/file/8531957.pdf	English	null	Intake of Fat-Soluble Vitamins in the Belgian Population: Adequacy and Contribution of Foods, Fortified Foods and Supplements	Nutrients	2,017	cc-by	14,490	Isabelle Moyersoen 1,2,* ID , Brecht Devleesschauwer 1, Arnold Dekkers 3, Karin de Ridder 1, Jean Tafforeau 1, John van Camp 2, Herman van Oyen 1,4 and Carl Lachat 2 1 Department of Public Health and Surveillance, Scientiﬁc Institute of Public Health (WIV-ISP), Juliette Wytsmanstraat 14, 1050 Brussels, Belgium; brecht.devleesschauwer@wiv-isp.be (B.D.); Karin.DeRidder@wiv-isp.be (K.d.R.); Jean.Tafforeau@wiv-isp.be (J.T.); herman.vanoyen@wiv-isp.be (H 1 Department of Public Health and Surveillance, Scientiﬁc Institute of Public Health (WIV-ISP), Juliette Wytsmanstraat 14, 1050 Brussels, Belgium; brecht.devleesschauwer@wiv-isp.be (B.D.); Karin.DeRidder@wiv-isp.be (K.d.R.); Jean.Tafforeau@wiv-isp.be (J.T.); herman.vanoyen@wiv-isp 2 Department of Food Safety and Food Quality, Ghent University, Coupure links 653, 9000 Ghent, john.vancamp@ugent.be (J.v.C.); Carl.lachat@Ugent.be (C.L.) 3 p Juliette Wytsmanstraat 14, 1050 Brussels, Belgium; brecht.devleesschauwer@wiv-isp.be (B.D.); Karin.DeRidder@wiv-isp.be (K.d.R.); Jean.Tafforeau@wiv-isp.be (J.T.); herman.vanoyen@wiv-isp.be (H.v.O 2 Department of Food Safety and Food Quality, Ghent University, Coupure links 653, 9000 Ghent, Belgium; john.vancamp@ugent.be (J.v.C.); Carl.lachat@Ugent.be (C.L.) p ( ) p ( ) y p ( 2 Department of Food Safety and Food Quality, Ghent University, Coupure links 653, 9000 Ghent, Belg john.vancamp@ugent.be (J.v.C.); Carl.lachat@Ugent.be (C.L.) 3 Department for Statistics, Informatics and Modelling, National Institute for Public Health and the Environment (RIVM), P.O. BOX 1, 3720 BA Bilthoven, The Netherlands; arnold.dekkers@rivm.nl Department for Statistics, Informatics and Modelling, National Institute for Public Health and the Environment (RIVM), P.O. BOX 1, 3720 BA Bilthoven, The Netherlands; arnold.dekkers@rivm.nl 4 Department of Public Health, Ghent University, De Pintelaan 185, 9000 Gent, Belgium * Correspondence: isabelle moyersoen@wiv-isp be; Tel : +32-264-256-94 ( ), , , ; 4 Department of Public Health, Ghent University, De Pintelaan 185, 9000 Gent, Belgium ( ) 4 Department of Public Health, Ghent University, De Pintelaan 185, 9000 Gent, Belgium * Correspondence: isabelle.moyersoen@wiv-isp.be; Tel.: +32-264-256-94 Department of Public Health, Ghent University, De Pintelaan 185, 9000 Gent, Belgium * Correspondence: isabelle.moyersoen@wiv-isp.be; Tel.: +32-264-256-94 * Correspondence: isabelle.moyersoen@wiv-isp.be; Tel.: +32-264-256-94 Received: 29 June 2017; Accepted: 2 August 2017; Published: 11 August 2017 Abstract: A key challenge of public health nutrition is to provide the majority of the population with a sufﬁcient level of micronutrients while preventing high-consumers from exceeding the tolerable upper intake level. Data of the 2014 Belgian food consumption survey (n = 3200) were used to assess fat-soluble vitamin (vitamins A, D, E and K) intake from the consumption of foods, fortiﬁed foods and supplements. This study revealed inadequate intakes for vitamin A, from all sources, in the entire Belgian population and possible inadequacies for vitamin D. The prevalence of inadequate intake of vitamin A was lowest in children aged 3–6 (6–7%) and highest in adolescents (girls, 26%; boys, 34–37%). Isabelle Moyersoen 1,2,* ID , Brecht Devleesschauwer 1, Arnold Dekkers 3, Karin de Ridder 1, Jean Tafforeau 1, John van Camp 2, Herman van Oyen 1,4 and Carl Lachat 2 Except for women aged 60–64 years, more than 95% of the subjects had vitamin D intake from all sources below the adequate intake (AI) of 15 µg/day. The risk for inadequate intake of vitamins K and E was low (median > AI). Belgian fortiﬁcation and supplementation practices are currently inadequate to eradicate suboptimal intakes of vitamins A and D, but increase median vitamin E intake close to the adequate intake. For vitamin A, a small proportion (1–4%) of young children were at risk of exceeding the upper intake level (UL), while for vitamin D, inclusion of supplements slightly increased the risk for excessive intakes (% > UL) in adult women and young children. The results may guide health authorities when developing population health interventions and regulations to ensure adequate intake of fat-soluble vitamins in Belgium. Keywords: dietary intake; micronutrient adequacy; fat-soluble vitamins; fortiﬁed foods; supplements; Belgian population Intake of Fat-Soluble Vitamins in the Belgian Population: Adequacy and Contribution of Foods, Fortiﬁed Foods and Supplements Isabelle Moyersoen 1,2,* ID , Brecht Devleesschauwer 1, Arnold Dekkers 3, Karin de Ridder 1, Jean Tafforeau 1, John van Camp 2, Herman van Oyen 1,4 and Carl Lachat 2 nutrients nutrients nutrients nutrients 1. Introduction Fat-soluble vitamins are indispensable to ascertain optimum health in all life stages [1]. Except for vitamin D, which is mainly produced under the inﬂuence of ultraviolet B (UVB) radiation on the skin, dietary intake is the main source for all vitamins. A well-balanced and healthy diet is therefore essential to prevent disease and reduce the burden of disease [2]. Over the last few decades, diets in high income countries have shifted to more energy-dense and nutrient-poor diets, resulting in increasing prevalence of inadequate intake and suboptimal status of micronutrients [3–5]. Inadequate intakes of vitamins A and D are a general concern in Nutrients 2017, 9, 860; doi:10.3390/nu9080860 www.mdpi.com/journal/nutrients www.mdpi.com/journal/nutrients 2 of 22 Nutrients 2017, 9, 860 different populations and population subgroups in Europe [4,5]. The reduction of the prevalence of micronutrient inadequacies should therefore be a priority in food and nutrition policies in Europe [6]. Adequate intake of micronutrients can be achieved through health promotion, as well as through fortiﬁcation and supplementation practices [1,7]. In the USA, fortiﬁed foods and/or supplements contribute largely to the intake of fat-soluble vitamins and help achieve the recommended intakes for vitamins A, D and E for some part, but not all of the population [8,9]. However, while fortiﬁed foods are well adopted in the USA, Europeans are more skeptical of fortiﬁcation [10]. In Europe, non-fortiﬁed foods are the main source for most vitamins. Despite a uniform regularization within the EU fortiﬁcation and supplementation practices differ substantially between countries [1] p g EU, fortiﬁcation and supplementation practices differ substantially between countries [1]. It has been repeatedly reported in European studies that voluntarily-fortiﬁed foods can have a signiﬁcant impact on reducing the proportion of inadequate intakes within a population [1,11,12]. Supplements generally have a more important contribution to total vitamin intake than voluntarily-fortiﬁed foods, but are often less effective at reducing the proportion of suboptimal intakes due to insufﬁcient adherence [3,12]. In response to very low intakes (e.g., vitamin D), national encouraged fortiﬁcation can play an important role in the reduction of inadequacies. In Finland, vitamin D fortiﬁcation of dairy products and fats was successfully introduced as a strategy to improve vitamin D status in the entire population [13]. Despite the positive effects of fortiﬁed foods and supplements, excessive intake of fat-soluble vitamins can lead to adverse health effects [14]. 1. Introduction In general, however, only a small proportion of the population, mainly children, exceeds the upper intake level (UL) [1,8]. A general concern of excessive intakes remains when for certain nutrients, both fortiﬁcation and supplementation practices are common [1,3,15]. Vitamins with a relative low UL in comparison to the recommended intakes, like vitamins A, D and E, need special attention [3]. In Belgium, fortiﬁed foods and supplements require notiﬁcation, and the level of vitamins added is governed by the Belgian Royal Decree (Koninklijk besluit (KB) 3/3/1992). Fortiﬁcation in Belgium is mandatory for margarines (fat content (FC) > 80%) and spreadable fats (39% < FC < 41%) (KB 02/10/1980) and ofﬁcially encouraged for spreadable fats with other fat contents. Nutritive fortiﬁcation values of margarines and spreadable fats however are currently based on nutritive values of butter (Table 1). Similar to other countries, the Belgian Superior Health Council advises a vitamin D supplement for the entire population in the case of minimal exposure to sunlight [15,16]. Independently of their intake from food, a supplementation of 10 µg/day for children and 15 µg/day for adolescents and adults is recommended. This supplementation advice however is currently based on the population reference intake. Dietary intake data are needed to ﬁne-tune these recommendations in correspondence with Belgian dietary habits, as well as with intake from fortiﬁed foods and supplements [17]. When designing strategies to combat inadequate or excessive intakes, it is important to evaluate the efﬁcacy of the actual fortiﬁcation and supplementation programs. Knowledge of the intake distribution of fat-soluble vitamins and the contribution of all sources is therefore required [1,3,8]. In Belgium, those studies are scarce. One study evaluated the intake of micronutrients in Flemish preschool children (2.5–6.5 years). Unfortunately analyses of fat-soluble vitamins were conﬁned to vitamin D, and dietary supplements were hereby not taken into account [18]. Nevertheless, in the review study of Mensink et al., raw data from eight countries were uniformly re-analyzed to evaluate inadequacies of micronutrients in Europe. This study included results for vitamin A, D and E from the Flemish children study [4]. The current study aims to evaluate the contribution of mandatorily fortiﬁed foods, voluntarily fortiﬁed foods and supplements to the intake of fat-soluble vitamins in the general Belgian population (3–64 years) and to compare usual intake to dietary reference values. As food consumption varies by age and gender, the intake distributions were stratiﬁed by age and gender. (1) Fortiﬁcation under the form of provitamin A, i.e., β-carotene. 2.2.1. Original Food Composition Table Fat-soluble vitamin intake was calculated by combining the consumption data of the BFCS 2014 with food and supplement composition data. The latter were retrieved from the Belgian food composition table NUBEL and the Dutch composition table NEVO [19,21,22]. Data on supplement composition were obtained either from Farmacompendium 2014 [23] (an online database for pharmaceutical products in Belgium), or from label information, or the Internet. 1. Introduction 3 of 22 Nutrients 2017, 9, 860 Table 1. Overview of the inventory of foods fortiﬁed with vitamins A, D, E and/or K and corresponding fortiﬁed value, suitable for the target population, Belgian study on the intake of vitamin A, D, E and K (VITADEK-study) 2015. Vitamin A Vitamin D Vitamin E Vitamin K Food Item (µg/100 g) Food Item (µg/100 g) Food Item (mg/100 g) Food Item (µg/100 g) Voluntarily-fortiﬁed foods Fortiﬁed milk 120 Fortiﬁed milk 0.75–1 Fortiﬁed milk 1.8–2.4 Fortiﬁed milk 11.5 Growing-up milk 63–87 Growing-up milk 1.1–3.1 Growing-up milk 0.83–1.1 Dairy drinks, probiotic drinks 11.5 Dairy drinks, probiotic drinks 120 Dairy drinks, probiotic drinks 0.75–3.1 Milk substitutes 1.4–1.8 Growing-up milk 4–5.5 Spreadable cheese 180 Milk substitutes 0.75–1.8 Margarines and spreadable fats 9.1–34 Fruit juices and lemonades 2 Cacao powder 800–1263 Dairy desserts and substitutes 0.75–3.1 Cereals 10 Fruit juices and lemonades 100–400 (1) Cereals 2.9–7.5 Biscuits 3.6–7.2 Weight-loss products 233–1000 Biscuits 1.3–5.0 Fruit juices and lemonades 0.9–6 Fruit juices and lemonades 0.38 Cacao powder 12–15.8 Cacao powder 7.9–11 Weight-loss products 3.3–19 Weight-loss products 1.7–6.6 Mandatorily-fortiﬁed foods Margarines and spreadable fats 700–900 Margarines and spreadable fats 6–7.5 (1) Fortiﬁcation under the form of provitamin A, i.e., β-carotene. Table 1. Overview of the inventory of foods fortiﬁed with vitamins A, D, E and/or K and corresponding fortiﬁed value, suitable for the target population, Belgian study on the intake of vitamin A, D, E and K (VITADEK-study) 2015. A, D, E and/or K and corresponding fortiﬁed value, suitable for the target population, Belgian Table 1. Overview of the inventory of foods fortiﬁed with vitamins A, D, E and/or K and corresponding fortiﬁed value, suitable for the target population, Belgian study on the intake of vitamin A, D, E and K (VITADEK-study) 2015. 4 of 22 Nutrients 2017, 9, 860 2.1. Data Collection Data of the Belgian food consumption survey (BFCS) 2014 were used to assess the intake of fat-soluble vitamins in the Belgian population. The survey is described in detail by Bel et al. [19]. In brief, data were collected among a representative sample of the Belgian population (n = 3200, 3–64 years old) following a multistage stratiﬁed sampling design. This included a geographical stratiﬁcation according to the provinces (10 Belgian provinces and the Brussels Capital Region), a selection of municipalities within each geographical stratum and a selection of individuals within each municipality. The national population register was used as the sampling frame. The sample was stratiﬁed into ﬁve sex-age groups, i.e., 3–5, 6–9, 10–17, 18–39 and 40–64 years [20]. Seasonal effects and day-to-day variation in food consumption were taken into account by equally dividing the interviews between all weekdays and seasons. Food and supplement consumption data in adults and adolescents (10–64 years old) were collected by a dietician using two non-consecutive 24-h dietary recalls (using the GloboDiet® software) and a self-administered food frequency questionnaire (FFQ) [20]. In children (3–9 years old), consumption data were collected using two non-consecutive one-day food diaries, a completion interview by a dietician using GloboDiet® and a self-administered frequency questionnaire. Data in children were collected through their proxy (i.e., one of the parents or legal guardians). The self-administered frequency questionnaire included questions on supplements’ consumption with a recall period of one year. In all subjects, socio-demographic characteristics were collected by interview, while height, weight and waist circumference were measured by the dietician during the second home-visit. The participation rate of the survey was 37%. Weighting factors were used to ensure the representativeness of the sample for age, gender and place of residency [19]. All participants or their proxies provided written informed consent. Approvals of the Ethics Committee of Ghent University and the Commission for the Protection of Privacy were obtained. The study was conducted in accordance with the ethical principles for medical research involving human subjects [19]. 2.2. Food Composition 2.2. Food Composition 2.2.2. Adapted Food Composition Table Fortiﬁed foods are subject to permanent modiﬁcations in terms of composition and brands [24]. Furthermore, food codes in national food composition tables are often not brand-speciﬁc and therefore lacking information on fortiﬁed nutrient content. At the beginning of 2015, a market research was conducted to make an inventory of the actual supply of fortiﬁed foods on the Belgian market. This inventory was done in supermarkets and grocery stores by physical visits, by means of supermarket websites and via manufacturers’ websites. The fortiﬁed nutrient content as labelled on the packages was obtained for all foods fortiﬁed with vitamins A, D, E and/or K. This information, however, does not differentiate between the naturally-occurring amount and the amount added during fortiﬁcation. The nutrient content on the label hence reﬂects the total content of the fortiﬁed nutrient [3]. y g g nutrient content on the label hence reﬂects the total content of the fortiﬁed nutrient [3]. In case of missing data, four extra national food composition tables were used in the following order: (1) McCance and Widowson’s, the U.K. food composition table [25]; (2) Ciqual, the French 5 of 22 Nutrients 2017, 9, 860 composition table [26]; (3) the Danish food composition table [27] and (4) the American food composition table [28]. When the missing vitamin value was not found in the ﬁrst source, the next source was consulted till the appropriate information was retrieved. When data were still missing, values were retrieved from ingredient-based or recipe-based calculations, through similarities or as a median value [29]. This was especially the case for vitamin K and D. More details on the market research and the compilation of the adapted food composition table can be found elsewhere [30]. 3.2. Intake Modelling The Statistical Program to Assess Dietary Exposure (SPADE) was used to estimate the habitual intake distributions for vitamins A, D, E and K from all sources. SPADE.RIVM is a freely available R package with different modelling options [32]. The multipart model was used, which allowed estimating the habitual intake from food, mandatorily fortiﬁed foods, voluntarily fortiﬁed food and supplements separately. At the end, the habitual amounts were added to the habitual intake. Speciﬁc challenges such as multimodality, a spiked distribution of vitamin dosages in supplements and heterogeneity in variances due to the differing intakes (daily versus episodically) from foods versus fortiﬁed foods and supplements were taken into account [32]. SPADE further accounts for the within-person variability typical for short-term assessment methods like the 24-h recall, by means of a “ﬁrst shrink then add” method. Intake data were modelled as a function of age and reported in the age ranges used by the European Food Safety Authority (EFSA) in setting their dietary reference values, i.e., children 3–6 and 7–10, adolescents 11–14 and 15–17 and adults 18–39 and 40–64 years. The 5th, 50th, 95th and 97.5th percentiles, as well as the arithmetic mean intakes of vitamins A, D, E and K were calculated for foods only, foods plus fortiﬁed foods and foods plus fortiﬁed foods plus supplements. For vitamins A and D for which mandatory fortiﬁcation is applied on margarines and spreadable fats, an additional intake from mandatorily fortiﬁed foods was calculated. Uncertainty of mean intake is quantiﬁed with bootstrap 95% conﬁdence intervals [32]. For the purpose of this study, supplement users were deﬁned as subjects that indicated the use of supplements on one of the 24-h recalls or on the FFQ. Subjects that had not completed the FFQ (n = 900) were treated as users when they consumed a supplement on one of the 24-h recalls or never-users when they did not consume supplements on both 24-h recalls. Supplements comprised single vitamins, multivitamins or mineral-vitamin supplements containing the respective vitamin. 3.1. Contribution of Food Groups Contributions of food groups to total vitamins A, D, E or K intake were calculated following the GloboDiet® classiﬁcation system based on 18 food groups (Supplementary Table S1). Calculations were based on the ﬁrst 24-h recall. For each subject, the proportion of vitamin intake between the respective food group and the total vitamin intake was calculated. The mean contribution of a speciﬁc food group to the total vitamin intake of the Belgian population was calculated as the survey-weighted mean of these individual proportions [31]. Analyses were conducted in SAS 9.3 (SAS Institute Inc., Cary, NC, USA). 3.3. Adequacy of Vitamin Intake To evaluate the adequacy of the intake of fat-soluble vitamins, the intake distribution of the Belgian population was evaluated against the dietary reference values set by EFSA [14,33–36]. For vitamin A and retinol, insufﬁcient intakes were evaluated with the estimated average requirement (EAR) as cut-off value. The EAR is deﬁned as an intake level adequate for 50% of the population [37]. The risk of insufﬁcient intakes in the population was determined as the proportion of the population with usual vitamin A intake below the EAR. Vitamin A intake was expressed in Nutrients 2017, 9, 860 6 of 22 microgram retinol equivalents (µg RE), following the conversion factors adopted by EFSA, i.e., 1 µg RE equals 1 µg of retinol, 6 µg of β-carotene and 12 µg of other pro-vitamin A carotenoids [35]. In this study, only retinol and β-carotene were considered [35]. The reported vitamin K intake comprises total vitamin K intake (vitamin K1 + K2) since some composition tables distinguish phylloquinones (vitamin K1) from menaquinones (vitamin K2), while others only report the total vitamin K value. The dietary reference values for vitamin K (adequate intake (AI) and UL) are based on phylloquinones only. Despite phylloquinones being the major form of “total vitamin K”, this implies some uncertainties in the usual vitamin K intake estimation [36]. When the distribution of requirement of a certain vitamin is not known, an AI is used instead of an EAR. The AI value is based on the median daily intake of an apparently healthy population [37]. The prevalence of inadequate intakes evaluated with an AI was stated as “low” if the median intake of the population for the respective vitamin was above the AI. If the median intake was below the AI, the adequacy for a certain vitamin could not be evaluated (no statement) [38]. Possible excessive intakes for all vitamins were estimated as the proportion of the population with usual intakes above the UL [37]. The UL for vitamin A was based on retinol only. Therefore excessive intakes for vitamin A were expressed as the percentage of the population with habitual retinol intake exceeding the UL [14]. 4.1. Inventory of Fortiﬁed Foods Table 1 gives an overview of the inventory of foods fortiﬁed with vitamins A, D, E and K available on the Belgian market in 2015 and suitable for the target population. The inventory revealed a considerable amount of fortiﬁed foods specially designed for children, i.e., growing-up milk, fortiﬁed milk, cereals, cacao, fruit juices, spreadable cheese and dairy desserts. Fortiﬁcation with vitamin E and especially vitamin D was most common. 3.4. Misreporting Misreporting of dietary intake affects the estimates of both the low and high percentiles of the intake distributions. Misreporting was identiﬁed using Goldberg cut-off values with age-speciﬁc physical activity level (PAL) [39,40]. Additional analyses were performed with exclusion of under- and over-reporters. These results are presented in the Supplementary ﬁles. 4.2. Supplement Consumption The consumption of supplements with vitamins A, D, E or K was low in the Belgian population, respectively 16%, 25%, 17% and 4%. Vitamin A, E and K supplements were mostly consumed as multivitamins, while 44% of vitamin D supplements were consumed as a single vitamin [41]. 4.3. Contribution of Food Groups and Supplements 4.4.1. Vitamin A Usual vitamin A intake in the Belgian population from foods varied from 606 µg/day (girls 3–6 years) to 937 µg/day (men 40–64 years). Foods were the greatest contributors to total vitamin A intake. Contributions from “foods only” varied from 83% in adult women (18–39 years) to 91% in boys (7–10 years) (Tables 3 and 4). Inadequate intakes for vitamin A (% <EAR) were found in all sex-age groups. When considering foods only, the proportion of the population with inadequate intake ranged from 5–47%. Mandatorily fortiﬁed foods, namely margarines and spreadable fats, were the second largest contributor to usual vitamin A intake and reduced inadequacy proportions by 2–10%. Mandatorily fortiﬁed foods, namely margarines and spreadable fats, were the second largest contributor to usual vitamin A intake and reduced inadequacy proportions by 2–10%. Only limited amounts of foods were voluntarily fortiﬁed with vitamin A. The contribution of voluntarily fortiﬁed foods was consequently low and varied from 0.3–5% and had a minor effect on the reduction of the prevalence of inadequate intakes (1–2% reduction). Only limited amounts of foods were voluntarily fortiﬁed with vitamin A. The contribution of voluntarily fortiﬁed foods was consequently low and varied from 0.3–5% and had a minor effect on the reduction of the prevalence of inadequate intakes (1–2% reduction). The contribution of supplements to total vitamin A intake varied from 0.9% in boys (7–10 years) to 10% in adult women (18–39 years), reducing the proportion of the population with inadequate intake up to 4%. When considering all sources, a substantial proportion of inadequate vitamin A intakes remained in all sex-age groups of the Belgian population. The prevalence of inadequate vitamin A intake was lowest in children (3–6 years), with levels ranging from 1–2% and highest in adolescents, with levels ranging from 26% in adolescent girls and 34–37% in adolescent boys. The risk of excessive vitamin A intake (based on retinol only [14]) was nil in almost all sex-age groups. In young boys (3–6 years), however, 4% had a risk of excessive intakes from all sources combined. High intakes originated from the consumption of cream pâté (7797 µg/100 g) [22]. The intake distribution from retinol is illustrated in Supplementary Tables S2 and S3. Excluding miss-reporters did not change the proportion with excessive intakes, but substantially decreased the proportion of subjects with intakes below the EAR. However, the conclusions within age-groups remained the same (Supplementary Tables S4 and S5). 4.3. Contribution of Food Groups and Supplements 4.3. Contribution of Food Groups and Supplements 4.3. Contribution of Food Groups and Supplements Table 2 gives an overview of the food groups that contributed most to the total vitamin, A, D, E and K intake. Food sources of animal origin, as well as fat and oils were important contributors for all fat-soluble vitamin intakes, while vegetables were the major contributors for vitamins A, E and K. The high contribution of fat and oils for vitamins A and D originates from margarines and spreadable fats that are mandatorily fortiﬁed in Belgium to levels similar as in butter, i.e., 700–900 µg/100 g for vitamin A and 6–7.5 µg/100 g for vitamin D. In Belgium, the intake of vitamins A, D, E and K from supplements was generally low and varied from 0.9% (vitamin K) to 5.8% (vitamin D) of total intake. In Belgium, the intake of vitamins A, D, E and K from supplements was generally low and varied from 0.9% (vitamin K) to 5.8% (vitamin D) of total intake. 7 of 22 Nutrients 2017, 9, 860 Table 2. Overview of the 5 most contributing food groups and the contribution of supplements to total vitamin A, D, E and K intake in the Belgian population 2014, Belgian food consumption survey 2014. Vitamin A Vitamin D Vitamin E Vitamin K Food Group Contribution (%) Food Group Contribution (%) Food Group Contribution (%) Food Group Contribution (%) Vegetables 31 Meat and meat products 27 Fat and oils 13 Vegetables 54 Dairy products 22 Dairy products 20 Vegetables 13 Dairy products 16 Fat and oils 13 Fat and oils 19 Sauces, spices, herbs and condiments 11 Fruits and nuts 11 Meat and meat products 8.0 Fish and shellﬁsh 8.5 Meat and meat products 9.1 Fat and oils 9.0 Cakes and sweet biscuits 6.4 Cakes and sweet biscuits 7.4 Cakes and sweet biscuits 8.4 Sauces, spices, herbs and condiments 3.8 Supplements 2.5 Supplements 5.8 Supplements 3.5 Supplements 0.9 Table 2. Overview of the 5 most contributing food groups and the contribution of supplements to total vitamin A, D, E and K intake in the Belgian population 2014, Belgian food consumption survey 2014. and the contribution of supplements to total vitamin A, D, E and K intake in the Belgian population 2014, Nutrients 2017, 9, 860 8 of 22 4.4. Vitamin Intake and Adequacy Tables 3–10 summarize the intake distributions and estimates of inadequacy (% <EAR or AI and excessive intakes % >UL) for vitamins A, D, E and K in each sex-age group of the Belgian population (3–64 years). 4.4.2. Vitamin D Usual vitamin D intake from the consumption of “foods only” was generally low in the Belgian population (Tables 5 and 6). The mean usual vitamin D intake varied from 2.34 µg/day in boys (3–6 years) to 3.71 µg/day in adult men (40–64 years). With the inclusion of vitamin D from fortiﬁed foods and especially from supplements, intake levels increased signiﬁcantly. The mean usual vitamin D intake from all sources was lowest in adolescents aged 11–14 years (4.85 µg/day in boys; 5.6 µg/day in girls) and highest in children aged 3–6 years (11.61 µg/day in boys, 9.3 µg/day in girls) and adult women aged 18–39 and 40–64 years (9.1 µg/day, respectively 13.1 µg/day). In Belgium, margarines and spreadable fats are mandatorily fortiﬁed with vitamin D with levels of vitamin D ranging from 6–7.5 µg/day (Table 1). Mandatorily-fortiﬁed foods contributed between 3.2% and 13.9% to total vitamin D intake. Contribution of voluntarily fortiﬁed foods to total vitamin D intake was more important in children and adolescents than in adults (Tables 5 and 6). In children and adolescents, fortiﬁed foods contributed in a range from 6.6–14.8%. In adults, the contribution of voluntarily fortiﬁed foods ranged from 2.7–5.7%. 9 of 22 Nutrients 2017, 9, 860 Table 3. Usual intake of vitamin A (µg/day) from food, fortiﬁed food and supplements in Belgian men (3–64 years), Belgian study on the intake of vitamins A, D, E and K (VITADEK-study) 2015. s and 245 µg/day in children aged 4–6 years old. The UL for vitamin A (retinol) is 800 µg day in children aged 1–3 years average requirement; UL: upper intake level. 4.4.2. Vitamin D Percentiles Age Usual Intake (CI) % Contribution 5 50 95 97.5 EAR % <EAR UL % >UL 3–6 Food 673 (615–762) 89.3 227 574 1452 1734 205–245 (1) 6 800–1100 (1) 2 + mandatorily fortiﬁed food 706 (657–783) 4.4 257 613 1468 1736 3 3 + voluntarily fortiﬁed food 744 (679–805) 5.0 277 646 1560 1827 2 3 + supplements 754 (698–831) 1.3 285 659 1554 1841 2 4 7–10 Food 693 (647–766) 90.7 233 591 1494 1784 320 14 1500 0 + mandatorily fortiﬁed food 732 (689–792) 5.1 271 631 1540 1813 9 0 + voluntarily fortiﬁed food 757 (706–809) 3.3 282 652 1572 1847 8 0 + supplements 764 (721–831) 0.9 289 669 1578 1851 7 1 11–14 Food 713 (667–774) 89.9 240 608 1537 1835 580 47 2000 0 + mandatorily fortiﬁed food 748 (712–809) 4.4 277 646 1578 1839 41 0 + voluntarily fortiﬁed food 771 (725–826) 2.9 289 667 1593 1929 40 0 + supplements 793 (739–844) 2.8 293 687 1656 1933 37 0 15–17 Food 731 (681–788) 90.1 246 624 1576 1882 570 44 2600 0 + mandatorily fortiﬁed food 775 (728–827) 5.4 287 664 1651 1948 38 0 + voluntarily fortiﬁed food 786 (740–845) 1.4 289 675 1635 1937 37 0 + supplements 811 (761–870) 3.1 304 702 1679 2005 34 0 18–39 Food 803 (728–856) 90.4 270 685 1735 2074 570 37 3000 0 + mandatorily fortiﬁed food 851 (774–908) 5.4 314 736 1776 2101 31 0 + voluntarily fortiﬁed food 862 (785–923) 1.2 320 748 1798 2115 30 0 + supplements 888 (809–937) 2.9 327 771 1844 2183 28 0 40–64 Food 937 (850–1033) 88.0 315 799 2026 2422 570 28 3000 0 + mandatorily fortiﬁed food 1050 (952–1143) 10.6 400 914 2154 2553 18 0 + voluntarily fortiﬁed food 1053 (960–1151) 0.3 403 919 2155 2542 17 0 + supplements 1065 (975–1163) 1.1 409 931 2172 2565 17 0 (1) The EAR for vitamin A is 205 µg/day in children aged 1–3 years and 245 µg/day in children aged 4–6 years old. The UL for vitamin A (retinol) is 800 µg day in children aged 1–3 years and 1100 µg/day in children aged 4–6 years old. EAR: estimated average requirement; UL: upper intake level. 4.4.2. Vitamin D Usual intake of vitamin A (µg/day) from food, fortiﬁed food and supplements in Belgian men (3–64 years), Belgian study on the intake of vitamins A, D, E VITADEK t d ) 2015 Table 3. Usual intake of vitamin A (µg/day) from food, fortiﬁed food and supplements in Belgian men (3–64 years), Belgian study on the intake of vitamins A, D, E and K (VITADEK-study) 2015. 10 of 22 Nutrients 2017, 9, 860 Table 4. Usual intake of vitamin A (µg/day) from food, fortiﬁed food and supplements in Belgian women (3–64 years), Belgian study on the intake of vitamins A, D, E and K (VITADEK-study) 2015. 4.4.2. Vitamin D Percentiles Age Usual Intake (CI) % Contribution 5 50 95 97.5 EAR % <EAR UL % >UL 3–6 Food 606 (563–649) 86.4 238 540 1196 1388 205–245 (1) 5 800–1000 (1) 0 + mandatorily fortiﬁed food 659 (612–696) 7.6 273 594 1275 1468 3 0 + voluntarily fortiﬁed food 671 (635–718) 1.7 290 608 1261 1461 2 0 + supplements 701 (645–747) 4.3 299 636 1316 1521 1 1 7–10 Food 621 (585–659) 87.3 244 554 1225 1422 320 13 1500 0 + mandatorily fortiﬁed food 667 (633–705) 6.5 278 599 1282 1488 9 0 + voluntarily fortiﬁed food 693 (651–723) 3.7 297 629 1335 1548 7 0 + supplements 711 (663–740) 2.5 306 648 1356 1534 6 0 11–14 Food 636 (600–673) 88.2 250 568 1255 1456 490 38 2000 0 + mandatorily fortiﬁed food 682 (646–719) 6.4 284 615 1316 1521 31 0 + voluntarily fortiﬁed food 698 (663–734) 2.2 298 628 1337 1528 29 0 + supplements 721 (675–753) 3.2 311 653 1352 1567 26 0 15–17 Food 650 (613–687) 89.5 256 580 1281 1487 490 37 2600 0 + mandatorily fortiﬁed food 694 (657–734) 6.1 295 626 1308 1503 30 0 + voluntarily fortiﬁed food 709 (673–746) 2.1 296 637 1364 1578 28 0 + supplements 726 (688–770) 2.3 311 659 1358 1544 26 0 18–39 Food 705 (654–754) 82.6 277 629 1388 1612 490 31 3000 0 + mandatorily fortiﬁed food 749 (701–797) 5.2 319 675 1429 1649 24 0 + voluntarily fortiﬁed food 768 (723–816) 2.2 327 693 1462 1690 22 0 + supplements 853 (790–931) 10.0 349 753 1698 2006 18 0 40–64 Food 805 (740–875) 86.6 319 719 1582 1833 490 22 3000 0 + mandatorily fortiﬁed food 868 (803–947) 6.8 370 784 1655 1898 15 0 + voluntarily fortiﬁed food 881 (810–958) 1.4 379 796 1669 1930 14 0 + supplements 930 (848–1014) 5.3 404 845 1742 1988 11 0 (1) EAR for vitamin A is 205 µg/day in children aged 1–3 years and 245 µg/day in children aged 4–6 years old. The UL for vitamin A (retinol) is 800 µg day in children aged 1–3 years and 1100 µg/day in children aged 4–6 years old. EAR: estimated average requirement; UL: upper intake level. d 245 µg/day in children aged 4–6 years old. The UL for vitamin A (retinol) is 800 µg day in children aged 1–3 years and age requirement; UL: upper intake level. 4.4.2. Vitamin D Usual intake of vitamin A (µg/day) from food, fortiﬁed food and supplements in Belgian women (3–64 years), Belgian study on the intake of vitamins A, D, E VITADEK t d ) 2015 Table 4. Usual intake of vitamin A (µg/day) from food, fortiﬁed food and supplements in Belgian women (3–64 years), Belgian study on the intake of vitamins A, D, E and K (VITADEK-study) 2015. 11 of 22 Nutrients 2017, 9, 860 Table 5. Usual intake of vitamin D (µg/day) from food, fortiﬁed food and supplements in Belgian men (3–64 years), Belgian study on the intake of vitamins A, D, E and K (VITADEK-study) 2015. AI, adequate intake. 4.4.2. Vitamin D Percentiles Age Usual Intake (CI) % Contribution 5 50 95 97.5 AI % Inadequate (1) UL % >UL 3–6 Food 2.34 (2.08–2.95) 20.2 0.72 2.01 5.11 6.06 15 ns (2) 50 0.0 + mandatorily fortiﬁed food 2.71 (2.4–3.31) 3.2 0.99 2.39 5.48 6.49 15 ns 0.0 + voluntarily fortiﬁed food 3.69 (3.4–4.31) 8.4 1.44 3.37 7.01 7.99 15 ns 0.0 + supplements 11.61 (4.4–19.97) 68.2 1.56 3.89 13.91 51.76 15 ns 2.5 7–10 Food 2.82 (2.5–3.2) 53.4 0.90 2.44 6.06 7.16 15 ns 50 0.0 + mandatorily fortiﬁed food 3.20 (2.9–3.63) 7.2 1.18 2.82 6.58 7.63 15 ns 0.0 + voluntarily fortiﬁed food 3.98 (3.7–4.41) 14.8 1.56 3.58 7.80 8.89 15 ns 0.0 + supplements 5.28 (4.16–7.12) 24.6 1.61 3.84 9.09 11.26 15 ns 0.4 11–14 Food 3.07 (2.74–3.39) 63.3 0.99 2.65 6.55 7.72 15 ns 100 0.0 + mandatorily fortiﬁed food 3.46 (3.13–3.83) 8.0 1.30 3.05 7.01 8.00 15 ns 0.0 + voluntarily fortiﬁed food 4.04 (3.69–4.41) 12.0 1.57 3.64 7.84 9.04 15 ns 0.0 + supplements 4.85 (4.05–11.51) 16.7 1.66 3.81 8.92 10.81 15 ns 0.2 15–17 Food 3.21 (2.83–3.47) 60.9 1.04 2.78 6.83 8.05 15 ns 100 0.0 + mandatorily fortiﬁed food 3.65 (3.24–3.97) 8.3 1.31 3.19 7.45 8.01 15 ns 0.0 + voluntarily fortiﬁed food 4.08 (3.68–4.41) 8.2 1.57 3.65 8.04 9.39 15 ns 0.0 + supplements 5.27 (3.98–7.64) 22.6 1.66 3.85 9.15 11.21 15 ns 0.3 18–39 Food 3.49 (3.10–3.68) 56.7 1.14 3.02 7.39 8.70 15 ns 100 0.0 + mandatorily fortiﬁed food 3.99 (3.61–4.24) 8.1 1.5 3.54 8.01 9.28 15 ns 0.0 + voluntarily fortiﬁed food 4.34 (3.92–4.58) 5.7 1.68 3.89 8.47 9.82 15 ns 0.0 + supplements 6.15 (4.51–8.07) 29.4 1.73 4.09 9.86 12.26 15 ns 0.5 40–64 Food 3.71 (3.38–4.04) 51.6 1.23 3.23 7.85 9.23 15 ns 100 0.0 + mandatorily fortiﬁed food 4.71 (4.27–5.19) 13.9 1.90 4.24 9.14 10.56 15 ns 0.0 + voluntarily fortiﬁed food 4.88 (4.48–5.37) 2.4 1.99 4.41 9.34 10.76 15 ns 0.0 + supplements 7.19 (4.86–10.67) 32.1 2.05 4.61 10.68 13.58 15 ns 0.6 (1) When not enough evidence is available to set an EAR, an AI is set. The AI is based on the average intake of an apparently healthy population. (2) When the median intake of the population > the AI, the risk for inadequate intake is low. 4.4.2. Vitamin D No statement (ns) can be formulated on the adequacy of vitamin D when the median intake < AI. EAR: Estimated average requirement; UL: upper intake level; AI: adequate intake. Usual intake of vitamin D (µg/day) from food, fortiﬁed food and supplements in Belgian men (3–64 years), Belgian study on the intake of vitamins A, D, E VITADEK-study) 2015 AI adequate intake Table 5. Usual intake of vitamin D (µg/day) from food, fortiﬁed food and supplements in Belgian men (3–64 years), Belgian study on the intake of vitamins A, D, E and K (VITADEK-study) 2015. AI, adequate intake. Table 5. Usual intake of vitamin D (µg/day) from food, fortiﬁed food and supplements in Belgian men (3–64 years), Belgian study on the intake of vitamins A, D, E and K (VITADEK-study) 2015. AI, adequate intake. 12 of 22 Nutrients 2017, 9, 860 Table 6. Usual intake of vitamin D (µg/day) from food, fortiﬁed food and supplements in Belgian women (3–64 years), Belgian study on the intake of vitamins A, D, E and K (VITADEK-study) 2015. 4.4.2. Vitamin D Percentiles Age Usual Intake (CI) % Contribution 5 50 95 97.5 AI % Inadequate (1) UL % >UL 3–6 Food 2.75 (2.32–3.02) 29.7 0.95 2.41 5.69 6.66 15 ns (2) 25 0.0 + mandatorily fortiﬁed food 3.05 (2.65–3.33) 3.2 1.19 2.73 6.03 7.06 15 ns 0.0 + voluntarily fortiﬁed food 3.79 (3.39–4.11) 8.0 1.60 3.48 6.98 8.05 15 ns 0.0 + supplements 9.27 (4.49–18.1) 59.1 1.73 3.84 14.54 25.08 15 ns 1.7 7–10 Food 2.75 (2.47–3.03) 38.1 0.95 2.41 5.70 6.67 15 ns 25 0.0 + mandatorily fortiﬁed food 3.04 (2.79–3.33) 4.0 1.21 2.71 6.04 6.96 15 ns 0.0 + voluntarily fortiﬁed food 3.66 (3.39–3.95) 8.6 1.51 3.33 6.89 7.92 15 ns 0.0 + supplements 7.21 (4.54–11.5) 49.2 1.56 3.64 10.85 16.43 15 ns 1.1 11–14 Food 2.76 (2.49–3.03) 49.3 0.95 2.42 5.72 6.69 15 ns 50 0.0 + mandatorily fortiﬁed food 3.04 (2.81–3.33) 5.0 1.19 2.74 5.98 6.91 15 ns 0.0 + voluntarily fortiﬁed food 3.51 (3.28–3.83) 8.4 1.43 3.22 6.58 7.48 15 ns 0.0 + supplements 5.62 (3.81–7.4) 37.3 1.45 3.43 9.48 13.69 15 ns 0.5 15–17 Food 2.76 (2.51–3.04) 46.9 0.95 2.43 5.73 6.70 15 ns 50 0.0 + mandatorily fortiﬁed food 3.07 (2.83–3.35) 5.3 1.21 2.73 5.98 7.06 15 ns 0.0 + voluntarily fortiﬁed food 3.46 (3.21–3.80) 6.6 1.39 3.15 6.58 7.50 15 ns 0.0 + supplements 5.89 (3.71–7.0) 41.3 1.43 3.33 9.04 13.13 15 ns 0.5 18–39 Food 2.79 (2.54–3.07) 30.8 0.96 2.45 5.77 6.75 15 ns 50 0.0 + mandatorily fortiﬁed food 3.20 (2.95–3.47) 4.5 1.27 2.87 6.24 7.21 15 ns 0.0 + voluntarily fortiﬁed food 3.51 (3.26–3.85) 3.4 1.42 3.18 6.71 7.67 15 ns 0.0 + supplements 9.07 (4.88–13.4) 61.3 1.55 3.64 14.39 26.23 15 ns 1.4 40–64 Food 2.82 (2.54–3.12) 21.5 0.98 2.48 5.84 6.84 15 ns 50 0.0 + mandatorily fortiﬁed food 3.45 (3.11–3.77) 4.8 1.40 3.12 6.63 7.64 15 ns 0.0 + voluntarily fortiﬁed food 3.80 (3.48–4.17) 2.7 1.59 3.47 7.09 8.16 15 ns 0.0 + supplements 13.15 (6.09–24.1) 71.0 1.77 4.17 22.59 44.0 15 ns 1.9 (1) When not enough evidence is available to set an EAR, an AI is set. The AI is based on the average intake of an apparently healthy population. (2) When the median intake of the population >the AI, the risk for inadequate intake is low. No statement (ns) can be formulated on the adequacy of vitamin D when the median intake <AI. 4.4.2. Vitamin D EAR: estimated average requirement; UL: upper intake level; AI: adequate intake. Usual intake of vitamin D (µg/day) from food, fortiﬁed food and supplements in Belgian women (3–64 years), Belgian study on the intake of vitamins A, D, E VITADEK study) 2015 Table 6. Usual intake of vitamin D (µg/day) from food, fortiﬁed food and supplements in Belgian women (3–64 years), Belgian study on the intake of vitamins A, D, E and K (VITADEK-study) 2015. 13 of 22 Nutrients 2017, 9, 860 Table 7. Usual intake of vitamin E (mg/day) from food, fortiﬁed food and supplements in Belgian men (3–64 years), Belgian study on the intake of vitamins A, D, E and K (VITADEK-study) 2015. Percentiles Age Usual Intake (CI) % Contribution 5 50 95 97.5 AI (2) % Inadequate (2) UL % >UL 3–6 Food 7.6 (7.1–8.1) 82.6 3.4 7.0 14.0 16.0 6–9 (1) Ns (3) 60 0 + fortiﬁed food 9.0 (8.3–9.3) 15.2 4.1 8.3 16.2 18.2 Ic (4) 0 + supplements 9.2 (8.6–9.6) 2.2 4.2 8.5 16.7 18.8 ic 0 7–10 Food 9.9 (9.4–10.6) 88.4 4.6 9.2 18.0 20.4 9 Low (3) 100 0 + fortiﬁed food 10.8 (10.3–11.4) 8.0 5.1 10.0 19.0 21.7 low 0 + supplements 11.2 (10.6–11.7) 3.6 5.1 10.3 20.0 22.6 low 0 11–14 Food 11.4 (10.76–12.1) 91.9 5.3 10.5 20.5 23.2 13 ns 120 0 + fortiﬁed food 12.1 (11.5–12.7) 5.6 5.8 11.3 21.4 24.5 ns 0 + supplements 12.4 (11.8–13.0) 2.4 5.9 11.6 22.2 25.0 ns 0 15–17 Food 12.2 (11.54–12.8) 92.4 5.7 11.3 21.9 24.8 13 ns 130 0 + fortiﬁed food 12.9 (12.2–13.4) 5.3 6.1 11.9 22.9 25.7 ns 0 + supplements 13.2 (12.5–13.8) 2.3 6.2 12.3 23.4 26.7 ns 0 18–39 Food 13.0 (12.17–13.6) 92.2 6.1 12.0 23.3 26.4 13 ns 150 0 + fortiﬁed food 13.6 (12.8–14.3) 4.3 6.4 12.6 24.1 27.2 ic 0 + supplements 14.1 (13.2–14.8) 3.5 6.6 13.0 25.0 28.3 ic 0 40–64 Food 11.7 (11.01–12.4) 87.3 5.4 10.8 21.1 24.0 13 ns 150 0 + fortiﬁed food 12.8 (11.9–13.5) 8.2 6.0 11.8 22.7 25.5 ic 0 + supplements 13.4 (12.6–14.3) 4.5 6.2 12.2 24.4 28.2 ic 0 (1) The AI for vitamin E is 6 mg/day in children aged 1–3 years and 9 mg/day in children aged 4–6 years old. (2) When not enough evidence is available to set an EAR, an AI is set. 4.4.2. Vitamin D The AI is based on the average intake of an apparently healthy population. (3) When the median intake of the population lies above the AI, the risk for inadequate intake is low. When the median intake <AI, no statement (ns) can be formulated on the the adequacy of vitamin E. (4) ic: inconclusive within a certain age-group. EAR: estimated average requirement; UL: upper intake level; AI: adequate intake. Usual intake of vitamin E (mg/day) from food, fortiﬁed food and supplements in Belgian men (3–64 years), Belgian study on the intake of vitamins A, D, E VITADEK stud ) 2015 14 of 22 Nutrients 2017, 9, 860 Table 8. Usual intake of vitamin E (mg/day) from food, fortiﬁed food and supplements in Belgian women (3–64 years), Belgian study on the intake of vitamins A, D, E and K (VITADEK-study) 2015. Percentiles Age Usual Intake (CI) % Contribution 5 50 95 97.5 AI (2) % Inadequate (3) UL % >UL 3–6 Food 7.0 (6.6–7.6) 82.4 3.4 6.6 12.2 13.7 6–9 (1) ns 60 0 + fortiﬁed food 8.0 (7.5–8.5) 11.8 3.9 7.5 13.9 15.7 ic (4) 0 + supplements 8.5 (8.0–9.1) 5.9 4.1 7.9 15.3 17.6 ic 0 7–10 Food 8.8 (8.4–9.3) 87.1 4.5 8.3 14.9 16.6 9 ns 100 0 + fortiﬁed food 9.7 (9.2–10.1) 8.9 4.9 9.1 16.4 18.1 ic 0 + supplements 10.1 (9.5–10.5) 4 5.0 9.5 17.3 19.4 low 0 11–14 Food 9.5 (8.8–9.9) 89.6 4.9 9.0 16.0 17.8 11 ns 120 0 + fortiﬁed food 10.3 (9.7–10.7) 7.5 5.3 9.7 7.2 19.2 ns 0 + supplements 10.6 (9.9–11.0) 2.8 5.3 9.9 18.1 20.3 ns 0 15–17 Food 9.8 (9.0–10.1) 90.7 5.0 9.2 16.4 18.2 11 ns 130 0 + fortiﬁed food 10.5 (9.8–10.9) 6.5 5.5 9.9 17.5 19.4 ns 0 + supplements 10.8 (10.1–11.3) 2.8 5.5 10.2 18.3 20.6 ns 0 18–39 Food 9.7 (9.1–10.1) 83.6 5.0 9.1 16.2 18.1 11 ns 150 0 + fortiﬁed food 10.4 (9.9–11.0) 6 5.4 9.8 17.4 19.3 ns 0 + supplements 11.6 (10.9–13.1) 10.3 5.6 10.5 20.4 23.6 ns 0 40–64 Food 8.9 (8.5–9.6) 80.2 4.5 8.4 15.0 16.7 11 ns 150 0 + fortiﬁed food 9.5 (9.0–10.4) 5.4 4.9 9.0 16.0 17.8 ns 0 + supplements 11.1 (10.3–12.0) 14.4 5.1 9.8 21.1 25.3 ns 0 (1) AI for vitamin E is 6 mg/day in children aged 1-3 years and 9 mg/day in children aged 4–6 years old. (1) AI for vitamin E is 6 mg/day in children aged 1-3 years and 9 mg/day in children aged 4–6 years old. (2) When not enough evidence is available to set an EAR, an AI is set. The AI is based on the average intake of an apparently healthy population. (3) When the median intake of the population lies above the AI, the risk for inadequate intake is low. Since the distribution of requirements is not known, no statement (ns) can be formulated on the adequacy of vitamin E when the median intake is lower than the AI. (4) ic: inconclusive within a certain age-group. EAR: Estimated average requirement; UL: upper intake level; AI: adequate intake. 4.4.2. Vitamin D Usual intake of vitamin K (µg/day) from foods only in Belgian women (3–64 years), Belgian study on the intake of vitamins A, D, E and K (VITADEK-study) 2015. Percentiles Age Usual Intake (CI) 5 50 95 97.5 AI (2) % Inadequate (3) UL % >UL 3–6 Food 54 (47–61) 21 47 109 128 12–20 (1) low 200–300 (1) 0.1 7–10 Food 58 (55–64) 22 51 118 139 30 low 450 0.0 11–14 Food 65 (61–71) 25 57 131 154 45 low 750 0.0 15–17 Food 71 (66–79) 27 63 145 170 65 ns 900 0.0 18–39 Food 108 (99–116) 38 94 228 270 70 ic(4) 1000 0.0 40–64 Food 176 (164–192) 66 154 360 424 70 low 1000 0.0 (1) AI for vitamin K is 12 µg/day in children aged 1–3 years and 20 µg/day in children aged 4–6 years old. The UL for vitamin K is 200 µg/day in children aged 1–3 years and 300 µg/day in children aged 4–6 years old. (2) When not enough evidence is available to set an EAR, an AI is set. The AI is based on the average intake of an apparently healthy population. (3) When the median intake of the population lies above the AI, the risk for inadequate intake is low. Since the distribution of requirements is not known, no statement (ns) can be formulated on the adequacy of vitamin K when the median intake is lower than the AI (4) ic: inconclusive within a certain age-group EAR: estimated average requirement; UL: upper intake level; Table 10. Usual intake of vitamin K (µg/day) from foods only in Belgian women (3–64 years), Belgian study on the intake of vitamins A, D, E and K (VITADEK-study) 2015. Percentiles Age Usual Intake (CI) 5 50 95 97.5 AI (2) % Inadequate (3) UL % >UL 3–6 Food 54 (47–61) 21 47 109 128 12–20 (1) low 200–300 (1) 0.1 7–10 Food 58 (55–64) 22 51 118 139 30 low 450 0.0 11–14 Food 65 (61–71) 25 57 131 154 45 low 750 0.0 15–17 Food 71 (66–79) 27 63 145 170 65 ns 900 0.0 18 39 F d 108 (99 116) 38 94 228 270 70 i (4) 1000 0 0 Table 10. Usual intake of vitamin K (µg/day) from foods only in Belgian women (3–64 years), Belgian study on the intake of vitamins A, D, E and K (VITADEK-study) 2015. 4.4.2. Vitamin D (2) When not enough evidence is available to set an EAR, an AI is set. The AI is based on the average intake of an apparently healthy population. (3) When the median intake of the population lies above the AI, the risk for inadequate intake is low. Since the distribution of requirements is not known, no statement (ns) can be formulated on the adequacy of vitamin E when the median intake is lower than the AI. (4) ic: inconclusive within a certain age-group. EAR: Estimated average requirement; UL: upper intake level; AI: adequate intake. intake of vitamin E (mg/day) from food, fortiﬁed food and supplements in Belgian women (3–64 years), Belgian study on the intake of vitamins A, D, E EK t d ) 2015 Table 8. Usual intake of vitamin E (mg/day) from food, fortiﬁed food and supplements in Belgian women (3–64 years), Belgian study on the intake of vitamins A, D, E and K (VITADEK-study) 2015. 15 of 22 Nutrients 2017, 9, 860 Table 9. Usual intake of vitamin K (µg/day) from foods only in Belgian men (3–64 years), Belgian study on the intake of vitamins A, D, E and K (VITADEK-study) 2015. Percentiles Age Usual Intake (CI) 5 50 95 97.5 AI (2) % Inadequate (3) UL % >UL 3–6 Food 64 (56–73) 21 54 140 168 12–20 (1) low 200–300 (1) 0.5 7–10 Food 63 (57–68) 21 53 136 163 30 low 450 0.0 1–14 Food 69 (64–74) 23 58 150 180 45 low 750 0.0 15–17 Food 76 (70–82) 25.1 65 166 199 65 ic(4) 900 0.0 18–39 Food 111 (103–121) 35 93 249 301 70 ic 1000 0.0 40–64 Food 185 (172–202) 59 156 412 496 70 low 1000 0.1 (1) AI for vitamin K is 12 µg/day in children aged 1–3 years and 20 µg/day in children aged 4–6 years old. The UL for vitamin K is 200 µg/day in children aged 1–3 years and 300 µg/day in children aged 4–6 years old. (2) When not enough evidence is available to set an EAR, an AI is set. The AI is based on the average intake of an apparently healthy population. (3) When the median intake of the population lies above the AI, the risk for inadequate intake is low. 4.4.2. Vitamin D Percentiles Age Usual Intake (CI) 5 50 95 97.5 AI (2) % Inadequate (3) UL % >UL 3–6 Food 54 (47–61) 21 47 109 128 12–20 (1) low 200–300 (1) 0.1 7–10 Food 58 (55–64) 22 51 118 139 30 low 450 0.0 11–14 Food 65 (61–71) 25 57 131 154 45 low 750 0.0 15–17 Food 71 (66–79) 27 63 145 170 65 ns 900 0.0 18–39 Food 108 (99–116) 38 94 228 270 70 ic(4) 1000 0.0 40–64 Food 176 (164–192) 66 154 360 424 70 low 1000 0.0 (1) AI for vitamin K is 12 µg/day in children aged 1–3 years and 20 µg/day in children aged 4–6 years old. The UL for vitamin K is 200 µg/day in children aged 1–3 years and 300 µg/day in children aged 4–6 years old. (2) When not enough evidence is available to set an EAR, an AI is set. The AI is based on the average intake of an apparently healthy population. (3) When the median intake of the population lies above the AI, the risk for inadequate intake is low. Since the distribution of requirements is not known, no statement (ns) can be formulated on the adequacy of vitamin K when the median intake is lower than the AI. (4) ic: inconclusive within a certain age-group. EAR: estimated average requirement; UL: upper intake level; AI: adequate intake; ns: no statement. oods only in Belgian women (3–64 years), Belgian study on the intake of vitamins A, D, E and K Table 10. Usual intake of vitamin K (µg/day) from foods only in Belgian women (3–64 years), Belgian study on the intake of vitamins A, D, E and K (VITADEK-study) 2015. Table 10. Usual intake of vitamin K (µg/day) from foods only in Belgian women (3–64 years), B (VITADEK-study) 2015. (1) AI for vitamin K is 12 µg/day in children aged 1–3 years and 20 µg/day in children aged 4–6 years old. The UL for vitamin K is 200 µg/day in children aged 1–3 years and 300 µg/day in children aged 4–6 years old. (2) When not enough evidence is available to set an EAR, an AI is set. The AI is based on the average intake of an apparently healthy population. (3) When the median intake of the population lies above the AI, the risk for inadequate intake is low. 4.4.2. Vitamin D Since the distribution of requirements is not known, no statement (ns) can be formulated on the adequacy of vitamin K when the median intake is lower than the AI. (4) ic: inconclusive within a certain age-group. EAR: estimated average requirement; UL: upper intake level; AI: adequate intake. Table 10. Usual intake of vitamin K (µg/day) from foods only in Belgian women (3–64 years), Belgian study on the intake of vitamins A, D, E and K (VITADEK-study) 2015. Percentiles Age Usual Intake (CI) 5 50 95 97.5 AI (2) % Inadequate (3) UL % >UL 3–6 Food 54 (47–61) 21 47 109 128 12–20 (1) low 200–300 (1) 0.1 7–10 Food 58 (55–64) 22 51 118 139 30 low 450 0.0 11–14 Food 65 (61–71) 25 57 131 154 45 low 750 0.0 15–17 Food 71 (66–79) 27 63 145 170 65 ns 900 0.0 18–39 Food 108 (99–116) 38 94 228 270 70 ic(4) 1000 0.0 40–64 Food 176 (164–192) 66 154 360 424 70 low 1000 0.0 (1) AI for vitamin K is 12 µg/day in children aged 1–3 years and 20 µg/day in children aged 4–6 years old. The UL for vitamin K is 200 µg/day in children aged 1–3 years and 300 µg/day in children aged 4–6 years old. (2) When not enough evidence is available to set an EAR, an AI is set. The AI is based on the average intake of an apparently healthy population. (3) When the median intake of the population lies above the AI, the risk for inadequate intake is low. Since the distribution of requirements is not known, no statement (ns) can be formulated on the adequacy of vitamin K when the median intake is lower than the AI. (4) ic: inconclusive within a certain age-group. EAR: estimated average requirement; UL: upper intake level; AI: adequate intake; ns: no statement. 4.4.2. Vitamin D Percentiles Age Usual Intake (CI) 5 50 95 97.5 AI (2) % Inadequate (3) UL % >UL 3–6 Food 64 (56–73) 21 54 140 168 12–20 (1) low 200–300 (1) 0.5 7–10 Food 63 (57–68) 21 53 136 163 30 low 450 0.0 1–14 Food 69 (64–74) 23 58 150 180 45 low 750 0.0 15–17 Food 76 (70–82) 25.1 65 166 199 65 ic(4) 900 0.0 18–39 Food 111 (103–121) 35 93 249 301 70 ic 1000 0.0 40–64 Food 185 (172–202) 59 156 412 496 70 low 1000 0.1 (1) AI for vitamin K is 12 µg/day in children aged 1–3 years and 20 µg/day in children aged 4–6 years old. The UL for vitamin K is 200 µg/day in children aged 1–3 years and 300 µg/day in children aged 4–6 years old. (2) When not enough evidence is available to set an EAR, an AI is set. The AI is based on the average intake of an apparently healthy population. (3) When the median intake of the population lies above the AI, the risk for inadequate intake is low. Since the distribution of requirements is not known, no statement (ns) can be formulated on the adequacy of vitamin K when the median intake is lower than the AI. (4) ic: inconclusive within a certain age-group. EAR: estimated average requirement; UL: upper intake level; AI: adequate intake. Table 10 Usual intake of vitamin K (µg/day) from foods only in Belgian women (3 64 years) Belgian study on the intake of vitamins A D E and K µg/ y g y µg/ y g y µg/ y g y µg/ y in children aged 4–6 years old. (2) When not enough evidence is available to set an EAR, an AI is set. The AI is based on the average intake of an apparently healthy population. (3) When the median intake of the population lies above the AI, the risk for inadequate intake is low. Since the distribution of requirements is not known, no statement (ns) can be formulated on the adequacy of vitamin K when the median intake is lower than the AI. (4) ic: inconclusive within a certain age-group. EAR: estimated average requirement; UL: upper intake level; AI: adequate intake. Table 10. 4.4.2. Vitamin D Since the distribution of requirements is not known, no statement (ns) can be formulated on the adequacy of vitamin K when the median intake is lower than the AI. (4) ic: inconclusive within a certain age-group. EAR: estimated average requirement; UL: upper intake level; AI: adequate intake; ns: no statement. Nutrients 2017, 9, 860 16 of 22 16 of 22 The contribution of supplements to the total vitamin D intake was considerable. Contribution levels were generally higher in women than in men and ranged from 16.7% in adolescent boys (11–14 years) to 71% in women (40–64 years). At the 97.5th percentiles, the increases in vitamin D levels were most pronounced, indicating high concentrations of vitamin D in supplements. In all sex-age groups, the median vitamin D intake from “foods only” was below the AI. Despite their important contribution to the mean usual intake, inclusion of fortiﬁed foods and supplements only slightly increased median vitamin D intake to levels around 4 µg/day and remained thus far below the AI (13–31% of the AI). Therefore, no statement was made on the adequacy of vitamin D. The risk of excessive intakes of vitamin D from the consumption of food was nil also when mandatorily and voluntarily fortiﬁed foods were considered. With the addition of supplements, the risk of excessive intake remained null, except in children and adult women. 2.5% of boys and 1.7% of girls aged 3–6 years, 1.1% of girls aged 7–10 and 1.4–2% of adult women ages 18–39, respectively 40–64 years had a risk of exceeding the UL for vitamin D. The median intake of vitamin D hardly changed and remained below the AI when miss-reporters were excluded. The proportion of the population with excessive intakes did not change substantially (Supplementary Tables S6 and S7). 4.4.3. Vitamin E Usual vitamin E intake from “foods only” in the Belgian population ranged from 7 mg/day in girls (3–6 years) to 13 mg/day in men (18–39 years) (Tables 7 and 8). The addition of fortiﬁed foods and supplements increased the mean intake of vitamin E from levels of 8 mg/day in girls (3–6 years) to 14 mg/100 g in men (18–39 years). Food was the major contributor to total vitamin E intake. Contribution of “foods only” ranged from 80–92%. Fortiﬁed foods had a low contribution to total vitamin E intake. Contributions varied from 4% in adult men (18–39 years) to 15% in boys (3–6 years). The contribution of supplements to total vitamin E intake was generally low with contributions between 2% and 6% except for adult women, where the contribution of supplements was more important and varied from 10–14%. The median vitamin E intake of foods only was below the AI (73–92% of the AI) in most sex-age groups except in boys 7–10 years old. Inclusion of fortiﬁed foods and supplements increased median intakes in those sex-age groups closer to but still below the AI (88–94% of the AI). As such, no statement could be made on the adequacy of vitamin E. When excluding miss-reporters, median vitamin E intake from all sources was above the AI in almost all sex-age groups, except in children aged 4, 5 and 11–14 years. Therefore, the risk for inadequate vitamin E intake could be stated to be low. In those groups were median intake was below the AI, differences were minor. Considering the fact that an AI is higher than an eventual population reference intake (intake level adequate for 97.5% of the population) when possible to establish, it could be concluded that the risk for inadequate intakes of vitamin E in the Belgian population was low (Supplementary Table S8 and S9). The risk for excessive intakes of vitamin E from the consumption of food, fortiﬁed food and supplements in the Belgian population was nil. 5. Discussion To our knowledge, the current study is the ﬁrst evaluation of the risk of inadequate and excessive intakes of vitamins A, D, E and K in the general Belgian population taking account all sources, i.e., foods, fortiﬁed foods and supplements. Additionally, this study differentiates between mandatorily and voluntarily fortiﬁed foods as such, providing better insights in the current Belgian fortiﬁcation and supplementation practices. Our study revealed considerable inadequate intakes for vitamin A, from all sources, in the entire Belgian population. Median vitamin D intake from all sources was less than one third of the AI in all subgroups. The lowest intake values for vitamin A and D were found in adolescents. The risk for inadequate intake from vitamin E and K was low. In Belgium, mandatory fortiﬁcation of margarines and spreadable fats slightly decreased vitamin A inadequacies, but did not improve suboptimal intakes of vitamin D. Voluntarily fortiﬁed foods and supplements made a substantial contribution to the usual intake of vitamins D and E. Nonetheless, the effect on inadequacy risk for vitamin D was minor while inadequacies of vitamin E were substantially reduced. A small proportion of young children were at risk of exceeding the upper intake level of vitamin A, which was mainly related to higher intakes from the base diet. A minor proportion of adult woman and young children exceeded the UL for vitamin D when including supplements. 4.4.4. Vitamin K The mean usual vitamin K intake from food in the Belgian population was between 60.3 µg/day (girls 3–6 years) and 130.6 µg/day (men 40–64 years) (Tables 9 and 10). Food was the main source for vitamin K. Fortiﬁed foods and supplements hardly contributed to total vitamin K intake. Intake data are presented for foods only, since aggregation with intake from fortiﬁed foods and supplements gave intake values that were, in some cases, lower than from foods only. This is due to the modelling effect and the low number of positive intakes from fortiﬁed foods and supplements. The median vitamin K intake from food and from the aggregated consumption of food, fortiﬁed food and supplements was above the AI in all sex-age groups. The risk for inadequate intake of vitamin K in the Belgian population was therefore considered to be low. Except in women aged 15–17 and for 17 of 22 17 of 22 Nutrients 2017, 9, 860 some ages of adolescent men (15–17) and adults 18–39 (ic: inconclusive within a certain age-group), the median intake was below the AI (95–99% of the AI). Nevertheless, as the difference with the AI is minor and considering the nature of an AI, we consider the risk for inadequate intake of vitamin K to be low for the entire population. Total vitamin K intake from the aggregated consumption of all sources was below the UL. As a consequence, there was no risk for excessive intakes of vitamin K in the Belgium population. Excluding miss-reporters (Supplementary Table S10 and S11) decreased the median intake in most sex-age groups. However, the conclusions within age groups remained the same. 5.1. Intake and Inadequacies Except for vitamin D, which is mainly produced under the inﬂuence of UVB-radiation, a well-balanced and healthy diet should provide the required amount of vitamins [2]. Inadequate vitamin A intake in the Belgium population could consequently be related to the unbalanced consumption of vegetables and dairy products, which are important food sources for vitamin A [35]. The consumption of vegetables and dairy products in 2014 was far below the reference values for the entire Belgian population, in particular in adolescents [41]. Because of the magnitude of the difference between median intake and the AI, we presume inadequate intake of vitamin D in all sex-age groups of the Belgian population. It should be kept in mind that these ﬁndings are valid under minimal sun exposure. Although, since Belgian life style is characterized by longer working days indoors and less outdoor activities, limited vitamin D body reserves from sun exposure might be more frequently occurring in Belgium than expected. Especially in winter-time when we rely on those body reserves, this might lead to inadequate status of vitamin D [5]. To our knowledge, no representative country data are available on vitamin D status in Belgium. The available studies are restricted to subgroups of the population and not always related to seasonal variability. However, an indication of the prevalence of low vitamin D status in Belgium is given by the studies of Sioen et al., Hoge et al. and Mac Farlane et al. that described high prevalence of vitamin D deﬁciency (25(OH)D <50 nmol/l) among Belgian children and adults [42–44]. Comparing dietary intake data with other European studies is hampered by the heterogeneity in assessment methods and by the difference in age classiﬁcations and in dietary reference values used to evaluate adequacy. Nevertheless, the present results are in line with European review studies when comparing intake levels. Higher intakes of vitamin A from foods are found in countries (Germany, Poland, Italy) with high consumption of sausages, liver and vegetables [3]. Higher vitamin D intake Nutrients 2017, 9, 860 18 of 22 levels from food are found in Scandinavian countries were oil-rich ﬁsh and milk are more frequently consumed [4]. Our results for vitamin D intake from foods and fortiﬁed foods in young children (3–6 years) were somewhat higher, but in the same order as the results of the Flemish preschoolers [18]. 5.2. Fortiﬁed Foods and Supplements The market of fortiﬁed foods and supplements in Europe is known to be a challenging market environment [10]. Despite a uniform European regulation on the addition of vitamins and minerals to foods (1925/2006), additional national legislations are adopted deﬁning limited amounts of the added nutrients or national policies are set on mandatory fortiﬁcation. As a result, fortiﬁcation practices and consumption of fortiﬁed foods differ greatly between European countries [1]. In Belgium, children and adolescents generally consume more fortiﬁed foods than adults [40]. This reﬂects the substantial amount of foods designed for children and adolescents found in our inventory. The age-related decline of fortiﬁed food consumption is also substantiated by different European studies [1,12]. Although studies including the intake from fortiﬁed foods are limited, the available evidence indicates that voluntarily fortiﬁed foods can make a substantial contribution to dietary intakes and can reduce the risk of sub-optimal intakes at the population level of a range of micronutrients, among which are vitamins D and E [1]. Our results for vitamin E are in line with German results reporting fortiﬁed foods to be effective in raising low intake values of vitamin E closer to the reference values [1]. For vitamin D, however, we did not observe the same effect. Indeed, inclusion of fortiﬁed foods barely increased median vitamin D levels in the Belgian population. In Europe, large differences exist in supplement consumption. Important contributions of supplements to total intake were frequently reported for vitamin D. Nevertheless, due to low uptake, supplements are often not effective as a public health strategy [12]. Our results are in line with these findings. 5.1. Intake and Inadequacies Comparable to our study, notable discrepancies between mean intake and dietary recommendations for vitamins A and D are common in different European populations and in all sex-age groups. However, the proportion of inadequate vitamin A intakes in the Flemish preschoolers (4–10) were almost twice as high as in our study [18]. This can be explained by the higher reference values used in the latter study. In contradiction to our results, Mensink et al. reported substantial proportions of inadequacies for vitamin E in all sex-age groups. However, intake data of fortiﬁed foods were generally lacking in this study [4]. Vitamin K intake data should be evaluated with caution due to uncertainties and limitation of food composition tables. Belgian intake data for vitamin K fell within the ranges reported in other European studies [36]. 5.4. Strengths and Limitations The importance of the evaluation of fortiﬁcation and supplementation practices has been repeatedly stressed in previous works. The need to adapt survey methodologies to include intake data from all sources was hereby highlighted. Studies evaluating intake from all sources in Europe are scarce. These studies have been hampered by the complex research task required. Food and supplement composition tables need to be in line with the supply and nutritive values of fortiﬁed products on the market today [1,3,4]. To facilitate this, automatic adaptation of food composition tables with labelled values of fortiﬁed nutrient contents through bar-codes (e.g., Global standard one (GS1)) would encompass a serious reduction of the comprehensive task required. An important strength of this study is that the contribution of fortiﬁed foods and supplements to total vitamin intake was evaluated. The survey methodology comprised a market inventory to assure that computation of vitamin intake was based on the most recent nutritive values. Furthermore, data were collected through the combination of two 24-h recalls and a supplementary FFQ, which is recommended by EFSA as a dietary assessment method [20,45]. Finally, vitamin intake was modelled with SPADE, a statistical program coping with multimodal distributions and heterogeneous variances between sources, typical for intake estimations from different sources [15,38]. This study however was conﬁned to subjects aged 3–64 years. Besides the fortiﬁed foods presented in Table 1, our market investigation revealed a considerable amount of foods designed for infants and toddlers, like infant milk, follow-up milk, growing-up milk, milk cereals and dairy desserts for babies. The contribution of fortiﬁed foods and supplements might consequently differ greatly in these youngest age groups. This will have an impact on the risk of inadequate and excessive intake in those population subgroups and consequently on the national recommendations to be made in nutrition policy. It would therefore be advisable to additionally conduct ad hoc surveys in speciﬁc target populations like infants and toddlers. A limitation of the study is that no biological samples were collected. For vitamin D, for which cutaneous synthesis excited by sun exposure is the main source, serum 25(OH)D levels would be required to give a picture on vitamin D status in the Belgian population and stress the magnitude of the evaluated vitamin D deﬁciency. 5.4. Strengths and Limitations Another limitation of the study was that subjects with missing data of FFQ on supplements were all treated as non-users when they did not consume supplements on both the 24-h recalls. This conservative approach implies a possible underestimation of the intakes from supplements. At last, the study did not include frequency questions on the consumption of dietary supplements. As a consequence, the intake values of subjects taking higher doses of supplements on a weekly or monthly basis had to be treated as users with missing data on the 24-h recall to enable modelling. This implies also a bias in the estimation of supplement intakes. 5.3. Excessive Intakes In Europe, the risk for excessive intake of micronutrients taking into account all sources is low. Excessive intakes are mainly found in children, but the UL is exceeded by only a small proportion of children [1]. Concerning fat-soluble vitamins, excessive intakes were reported for retinol. Excessive intakes for retinol were mainly related to higher intakes from the base diet (Poland) or from supplements (Ireland). Fortiﬁed foods had little effect on the higher intakes (P95) due to the limited amounts of foods fortiﬁed with retinol. These results are in line with our ﬁndings. The review studies of Hennessey and Flynn concluded that the risk of adverse health effects in individuals exceeding the UL was minor due to the safety margins used in establishing the UL and given the fact that the UL is exceeded by a modest amount [1,3]. In our study, the risk for high exposure of retinol is also limited in time, as it was only observed among the youngest age group. In Europe, the risk for exceeding the UL of vitamin D is low, even in countries with a wide use of supplements [3,5]. In our study, high supplement intakes were generated from highly-dosed substances. In Belgium, vitamins with daily doses higher than 50 µg/day are considered as drugs. These drugs however are available at the pharmacist without prescription and therefore also taken into account in the usual intake estimations. Nutrients 2017, 9, 860 19 of 22 19 of 22 6. Conclusions We present the ﬁrst evaluation of the risk of inadequate and excessive intakes of vitamins A, D, E and K in the Belgian population taking into account foods, fortiﬁed foods and supplements. Belgian fortiﬁcation and supplementation practices were insufﬁcient to eradicate inadequate intakes of vitamins A and D. Excessive intakes were observed for vitamin A in a small proportion of children and for vitamin D in a small proportion of adult women and young children, although the risks associated with these excessive intakes were considered to be minor. By providing the ﬁrst-ever dietary intake data of fat-soluble vitamins in Belgium from all sources, our study can support the development of adapted dietary strategies to increase vitamin A and D intakes in the general Belgian population while preventing excessive intakes. 6. Conclusions Supplementary Materials: The following are available online at www.mdpi.com/2072-6643/9/8/860/s1: Table S1: Contribution of food groups to total intake of vitamins A, D, E and K, Belgian population, 2014; Table S2: Usual intake of retinol (µg/day) from food, fortiﬁed food and supplements in Belgian men (3–64 years), according to age and gender, Belgian VITADEK-study 2015; Table S3: Usual intake of retinol (µg/day) from food, fortiﬁed food and supplements in Belgian women (3–64 years), according to age and gender, Belgium, 2014; Table S4: Usual intake of vitamin A (µg/dag) from food, fortiﬁed food and supplements in Belgian men (3–64 years), excluding miss-reporters, according to age and gender, Belgian VITADEK-study 2015; Table S5: Usual intake of vitamin A (µg/dag) from food, fortiﬁed food and supplements in Belgian women (3–64 years), excluding miss-reporters, according to age and gender, Belgian VITADEK-study 2015; Table S6: Usual intake of vitamin D (µg/day) from food, fortiﬁed food and supplements in Belgian men (3–64 years), excluding miss-reporters, according to age and gender, Belgian VITADEK-study 2015; Table S7: Usual intake of vitamin D (µg/day) from food, fortiﬁed food and supplements in Belgian women (3–64 years), excluding miss-reporters, according to age and gender, Belgian VITADEK-study 2015; Table S8: Usual intake of vitamin E (mg/day) from food, fortiﬁed food and supplements in Belgian men (3–64 years), excluding miss-reporters, according to age and gender, Belgian VITADEK-study 2015; Table S9: Usual intake of vitamin E (mg/day) from food, fortiﬁed food and supplements in Belgian women (3–64 years), excluding miss-reporters, according to age and gender, Belgian VITADEK-study 2015; Table S10: Usual intake of vitamin K (µg/day) from foods only in Belgian men (3–64 years), excluding miss-reporters, according to age and gender, Belgian VITADEK-study 2015; Table S11: Usual intake of vitamin K (µg/day) from foods only in Belgian women (3–64 years), excluding miss-reporters, according to age and gender, Belgian VITADEK-study 2015. Acknowledgments: The BNFCS2014 was initiated and ﬁnanced by the Belgian Federal Public Service Health, Food Chain Safety and Environment. The authors also acknowledge the ﬁnancial support of the Scientiﬁc Institute of Public Health and the European Food Safety Authority (EFSA). The Belgian study on the intake of vitamins A, D, E and K (VITADEK-study) was initiated by the Scientiﬁc Institute of Public Health. The study was carried out in collaboration with the department of Food Safety and Food Quality of Ghent University. 5.5. Recommendations It is clear that fortiﬁcation and supplementation practices in Belgium are insufﬁcient to overcome inadequacies of vitamins A and D in the Belgium population. There is a need for dietary strategies to increase vitamin A and D intakes in the entire Belgian population. Intake data of the Belgian population provide an ideal starting point for modelling of fortiﬁcation and supplementation scenarios. These scenario analyses will be very useful from a policy point of view. They should allow identifying which measures will ensure effective and safe increases in vitamin A and D intake. Fortiﬁcation of margarines and fats in Belgium with vitamins A and D is currently based on nutritive values of butter. These scenario studies might give an insight into how a potential change in policy may affect intake levels of vitamins A and D at a population level [15,45,46]. However, it should be stressed that there is a need for nationally-representative data on vitamin D status in the Belgian population prior to setting out the ﬁnal recommendations on vitamin D intake. Nutrients 2017, 9, 860 20 of 22 20 of 22 6. Conclusions The study was ﬁnanced by the Belgian Federal Public Service Health, Food Chain Safety and Environment. The authors wish to thank Stefanie Vandevijvere for initiating the study. We specially would like to thank Dr. Koenraad Cuypers for his great support at the onset of the study. We would also like to thank Kenneth Schippers for his contribution to the compilation of the food and supplement composition table. We are very grateful to Sarah Bel, Loes Brocatus, Theresa Lebacq, Charlotte Stievenart, Eveline Teppers and Soﬁe Van den Abeele for their advice and help during the execution of the survey. Finally, we wish to thank the members of the scientiﬁc steering committee for their advice. Author Contributions: C.L., H.v.O., I.M., J.T. and J.v.C. conceived of and designed the study. A.D., B.D., I.M. and K.d.R. analyzed the data. All authors critically interpreted the results. I.M. drafted the ﬁrst version of the manuscript. All authors critically revised and approved the ﬁnal manuscript. Conﬂicts of Interest: All authors declare that they have no competing interests. 1. Hennessy, A.; Walton, J.; Flynn, A. 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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/). © 2017 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/W4362478359	https://figshare.com/articles/journal_contribution/Supplementary_Figure_4_from_ATP_Citrate_Lyase_Knockdown_Induces_Growth_Arrest_and_Apoptosis_through_Different_Cell-_and_Environment-Dependent_Mechanisms/22497714/1/files/39956061.pdf	English	null	Supplementary Figure 3 from ATP Citrate Lyase Knockdown Induces Growth Arrest and Apoptosis through Different Cell- and Environment-Dependent Mechanisms	null	2,023	cc-by	65	26 Viable cells 12 77 51 Early Apoptotic cells Late Apoptotic/Dead cells 62 25 15 24 8 G IPTG No IPTG IPTG P62-shACLY-17 HepG2-shACLY-17 tosis in HOP62 and HepG2 cells after ACLY silencing. Viable cells 77 51 Early Apoptotic cells Late Apoptotic/Dead cells 25 15 24 8 No IPTG IPTG HepG2-shACLY-17 and HepG2 cells after ACLY silencing. 2 1 6 IP hACLY- s in H
https://openalex.org/W4390913709	https://journal.microbe.ru/jour/article/download/1909/1443	Russian	null	Possibility of Using Disposable Polymeric Containers in the Production Technology of Live Plague Vaccine	Problemy osobo opasnyh infekcij	2,024	cc-by	4,480	Corresponding author: Andrey A. Leshchenko, e-mail: 23527@mil.ru. Citation: Sharov D.A., Darmov I.V., Kosenkov I.V., Leshchenko A.A., Bagin S.V., Mokhov D.A., Lazykin A.G., Kovalenko E.A. Possibility of Using Disposable Polymeric Containers in the Production Technology of Live Plague Vaccine. Problemy Osobo Opasnykh Infektsii [Problems of Particularly Dangerous Infections]. 2023; 4:149–155. (In Russian). DOI: 10.21055/0370-1069-2023-4-149-155 Received 26.06.2023. Revised 04.07.2023. Accepted 26.07.2023. Problemy Osobo Opasnykh Infektsii [Problems of Particularly Dangerous Infections]. 2023; 4 Problemy Osobo Opasnykh Infektsii [Problems of Particularly Dangerous Infections]. 2023; 4 Problemy Osobo Opasnykh Infektsii [Problems of Particularly Dangerous Infections]. 2023; 4 Original articles Корреспондирующий автор: Лещенко Андрей Анатольевич, e mail: 23527@mil.ru. Для цитирования: Шаров Д.А., Дармов И.В., Косенков И.В., Лещенко А.А., Багин С.В., Мохов Д.А., Лазыкин А.Г., Коваленко Е.А. Оценка возможности использования одноразовых полимерных контейнеров в технологии производства вакцины чумной живой. Проблемы особо опасных инфекций. 2023; 4:149–155. DOI: 10.21055/0370-1069-2023-4-149-155 ценка возможности использования одноразовых полимерных контейнеров в технологии производства вакцины чумной живой Филиал ФГБУ «48 Центральный научно-исследовательский институт» Министерства обороны Россий Киров, Российская Федерация тральный научно-исследовательский институт» Министерства обороны Российской Федерации (г. Киров), Киров, Российская Федерация Цель работы – оценка возможности использования одноразовых полимерных контейнеров в технологии про- изводства вакцины чумной живой. Материалы и методы. Использовали вакцинный штамм Yersinia pestis ЕV линии НИИЭГ. Для глубинного выращивания посевной культуры чумного микроба применяли стандартный од- норазовый полимерный контейнер типа Flexboy объемом 10 л фирмы Sartorius Stedim Biotech, оборудованный фильтр-капсулами Sartoроrе 2. Сравнение проводили с регламентной технологией получения посевных культур с использованием стеклянных бутылей объемом 20 л. Контроль полученных посевных культур вакцинного штам- ма ЕV осуществляли в соответствии с ФС.3.3.1.0022.15. Выращивание посевной культуры в одноразовом поли- мерном контейнере проводили в жидкой питательной среде при температуре от 26 до 28 °С с непрерывным бар- ботажем и механическим перемешиванием с частотой от 80 до 90 колебаний в минуту. Для аэрации использовался сжатый воздух с давлением от 0,3 до 0,4 кгс/см2. Объемный расход стерильного воздуха, подаваемого на аэрацию, составлял от 0,9 до 1,0 л/мин. Результаты и обсуждение. Применение одноразовых полимерных контейнеров позволило сократить продолжительность технологической стадии получения посевной культуры в 1,7 раза и уве- личить выход живых микробов с единицы объема питательной среды, по сравнению с регламентной, в 2,8 раза. Таким образом, показана возможность и перспективность использования одноразовых полимерных контейнеров в технологии производства вакцины чумной живой на стадии приготовления посевных культур. Ключевые слова: одноразовый полимерный контейнер, вакцинный штамм Yersinia pestis ЕV, производство вакцины чумной живой, стадия получения посевных культур. Корреспондирующий автор: Лещенко Андрей Анатольевич, e-mail: 23527@mil.ru. Корреспондирующий автор: Лещенко Андрей Анатольевич, e-mail: 23527@mil.ru. Для цитирования: Шаров Д.А., Дармов И.В., Косенков И.В., Лещенко А.А., Багин С.В., Мохов Д.А., Лазыкин А.Г., Коваленко Е.А. Оценка возможности использования одноразовых полимерных контейнеров в технологии производства вакцины чумной живой. Проблемы особо опасных инфекций. 2023 4:149–155. DOI: 10.21055/0370-1069-2023-4-149-155 Поступила 26.06.2023. Отправлена на доработку 04.07.2023. Принята к публ. 26.07.2023. Поступила 26.06.2023. Отправлена на доработку 04.07.2023. Приня 23. Отправлена на доработку 04.07.2023. Принята к публ. 26.07.202 ( ) Received 26.06.2023. Revised 04.07.2023. Accepted 26.07.2023. D.A. Sharov, I.V. Darmov, I.V. Kosenkov, A.A. Leshchenko, S.V. Bagin, D.A. Mokhov, A.G. Lazyki E.A. Kovalenko Possibility of Using Disposable Polymeric Containers in the Production Technology of Live Plague Vaccine D.A. Sharov, I.V. Darmov, I.V. Kosenkov, A.A. Leshchenko, S.V. Bagin, D.A. Mokhov, A.G. Lazykin, E.A. Kovalenko Материалы и методы В экспериментальных исследованиях исполь- зовали вакцинный штамм Yersinia pestis ЕV линии НИИЭГ, полученный из Государственной коллекции возбудителей бактериальных инфекций, используе- мых для разработки и оценки эффективности меди- цинских средств биологической защиты, филиала ФГБУ «48 Центральный научно-исследовательский институт» Министерства обороны Российской Федерации (г. Киров). р) В соответствии с Государственной фармакопе- ей Российской Федерации (XIV изд. Т. IV. М., 2018. С. 5342–5350), производство вакцины предусматри- вает технологические стадии: получения посевных культур I, II и III генераций; накопления биомассы, выращенной для приготовления вакцинной взвеси с необходимой концентрацией; розлива, заморажива- ния, сублимационного высушивания, герметизации и упаковки препарата. Опыт выпуска вакцины чум- ной живой и развитие рынка биотехнологического оборудования способствовали совершенствованию основных стадий ее производства путем внедрения новых решений, направленных на повышение каче- ства готового препарата и эффективности техноло- гии в целом [1–4]. Глубинное выращивание посевной культуры чумного микроба штамма ЕV осуществляли двумя способами: в соответствии с требованиями суще- ствующего регламента и с применением одноразово- го полимерного контейнера. Использовали жидкую ПС, приготовленную из ферментативного гидроли- зата мяса. Для засева использовали стабилизирован- ную в защитной среде рабочую культуру с посевной дозой не менее 0,4  млрд м.к. на 1 мл ПС. Общую концентрацию микробов определяли по отраслево- му стандартному образцу мутности бактериальных взвесей на 10 международных единиц (МЕ), что эк- вивалентно 0,95·109 м.к./мл чумного микроба. Процесс приготовления посевных культур явля- ется одной из основных стадий в технологии произ- водства вакцины чумной живой, реализация которой определяет качество полуфабрикатов и готовой фор- мы препарата. Получение посевной культуры в рам- ках существующей технологии осуществляется спо- собом глубинного выращивания вакцинного штамма в жидкой питательной среде (ПС). Культивирование проводится в течение 48 ч в стеклянных бутылях вместимостью 20 л, оборудованных специальны- ми монтажами, при постоянной аэрации согласно ПР 08461522-23-14 «Промышленный регламент на производство вакцины чумной живой, лиофилизата для приготовления суспензии для инъекций, инга- ляций и накожного скарификационного нанесения». Практическим недостатком использования данных решений является продолжительность подготови- тельных работ (до 24 ч), сопровождающихся много операционностью и энергозатратностью. Кроме того, достаточно высоки риски получения брака продукта по причине контаминации или попадания в ПС ве- ществ, ингибирующих рост микробов. При реализации регламентного способа полу- чения посевной культуры чумного микроба штам- ма ЕV культивирование проводили в стеклянных бу- тылях вместимостью 20 л, содержащих 10 л жидкой ПС, в течение 48 ч при температуре от 26 до 28 °С и аэрации не менее 10 л/мин (промышленный регла- мент ПР 08461522-23-14). Проблемы особо опасных инфекций. 2023; 4 Оригинальные статьи Проблемы особо опасных инфекций. 2023; 4 Приоритетной задачей, согласно Указу Прези дента РФ от 2 июля 2021 г. № 400 «О Стратегии на- циональной безопасности Российской Федерации», является расширение производства лекарственных средств, медицинских изделий, отечественных вак- цин против актуальных инфекционных болезней. Решение данной задачи требует своевременного совершенствования технологий, внедрения в них современного инновационного оборудования и материалов. вания, изначальная гарантированная стерильность, возможность «гибкого» встраивания в технологиче- ский процесс обеспечивают сокращение подготови- тельных работ и повышают защищенность продукта на этапах его производства и хранения [5–8]. В связи с этим внедрение одноразовых полимерных контей- неров в технологию производства чумной вакцины на стадии получения посевной культуры является актуальной задачей. вания, изначальная гарантированная стерильность, возможность «гибкого» встраивания в технологиче- ский процесс обеспечивают сокращение подготови- тельных работ и повышают защищенность продукта на этапах его производства и хранения [5–8]. В связи с этим внедрение одноразовых полимерных контей- неров в технологию производства чумной вакцины на стадии получения посевной культуры является актуальной задачей. Цель работы – оценка возможности использо- вания одноразовых полимерных контейнеров в тех- нологии производства вакцины чумной живой. Одним из жизненно необходимых и важных ле- карственных препаратов для медицинского приме- нения является «Вакцина чумная живая, лиофилизат для приготовления суспензии для инъекций, инга- ляций и накожного скарификационного нанесения» (Распоряжение Правительства РФ от 12 октября 2019 г. № 2406-р). E.A. Kovalenko Possibility of Using Disposable Polymeric Containers in the Production Technology of Live Plague Vaccinei Affiliated Branch of the“48th Central Research Institute” of the Ministry of Defense of the Russian Federation, Kirov, Russian Federation Abstract. The aim of the work was to assess the possibility of using disposable polymeric containers in the production technology of the live plague vaccine. Materials and methods. We deployed the vaccine strain Yersinia pestis ЕV NIIEG for the work. A standard disposable 10 L Flexboy type polymeric container manufactured by Sartorius Stedim Biotech, equipped with Sartopore 2 filter capsules, was used for submerged cultivation of plague microbe inoculum. This method was compared to the regulated technology for obtaining seed cultures using glass bottles with a volume of 20 liters. The control of the produced seed cultures of the vaccine strain EV was performed in accordance with FS.3.3.1.0022.15. The cultivation of the seed culture in a disposable polymeric container was carried out in a liquid nutrient medium at a tem- perature of 26 to 28 °C with continuous barbotage and mechanical agitation with a platform oscillation frequency of 80 to 90 per minute. For aeration, compressed air with a pressure of 0.3 to 0.4 kgf/cm2 was used. The volumetric flow rate of sterile air supplied for aeration ranged from 0.9 to 1.0 l/min. Results and discussion. The use of disposable polymeric containers made it possible to reduce the duration of the technological stage of obtaining a seed culture by 1.7 times and increase the yield of live microbes per unit volume of the nutrient medium by 2.8 times, as compared to the regulated production technology. Thus, the possibility and prospects of using disposable polymeric containers in the production technology of live plague vaccine at the stage of preparation of sowing cultures is evidenced. Key words: disposable polymeric container, Yersinia pestis EV vaccine strain, live plague vaccine production, stage of obtaining sowing cultures. Conflict of interest: The authors declare no conflict of interest. Conflict of interest: The authors declare no conflict of interest. Funding: The authors declare no additional financial support for this study. 149 Результаты и обсуждение р Как следует из рис. 2, общая концентрация клеток при выращивании посевной культуры чумного микро- ба штамма ЕV в одноразовом полимерном контейнере более чем в 2 раза превысила таковую при культиви- ровании в бутылях. Значение этого показателя с при- менением одноразового полимерного контейнера до- стигло максимума на 27-й час роста, тогда как регла- ментный процесс был продолжительнее и составил 48 ч. Длительность лаг-фазы при культивировании в регламентном режиме превышала таковую в экспери- ментальном на 6 ч. Фаза экспоненциального роста в ре- гламентном режиме культивирования также являлась более продолжительной (на 12 ч), чем в эксперимен- тальном режиме. Различия во времени накопления бак- терий в культуре обусловлены особенностями гидро- динамических процессов в одноразовом полимерном контейнере и бутыли. Регламентный процесс преду- сматривал барботаж, а экспериментальный, дополни- тельно, – перемешивание, что значительно улучшало турбулизацию и насыщение кислородом микробной культуры. Для реализации экспериментальной техноло- гии получения посевной культуры чумного микроба штамма ЕV оборудовали лабораторный технологи- ческий участок. На платформу орбитального тер- мостатируемого шейкера ЛАБ-ПУ-01 вертикально устанавливали одноразовый полимерный контейнер, фиксировали пластинами, порты ориентировали пер- пендикулярно блоку управления. В качестве соеди- нительных коммуникаций применяли термопластич- ные шланги, оборудованные пластмассовыми зажи- мами для перекрывания воздушных и материальных потоков. Всего смонтировали три порта: первый (3/8ʺ×5/8ʺ×50 см), для выхода воздуха из внутрен- ней полости одноразового полимерного контейнера, фиксировали вертикально и оборудовали фильтр- капсулой Sartoроrе 2; второй (1/8ʺ×1/4ʺ×50 см) ис- пользовали для подачи воздуха под зеркало жидко- сти; третий (3/8ʺ×5/8ʺ×50 см) – резервный. Воздух в одноразовый полимерный контейнер подавали под давлением, достаточным для создания скоростного напора, а также преодоления сопротив- ления трения и гидродинамического сопротивления столба перемешиваемой посевной культуры [12]. у ур Орбитальные колебания способствовали эффек- тивному течению массообменных процессов при «мягком» динамическом воздействии на механола- бильные клетки посевной культуры чумного микро- ба штамма ЕV [14, 15]. Оптимальный гидродинамический режим опре- деляли по результатам изучения динамики накопле- ния в культуре бактерий чумного микроба штам- ма ЕV. Оценивали зависимость общей концентрации микробных клеток от времени культивирования по- севной культуры. Параллельно осуществляли выра- щивание посевной культуры в бутылях объемом 20 л (контроль). По завершении выращивания оценивали каче- ство посевной культуры, определяя следующие по- казатели: рН, общую концентрацию микробных кле- ток, количество живых микробных клеток, наличие посторонней микрофлоры. Результаты оценки качества посевной куль- туры чумного микроба штамма ЕV, полученной в 20-литровой бутыли и одноразовом полимерном контейнере, представлены в табл. 1. Материалы и методы В экспериментальных исследованиях использо- вался стандартный одноразовый полимерный кон- тейнер типа Flexboy объемом 10 л с коэффициентом заполнения жидкой ПС 0,5 [9]. Перемешивание осу- ществляли с применением орбитального термоста- тируемого шейкера ЛАБ-ПУ-01. Оценку показателей качества посевной куль- туры проводили в соответствии с ФС.3.3.1.0022.15 «Вакцина чумная живая, лиофилизат для приготов- ления суспензии для инъекций, ингаляций и накож- ного скарификационного нанесения». В современных условиях при организации асеп- тических производств микробиологических пре- паратов все чаще находит применение одноразовое емкостное оборудование из инертных полимерных материалов. Простота конструкции данного оборудо- Материальный баланс получения посевной куль- туры составлялся согласно требованиям ОСТ 64-02- 003-2002 «Продукция медицинской промышлен- ности. Технологические регламенты производства. 150 Problemy Osobo Opasnykh Infektsii [Problems of Particularly Dangerous Infections]. 2023; 4 Problemy Osobo Opasnykh Infektsii [Problems of Particularly Dangerous Infections]. 2023; 4 Original articles Питательную среду после холодной стерилиза- ции с помощью фильтр-капсулы Sartoроrе 2 асепти- чески вносили во внутреннюю полость одноразового полимерного контейнера в объеме 5 л, засевали, по- мещали заполненный контейнер на термостатируе мую платформу орбитального шейкера ЛАБ-ПУ-01 и подключали к аэрации. Культивирование осущест- влялось при температуре от 26 до 28 °С, с непрерыв- ным барботажем и механическим перемешиванием с частотой от 80 до 90 колебаний в минуту. Объемный расход стерильного воздуха, подаваемого на аэра- цию, контролировали с помощью ротаметра РМ-1; величина расхода составляла от 0,9 до 1,0 л/мин. Стерильность воздуха достигалась путем примене- ния в системе фильтр-капсулы Sartoроrе 2 с разме- ром пор 0,2 мкм [13]. Содержание, порядок разработки, согласования и утверждения». Расчет выполняли на основании ме- тодики, представленной в практикуме по технологии лекарственных форм [10]. Все этапы исследований выполнялись в соответ- ствии с СанПиН 3.3686-21 «Санитарно-эпидемио логические требования по профилактике инфекци- онных болезней». Утилизацию одноразовых полимерных контей- неров после использования проводили, руковод ствуясь СанПиН 2.1.7.728-99 «Правила сбора, хра нения и удаления отходов лечебно-профилактиче ских учреждений». Статистический анализ полученных результатов выполняли с помощью программы Microsoft Excel. Достоверность результатов оценивали с использова- нием критерия Стьюдента при уровне доверительной вероятности 0,95 [11]. Кривые линии роста культуры строили с величиной достоверности аппроксимации R2≥0,95. В экспериментальном и контрольном режимах провели по пять циклов культивирования. Результаты изучения динамики накопления чумного микроба штамма ЕV при культивировании в одноразовых полимерных контейнерах и бутылях вместимостью 20 л показаны на рис. 2. Результаты и обсуждение С целью приближения к регламентным усло- виям экспериментальный процесс выращивания посевной культуры в одноразовом полимерном кон- тейнере проводился в соответствии с разработанной технологической схемой, приведенной на рис. 1. Данные табл. 1 свидетельствуют о соответствии нормативным показателям качества изучаемых посев- 151 Проблемы особо опасных инфекций. 2023; 4 Оригинальные статьи Рис. 1. Технологическая схема получения посевных культур чумного микроба штамма ЕV при культивировании в одноразовом полимерном контейнере Технологическая схема получения посевных культур чумного микроба штамма ЕV при культивировании ерном контейнере Рис. 1. Технологическая схема получения посевных культур чумного микроба штамма ЕV при культивировании в одноразовом полимерном контейнере Fig. 1. Process flow diagram of cultivation seed culture of the plague microbe strain EV in disposable polymeric container Fig. 1. Process flow diagram of cultivation seed culture of the plague microbe strain EV in disposable polymeric container 152 Problemy Osobo Opasnykh Infektsii [Problems of Particularly Dangerous Infections]. 2023; 4 Original articles Рис. 2. Динамика нак штамма ЕV при культи лимерных контейнерах (X̅ ±I95, n=5) Fig. 2. Dynamics of microbe strain EV du polymeric containers an (X̅ ±I95, n=5) Рис. 2. Динамика накопления чумного штамма ЕV при культивировании в однораз лимерных контейнерах и бутылях вместимо (X̅ ±I95, n=5) Fig. 2. Dynamics of accumulation of th microbe strain EV during cultivation in d polymeric containers and bottles with a capac (X̅ ±I95, n=5) Рис. 2. Динамика накопления чумного микроба штамма ЕV при культивировании в одноразовых по- лимерных контейнерах и бутылях вместимостью 20 л (X̅ ±I95, n=5) Рис. 2. Динамика накопления чумного микроба штамма ЕV при культивировании в одноразовых по- лимерных контейнерах и бутылях вместимостью 20 л (X̅ ±I95, n=5) Рис. 2. Динамика накопления чумного микроба штамма ЕV при культивировании в одноразовых по- лимерных контейнерах и бутылях вместимостью 20 л (X̅ ±I95, n=5) Fig. 2. Dynamics of accumulation of the plague microbe strain EV during cultivation in disposable polymeric containers and bottles with a capacity of 20 l (X̅ ±I95, n=5) Fig. 2. Dynamics of accumulation of the plague microbe strain EV during cultivation in disposable polymeric containers and bottles with a capacity of 20 l (X̅ ±I95, n=5) Как следует из табл. 2, несмотря на меньший объем посевной культуры, выращенной в одноразо- вом полимерном контейнере, общее содержание живых микробных клеток в данном случае боль- ше, чем при использовании бутыли объемом 20 л, на 18,5·1012 ж.м.к., что составляет 29 %. Результаты и обсуждение Данный факт свидетельствует о том, что количества живых микробных клеток в посевной культуре, выращен- ной в одноразовом полимерном контейнере, доста- точно для осуществления дальнейшего технологи- ческого этапа – глубинного культивирования чум- ного микроба штамма ЕV в ферментере БИОР-0,25. Применение одноразового полимерного контейнера позволило сократить продолжительность стадии по- лучения посевной культуры в 1,7 раза и увеличить выход живых микробов с единицы объема питатель- ной среды в 2,8 раза. ных культур чумного микроба штамма ЕV, получен- ных как в бутылях объемом 20 л, так и в одноразовом полимерном контейнере. Значения общей концентра- ции микробов и количества живых микробных клеток в посевной культуре, приготовленной в одноразовом полимерном контейнере, превышали таковые при ис- пользовании бутылей объемом 20 л. В связи с тем, что объемы выращиваемой биомассы эксперимен- тальным способом и регламентным были разными (для одноразового полимерного контейнера объем со- ставил 5 л, для бутыли – 10 л), представлялось целе- сообразным оценить эффективность обоих процессов путем составления материального баланса. Результаты сравнительной оценки материаль- ного баланса на этапе приготовления разными спо- собами посевной культуры в производстве вакцины чумной живой представлены в табл. 2. Note: * Manufacturing specifications (master formula) 08461522-23-14. Manufacturing specifications for the production of a live plague vaccine, lyophilizate for the preparation of a suspension for injections, inhalations and skin scarification. Список литературы Список литературы 1. Микшис Н.И., Кутырев В.В. Современное состояние проблемы разработки вакцин для специфической профилактики чумы. Проблемы особо опасных инфекций. 2019; 1:50−63. DOI: 10.21055/0370-1069-2019-1-50-63. 2. Abzaeva N.V., Gostishcheva S.E., Startseva O.L., Katunina L.S., Kovalev D.A., Ivanova G.F., Kostrominov A.V., Kurilova A.A. [Studying the effect of experimental bases on the growth quality of liquid nutritional media for submerged cultivation of plague microbe vaccine strain]. Problemy Osobo Opasnykh Infektsii [Problems of Particularly Dangerous Infections]. 2022; (1):71–6. DOI: 10.21055/0370-1069-2022-1-71-76. 2. Абзаева Н.В., Гостищева С.Е., Старцева О.Л., Катунина Л.С., Ковалев Д.А., Иванова Г.Ф., Костроминов А.В., Курилова А.А. Изучение влияния экспериментальных основ на ростовые качества жидких питательных сред для глубинного культивиро- вания вакцинного штамма чумного микроба. Проблемы особо опасных инфекций. 2022; 1:71−6. DOI: 10.21055/0370-1069-2022- 1-71-76. 3. Sharov D.A., Leshchenko A.A., Bagin S.V., Logvinov S.V., Ezhov A.V., Lazykin A.G., Mokhov D.A., Krupin V.V., Ziganshin A.R. [The improvement of live plaque vaccine production technology]. [Bulletin of NBC Protection Corps]. 2017; 1(3):30−7. 3. Шаров Д.А., Лещенко А.А., Багин С.В., Логвинов С.В., Ежов А.В., Лазыкин А.Г., Мохов Д.А., Крупин В.В., Зиганшин А.Р. Совершенствование технологии производства вакцины чумной живой. Вестник войск РХБ защиты. 2017; 1(3):30−8. 4 V S K T j U Pl i d l gy] [ f p ] ; ( ) 4. Verma S.K., Tuteja U. Plague vaccine development: cur- rent research and future trends. Front. Immunol. 2016; 7:602. DOI: 10.3389/fimmu.2016.00602. [C i f h f di bl d i l d i 5. [Comparison of the use of disposable and conventional mod- ifications of fermenters]. (Cited 01 Mar 2023). [Internet]. Available from: https://bio-rus.ru/stati/sravnenie-primeneniya-odnorazovyix-i- klassicheskix-ispolnenij-fermenterov.html. 4. Verma S.K., Tuteja U. Plague vaccine development: cur- rent research and future trends. Front. Immunol. 2016; 7:602. DOI: 10.3389/fimmu.2016.00602. i 5. Сравнение применения одноразовых и классических ис- полнений ферментеров. [Электронный ресурс]. 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Таблица 1 / Table 1 Таблица 1 / Table 1 ц Результаты оценки качества посевной культуры чумного микроба штамма ЕV, полученной различными способами (X̅ ±I95, n=5) The results of assessing the quality of the seed culture of the plague microbe strain EV obtained by various methods (X̅ ±I95, n=5) Наименование показателя, единица измерения Indicator, unit of measure Требования нормативной документации* Requirements of regulatory documents* Значения показателя для посевной культуры, полученной способом … Values of the indicator for the seeding culture obtained using the … technology регламентным regulated экспериментальным experimental Общая концентрация микробных клеток, млрд м.к./мл The total concentration of microbial cells, billion m.c./ml 6,0, не менее 6.0, not less 13,0±1,0 24,0±2,0 Количество живых микробных клеток, млрд ж.м.к./мл The number of living microbial cells, billion m.c./ml 3,0, не менее 3.0, not less 4,5±0,9 12,7±1,3 рН, ед. pH, units от 7,4 до 7,9 7.4 to 7.9 7,6±0,2 7,5±0,1 Отсутствие посторонних микроорганизмов Absence of foreign microorganisms Не должна содержать посторонних микроорганизмов Should not contain foreign microorganisms Не содержит посторонних микроорганизмов Does not contain foreign microorganisms Примечание: * ПР 08461522-23-14. Промышленный регламент на производство вакцины чумной живой, лиофилизата для приготовления суспензии для инъекций, ингаляций и накожного скарификационного нанесения. Note: * Man fact ring specifications (master form la) 08461522 23 14 Man fact ring specifications for the prod ction of a li e plag e accine осевной культуры чумного микроба штамма ЕV, полученной различными способами (X̅ ±I95, n=5) ̅ Примечание: * ПР 08461522-23-14. Промышленный регламент на производство вакцины чумной живой, лиофилизата для приготовления суспензии для инъекций, ингаляций и накожного скарификационного нанесения. Note: * Manufacturing specifications (master formula) 08461522-23-14. Manufacturing specifications for the production of a live plague vaccine, lyophilizate for the preparation of a suspension for injections, inhalations and skin scarification. 153 Проблемы особо опасных инфекций. 2023; 4 Оригинальные статьи Результаты сравнительной оценки материального баланса получения посевной культуры экспериментальным и регламентным способами в производстве вакцины чумной живой (X̅ , n=5) The results of comparative assessment of the material balance of obtaining a seeding culture using experimental and regulated technologies in the production of live plague vaccine (X̅ , n=5) Способ получения посевной культуры, продолжительность Method of obtaining a seeding culture, duration Объем, л Volume, l Количество живых микробных клеток, ж.м.к./мл The number of live microbial cells, live m.c./ml Общее количество живых микробных клеток в объеме, ж.м.к. The total number of living microbial cells in the volume, live m.c. Таблица 1 / Table 1 Экспериментальный, 27 ч Experimental, 27 hours 5,0 12,7·109 63,5·1012 Регламентный, 48 ч Regulated, 48 hours 10,0 4,5·109 45,0·1012 Результаты сравнительной оценки материального баланса получения севной культуры экспериментальным и регламентным способами в производстве вакцины чумной живой (X̅ , n=5) Результаты сравнительной оценки материального баланса получения экспериментальным и регламентным способами в производстве вакцины чумной живой (X̅ , n=5) The results of comparative assessment of the material balance of obtaining a seeding culture using experimental and regulated technologies in the production of live plague vaccine (X̅ , n=5) Таким образом, результаты исследований по- казывают возможность и перспективность исполь- зования одноразовых полимерных контейнеров в технологии производства вакцины чумной живой на стадии получения посевной культуры. 13. Комиссаров А.В., Морозов К.М., Перепелица А.И., Ульянов А.Ю., Волох О.А., Никифоров А.К. 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https://openalex.org/W4304187521	https://jpro.springeropen.com/counter/pdf/10.1186/s41687-022-00513-3	English	null	The development of a glaucoma-specific health-related quality of life item bank supporting a novel computerized adaptive testing system in Asia	Journal of patient-reported outcomes	2,022	cc-by	9,828	Backgroundh The prevalence of glaucoma, a potentially blinding eye condition, is estimated to reach 111.8 million by 2040, with Asia accounting for the largest number of cases worldwide [1]. While primary open angle glaucoma (POAG) is the predominant form of the disease among Correspondence: ecosse.lamoureux@duke-nus.edu.sg 1 Singapore National Eye Centre, Singapore Eye Research Institute (SERI), The Academia, 20 College Road, Level 6, Singapore 169856, Singapore Full list of author information is available at the end of the article © 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/. Fenwick et al. Journal of Patient-Reported Outcomes (2022) 6:107 https://doi.org/10.1186/s41687-022-00513-3 Fenwick et al. Journal of Patient-Reported Outcomes (2022) 6:107 https://doi.org/10.1186/s41687-022-00513-3 Fenwick et al. Journal of Patient-Reported Outcomes (2022) 6:107 https://doi.org/10.1186/s41687-022-00513-3 Journal of Patient- Reported Outcomes Journal of Patient- Reported Outcomes Open Access Abstract Background: A glaucoma-specific health-related quality of life (HRQoL) item bank (IB) and computerized adaptive testing (CAT) system relevant to Asian populations is not currently available. We aimed to develop content for an IB focusing on HRQoL domains important to Asian people with glaucoma; and to compare the content coverage of our new instrument with established glaucoma-specific instruments. Methods: In this qualitative study of glaucoma patients recruited from the Singapore National Eye Centre (Novem- ber 2018-November 2019), items/domains were generated from: (1) glaucoma-specific questionnaires; (2) published articles; (3) focus groups/semi-structured interviews with glaucoma patients (n = 27); and (4) feedback from glaucoma experts. Data were analyzed using the constant comparative method. Items were systematically refined to a concise set, and pre-tested using cognitive interviews with 27 additional glaucoma patients. Results: Of the 54 patients (mean ± standard deviation [SD] age 66.9 ± 9.8; 53.7% male), 67 (62.0%), 30 (27.8%), and 11 (10.2%) eyes had primary open angle glaucoma, angle closure glaucoma, and no glaucoma respectively. Eighteen (33.3%), 11 (20.4%), 8 (14.8%), 12 (22.2%), and 5 (9.3%) patients had no, mild, moderate, severe, or advanced/end-stage glaucoma (better eye), respectively. Initially, 311 items within nine HRQoL domains were identified: Visual Symptoms, Ocular Comfort Symptoms, Activity Limitation, Driving, Lighting, Mobility, Psychosocial, Glaucoma management, and Work; however, Driving and Visual Symptoms were subsequently removed during the refinement process. During cognitive interviews, 12, 23 and 10 items were added, dropped and modified, respectively. Conclusion: Following a rigorous process, we developed a 221-item, 7-domain Asian glaucoma-specific IB. Once operationalised using CAT, this new instrument will enable precise, rapid, and comprehensive assessment of the HRQoL impact of glaucoma and associated treatment efficacy. Keywords: Glaucoma, Quality of life, Item bank, Computerized adaptive testing, Eyedrops, Qualitativ The development of a glaucoma‑specific health‑related quality of life item bank supporting a novel computerized adaptive testing system in Asia Eva K. Fenwick1,2, Belicia Lim1, Ryan E. K. Man1,2, Mani Baskaran1,5, Monisha E. Nongpiur1, Chelvin C. A. Sng1,3, Jayant V. Iyer1, Rahat Husain1, Shamira A. Perera1, Tina T. Wong1,2, Jin Rong Low1, Olivia Huang Shimin1,2, Katherine Lun5, Tin Aung1,2,3 and Ecosse L. Lamoureux1,2,4 Measurement of HRQoL is traditionally done using patient-reported outcome measures (PROMs), which are now mandated for use in clinical trials by reg- ulatory authorities [7, 8], and are becoming essential to guide clinical care in the era of value-based medicine [9]. on the generation (Phase 1) and refinement (Phase 2) of domains and items for this new IB. We also com- pare and contrast the content coverage of our new instrument with other available PROMs that assess the HRQoL impact of glaucoma, with a focus on how GlauCAT™-Asian differs from the GlauCAT™-Western instrument. Page 2 of 13 Fenwick et al. Journal of Patient-Reported Outcomes (2022) 6:107 Fenwick et al. Journal of Patient-Reported Outcomes Caucasians, primary angle-closure glaucoma (PACG), a visually destructive subtype, is a major form of glaucoma in Asians [2]. Independent of visual acuity, glaucoma and its associated treatments can have a negative impact on health-related quality of life (HRQoL) [3–6], which is defined by the International Society for Quality of Life Research (ISOQOL) as the impact of disease and treat- ment on disability and daily functioning, and the impact of perceived health on an individual’s ability to live a ful- filling life. Measurement of HRQoL is traditionally done using patient-reported outcome measures (PROMs), which are now mandated for use in clinical trials by reg- ulatory authorities [7, 8], and are becoming essential to guide clinical care in the era of value-based medicine [9]. While several paper–pencil PROMs are available to measure glaucoma-specific HRQoL [10, 11], a recent systematic review [12] has revealed that most PRO- instruments demonstrated poor developmental quality, particularly a lack of conceptual frameworks and item generation strategies involving the patients’ perspective, and psychometric evaluation based largely on classical test theory methods. Moreover, these PROMs usually capture only one or two HRQoL domains and lack con- tent relating to new treatment therapies (e.g. minimally invasive glaucoma surgery [MIGS] and nanotechnol- ogy [13]) and other modern trends (e.g. usage of ‘smart’ devices). These issues can be overcome by sophisticated psychometric methods of instrument development, such as item banking and computerized adaptive testing (CAT) [14]. CAT is a ‘smart’ technology that adapts the items (questions) asked based on participants’ responses to previous items [15]. CAT reduces test length without loss of precision by presenting to the respondent targeted items from a calibrated item bank that measures a latent HRQoL construct [15]. Caucasians, primary angle-closure glaucoma (PACG), a visually destructive subtype, is a major form of glaucoma in Asians [2]. Independent of visual acuity, glaucoma and its associated treatments can have a negative impact on health-related quality of life (HRQoL) [3–6], which is defined by the International Society for Quality of Life Research (ISOQOL) as the impact of disease and treat- ment on disability and daily functioning, and the impact of perceived health on an individual’s ability to live a ful- filling life. Study design and population English- and Mandarin-speaking patients aged ≥ 40 years with a primary diagnosis of glaucoma (i.e. POAG/PACG) in at least one eye were recruited from the Singapore National Eye Centre (SNEC). Those with other retinal comorbidities including second- ary glaucomas, severe cataract, neurological condi- tions affecting vision, hearing or cognitive impairment (assessed using the 6-CIT questionnaire [19]) were excluded. Patients were purposively recruited to ensure that the spectrum of ethnicity, gender, age, and glau- coma severity was represented. guide clinical care in the era of value-based medicine [9]. While several paper–pencil PROMs are available to measure glaucoma-specific HRQoL [10, 11], a recent systematic review [12] has revealed that most PRO- instruments demonstrated poor developmental quality, particularly a lack of conceptual frameworks and item generation strategies involving the patients’ perspective, and psychometric evaluation based largely on classical test theory methods. Moreover, these PROMs usually capture only one or two HRQoL domains and lack con- tent relating to new treatment therapies (e.g. minimally invasive glaucoma surgery [MIGS] and nanotechnol- ogy [13]) and other modern trends (e.g. usage of ‘smart’ devices). These issues can be overcome by sophisticated psychometric methods of instrument development, such as item banking and computerized adaptive testing (CAT) [14]. CAT is a ‘smart’ technology that adapts the items (questions) asked based on participants’ responses to previous items [15]. CAT reduces test length without loss of precision by presenting to the respondent targeted items from a calibrated item bank that measures a latent HRQoL construct [15].i Phases 1 and 2 of our study were conducted between November 2018 and November 2019 at the SNEC research clinic. The study protocol received approval from the SingHealth Centralised Institutional Review Board (CIRB #2018/2459) and was conducted in accord- ance with the Declaration of Helsinki. Prior to study par- ticipation, written informed consent was obtained from participants by study personnel. Participants were reim- bursed SGD $60 and $40 (for focus group and cognitive interviews, respectively) to defray the cost of their par- ticipation in this research. HRQoL construct [15]. A glaucoma-specific HRQoL item bank was recently developed by Matsuura and colleagues [16] in a Japa- nese population; however, it focused predominantly on activity limitation and a CAT system is not yet available. While our group has already produced an operational CAT for glaucoma (GlauCAT™) [17, 18]. its development was informed by qualitative work with glaucoma patients from Western populations. Phase 2: item refinement i Binning and winnowing Items generated during Phase 1 were systematically categorized into relevant HRQoL domains based on their content and meaning in a process known as binning. In order to reduce the initial large item pools to a more manageable minimally representative set, items were subsequently deleted using a process of win- nowing, where an expert panel (EF, BL, RM and EL) met face-to-face over two 3-h sessions to remove redundant or duplicate items, and those that did not fit well within the particular HRQoL domain. The panel used a system- atic set of criteria developed by the Patient-Reported Outcomes Measurement Information System (PROMIS) group[25] to guide item removal, including: We used a ‘‘top-down’’ (theoretical framework informed by our comprehensive literature review and previous experience generating IBs [18]), and ‘‘bottom-up’’ (data- driven) approach for domain and item generation. Literature review A literature review exploring the impact of HRQoL in patients with glaucoma was per- formed by authors EF and BL using Pubmed and Google Scholar databases and bibliographies of relevant papers. Keywords included ‘glaucoma’, ‘quality of life’, ‘impact’, ‘functioning’, ‘emotional well-being’, ‘questionnaire’, ‘patient-reported outcome measure’. Findings were used to generate the moderator’s guide for the focus group dis- cussions. (a) Item redundancy—identical wording or too similar in content to another item; Qualitative sessions Guided by the consolidated criteria for reporting qualitative research (COREQ) guidelines, [23] we conducted a qualitative study in 27 patients across six focus group sessions (four English, two Mandarin). Author EF moderated FGs 1–2 and author BL moderated FGs 3–6. Author BL was note-taker for FGs 1–2, while a Clinical Research Coordinator fluent in both English and Mandarin and trained in qualitative methods was note- taker for FGs 3–6. The composition of each group was arranged to ensure an equal mix of gender, ethnicity and glaucoma severity. Participants answered open-ended questions about how glaucoma had affected different aspects of their HRQoL, i.e. what things were difficult or inconvenient; how this had affected emotional well-being; and the type, frequency and severity of symptoms experi- enced (see Additional file 1). Participants were also asked which three areas of HRQoL they felt were most impor- tant in relation to their glaucoma, and these responses were recorded. Study design and population As such, the content may not be relevant to Asian populations, where glaucoma prevalence and pathophysiology (e.g. POAG vs. PACG) [1, 2], and healthcare systems, treatment regimens, and perceptions of disease burden differ. A time-efficient and focused glaucoma CAT for Asian settings is imperative. Against this background, our group aimed to develop a glaucoma-specific HRQoL item bank (IB) and CAT system that focuses on the key HRQoL domains most important to Asian patients with glau- coma (GlauCAT™-Asian). This study reports primarily A glaucoma-specific HRQoL item bank was recently developed by Matsuura and colleagues [16] in a Japa- nese population; however, it focused predominantly on activity limitation and a CAT system is not yet available. While our group has already produced an operational CAT for glaucoma (GlauCAT™) [17, 18]. its development was informed by qualitative work with glaucoma patients from Western populations. As such, the content may not be relevant to Asian populations, where glaucoma prevalence and pathophysiology (e.g. POAG vs. PACG) [1, 2], and healthcare systems, treatment regimens, and perceptions of disease burden differ. A time-efficient and focused glaucoma CAT for Asian settings is imperative. Assessment of type and severity of glaucoma Glaucoma subtype, Snellen visual acuity (VA) and visual field (VF) data (both eyes) were extracted from patients’ files. We also conducted binocular Esterman tests using the Humphrey Visual Field Analyzer-3 (Carl Zeiss AG, Jena, Germany). Grading of glaucoma severity was done by glaucoma clinicians and co-authors (MB, MN, and JL) using both the Glaucoma Staging System (GSS) and the Advanced Glaucoma Intervention Study (AGIS) proto- col [20, 21] using all available data into better/worse eye with no, mild, moderate, severe, advanced, or end-stage glaucoma. Due to the low number of end-stage glaucoma cases, we combined the advanced/end-stage categories. Snellen VA was converted into equivalent logarithm of the minimum angle of resolution (LogMAR) units and vision impairment (VI) was defined as present if VA ≤ 0.3 LogMAR [22]; and further categorized into mild (> 0.3 LogMAR ≤ 0.48) and moderate/severe (> 0.48 LogMAR). Against this background, our group aimed to develop a glaucoma-specific HRQoL item bank (IB) and CAT system that focuses on the key HRQoL domains most important to Asian patients with glau- coma (GlauCAT™-Asian). This study reports primarily Page 3 of 13 Fenwick et al. Journal of Patient-Reported Outcomes (2022) 6:107 Fenwick et al. Journal of Patient-Reported Outcomes Phase 2: item refinement (b) Item clarity—item confusing, poorly worded, dou- ble- or multi-barrelled; (c) Item applicability—item too specialised; lacked broad application; (d) Item frequency—item did not occur often, or was not well-represented across the four sources of con- tent development. (e) Item relevance—precedence was given to items from qualitative patient interviews, as these were considered most likely to accurately reflect patient experiences. Development of item stems, preceding statement and response options Based on previous instrument development work [26] and empirical evidence [27], items were rated on a 4- or 5-point Likert-type scale. The pre- ceding statement, “Because of your glaucoma or glaucoma treatment…”), timeframe (e.g. “In the past 1 month…”), and item stem to specify the attribute of the QoL con- struct being measured (e.g. How much difficulty do you have…?”) were also developed, along with short descrip- tions of each HRQoL domain (see Table 1 for more details on the attribute/timeframe for each HRQoL domain). Immediately after each focus group (mean = 67 min), the moderator and note-taker debriefed the session and noted if any new themes had emerged. Focus groups were conducted until thematic saturation was reached (i.e. no substantial new themes emerged after two subsequent sessions). Sessions were audio recorded and transcribed verbatim. For sessions conducted in Mandarin, the tran- scripts were professionally translated to English. Cognitive interviews Following the development of the item pools, cognitive interviews were conducted with glaucoma patients, who were recruited from SNEC using the same eligibility criteria as described above. Cognitive interviews allow any issues to be addressed prior to large- scale testing. The “think-aloud” and “verbal-probing” methods [28] were used, which allowed patients’ compre- hension of the item stems, items and response options to be tested using open-ended questions (e.g. “I noticed you had to take time to understand the question. Can you tell me why this was?”). As the preliminary instrument was Expert opinion on the impact of glaucoma on patients’ HRQoL was obtained from four glaucoma consultants (7 approached, response rate 57.1%) at SNEC (authors MB, MN, TW and JI). Three open-ended questions were posed and responses collected via email in April 2019. Patient transcripts and clinician feedback were ana- lysed separately by two researchers (EF and BL) using the constant comparative method [24], and disagreements in coding were adjudicated by a third researcher (EL). Page 4 of 13 Fenwick et al. Phase 2: item refinement Journal of Patient-Reported Outcomes (2022) 6:107 Table 1 Description of domains and items in our glaucoma-specific quality of life instrumenta a Brief descriptions of key terms were provided as part of initial participant instructions to ensure they were understood consistently by all participants Domain (n = items) Definition Timeframe Item stem Response options Example content Ocular comfort symptoms (n = 19) Refers to any sensations or feelings in and around your eyes arising from glaucoma and glaucoma treatment In the past 1 month… How much were you bothered by…? How often did you experi- ence…? How often did you feel like…? Not at All (4) to A lot (1) None of the time (4) to All of the time (1) Pain around eyes, itchy eyes, headache Activity limitation (n = 72) Difficulties performing daily activities because of glaucoma or poor vision None How much difficulty do you have…? None (5) to Unable to do because of my vision (1) Reading (i.e. from a computer screen, street signs, bus numbers), cooking, finding things dropped on the floor, playing different sports Lighting (n = 15) How glaucoma and associated vision problems affect ability to do things in different lighting conditions None How much difficulty do you have…? None (5) to Unable to do because of my vision (1) Seeing under fluorescent or indoor lighting, driving in the day/ night, going down steps in dim lighting Mobility (n = 19) The impact of glaucoma and associated vision loss in moving around the community indepen- dently None How much difficulty do you have…? None (5) to Unable to do because of my vision (1) Walking (i.e. around unfamiliar areas, on uneven ground), notic- ing things or people to the left or right, getting on or off public transport Psychosocial (n = 55) Describes any concerns about or emotional reactions to hav- ing glaucoma and associated vision loss; as well as the impact of glaucoma on social life and personal relationships In the past 1 month… How concerned were you about…? How often did you feel…? Item generation summary At the conclusion of Phase 1, the number of unique items generated from four separate sources was 311 (Table 4, row 1 ‘initial pools’), comprising 77, 44, 158 and 32 items from eight papers, 11 glaucoma-specific questionnaires, patient focus groups, and expert feedback, respectively. Table 2 Items generated across four sources of content development (Phase 1) Table 2 Items generated across four sources of content development (Phase 1) Source of content development Number of novel items generated Qualitative (n = 4) & review articles (n = 4) 77 Validated patient reported outcome measures (n = 11) 44 Qualitative sessions with patients (n = 27 patients) 158 Qualitative sessions with experts (n = 4 experts) 32 Total 311 Results i The most commonly reported visual symptoms by our glaucoma patients were blurred vision and ‘block- ing’ of vision (i.e. a sense of obstruction, vision being cut off). Commonly mentioned ocular comfort symptoms were feeling like there was something in their eyes (i.e. a foreign body sensation) and a sticky sensation around eyelashes or eyelids. Some patients reported that admin- istering eyedrops was tiresome, while others worried whether their treatment plan was effective. Many patients found reading small print (e.g. letters or bills), using internet banking, walking on uneven ground and seeing people or objects coming towards them daunting due to their vision. Several patients reported that dim light- ing and/or glare affected their ability to perform daily activities. Difficulties reading and working on a computer screen for long hours impacted the work performance of some patients, which often strained work relationships with colleagues or supervisors. A universal fear reported by glaucoma patients was further loss of vision and even- tual blindness. Most patients also expressed safety con- cerns, like falling, tripping, or bumping into people or objects. Phase 1: domain and item generation Literature review Based on our literature review, 77 and 44 items within multiple domains of HRQoL were extracted from four relevant qualitative papers [18, 29–31] and four reviews [32–35], and 11 questionnaires or IBs [10, 11, 16, 18, 36– 42], respectively (Table 2). Phase 2: item refinement Not at all (5) to Extremely (1) None of the time (5) to All of the time (1) Falling, your eyesight getting worse, being burden to your family Frustrated, depressed, helpless, socially isolated Glaucoma management (n = 28) Difficulties and concerns sur- rounding glaucoma treatment, such as financial impact, difficul- ties in constantly administering eyedrops, etc None How difficult is…? How concerned are you about…? Not at all (5) to Extremely (1) Consistently administering the right amount of eyedrops, attending frequent appointments, the ongoing costs of glaucoma eyedrops Work (n = 13) Work performance and financial impact of glaucoma Currently… How much difficulty do you have…? How concerned are you about…? None (5) to Unable to do because of my vision (1) Not at all (5) to Extremely (1) Keeping up at work, having limita- tions on the types of job Fenwick et al. Journal of Patient-Reported Outcomes (2022) 6:107 Page 5 of 13 Fenwick et al. Journal of Patient-Reported Outcomes (including challenges and concerns relating to glaucoma treatment and attending appointments); and Work. long (n = 232 items), it was not feasible to test the entire set. As such, 30 questions with high potential for response errors were shortlisted by the study team for testing. The three most important HRQoL domains listed by our focus group patients were Ocular Comfort Symp- toms, Mobility and Psychosocial, with Activity limitation and Glaucoma Management also frequently mentioned. However, Driving was rarely listed by patients as impor- tant, most likely due to the low number of elderly people driving in Singapore; as such, we dissolved the Driv- ing domain (moving some items to other domains like Activity limitation or Lighting). This resulted in 261 items across eight domains (Table 4, row 2 ‘After domain ranking’). Themes for each HRQoL domain are briefly outlined below, with more information and supporting quotes provided in Additional file 2. Interviews were conducted in rounds of 3–4 partici- pants and feedback was iteratively incorporated after each round. Changes were re-tested in a new batch of participants until no new issues emerged, resulting in a total of 19 glaucoma patients completing the cogni- tive interviews. Finally, an additional 8 participants completed the full questionnaire (i.e. all 232 items) as a ‘dry-run’ until no new issues emerged, resulting in a final sample of 27 patients. Qualitative sessions Of the 27 patients (mean ± standard deviation [SD] age 67.9 ± 8.2; 48.1% male, 81.5% Chinese), who participated in the focus groups, nine (33.3%), five (18.5%), two (7.4%), eight (29.6%) and three (11.1%) had no, mild, moderate, severe, advanced/end-stage glaucoma in the better eye, respectively (Table 3). Most participants (n = 26, 96.3%) had received topical medication in at least one eye, with nine (33.3%) and 11 (40.7%) receiving laser and surgery (e.g., trabeculectomy, minimally invasive glaucoma sur- gery, aqueous shunts), respectively. Following thematic analysis, we isolated 311 unique items, across nine domains (Table 4, row 1 ‘initial pools’), namely Visual Symptoms; Ocular Comfort Symptoms; Activity Limitation; Driving; Lighting; Mobility; Psy- chosocial (including concerns, emotional reactions and social well-being); and Glaucoma Management Phase 2: item refinement Binning and winnowing Th h d The eight domains were evaluated during two sessions of binning and winnowing, during which, the expert panel decided to remove Visual Symptoms because the items were deemed to function more as a checklist than a latent construct and could be quickly captured during history taking. Certain items from the Visual Symptoms domain Fenwick et al. Phase 2: item refinement Binning and winnowing Th h d Journal of Patient-Reported Outcomes (2022) 6:107 Table 3 (continued) Variable N % Married 20 74.1% Divorced/separated/widowed 4 14.8% Highest education levela Primary 5 20.8% Secondary 11 45.8% A Level 3 12.5% Polytechnic/Diploma/ Vocational Training 2 8.3% University or higher 2 8.3% Employment status Working 20 74.1% Not working 7 25.9% Chronic health conditionsa Hypertension 10 37.0% Dyslipidaemia 12 44.4% Diabetes 5 18.5% Heart attack 1 3.73% Stroke 1 3.7% Continuous variables Mean SD Age (years) 67.9 8.2 Presenting VA (better eye), LogMAR; Snellen 0.11; 20/25 0.11; 20/25 Presenting VA (worse eye), LogMAR; Snellen 0.37; 20/40 0.21; 20/32 Visual fields (better eye), mean deviation − 10.36 9.69 Visual fields (worse eye), mean deviation − 16.83 9.74 No. of topical treatments within past 6 months 1.8 0.9 a Percentages for some variables may not equal 100% due to missing data or participants selecting > 1 category LogMAR logarithm of the minimal angle of resolution; NTG normal tension glaucoma; PACG primary angle closure glaucoma; POAG primary open angle glaucoma; SD standard deviation; SGD Singapore dollars; VA visual acuity a Percentages for some variables may not equal 100% due to missing data or participants selecting > 1 category LogMAR logarithm of the minimal angle of resolution; NTG normal tension glaucoma; PACG primary angle closure glaucoma; POAG primary open angle glaucoma; SD standard deviation; SGD Singapore dollars; VA visual acuity LogMAR logarithm of the minimal angle of resolution; NTG normal tension glaucoma; PACG primary angle closure glaucoma; POAG primary open angle glaucoma; SD tandard deviation; SGD Singapore dollars; VA visual acuity Table 4 The process of refining the initial item pools to the final pilot instrument (Phase 2) VS visual symptoms; OS ocular comfort symptoms; AL activity limitation; DV driving; LT lighting; MB mobility; PS psychosocial; GM glaucoma management; WK work VS OS AL DV LT MB PS GM WK Total Initial pools 18 21 73 18 10 22 108 18 23 311 After domain ranking 18 21 79 – 16 20 69 18 20 261 Binning & winnowing #1 15 19 79 – 16 20 69 16 20 254 Binning & winnowing #2 – 20 77 - 16 20 70 16 13 232 Cognitive interviews – 19 72 – 15 19 55 28 13 221 Phase 2: item refinement Binning and winnowing Th h d Journal of Patient-Reported Outcomes (2022) 6:107 Page 6 of 13 Table 3 Sociodemographic and clinical cha Variable Gender Male Age (Years) 40—49 50—59 60—69 69 < Ethnicity Chinese Malay Indian Duration of glaucoma (years) 0–2 3–5 6–10 11–15 > 15 Allergic reactions/side effects to medication Yes No Glaucoma type (per eye) POAG/ NTG PACG None Glaucoma severity (better eye) None Mild Moderate Severe Advanced/End-stage Glaucoma severity (worse eye) Mild Moderate Severe Advanced/End-stage Glaucoma treatments (in at least one eye) Topical medication Laser Surgery Vision impairment (better eye) None (≤ 0.3 LogMAR or ≤ 20/40 Snellen) Mild (> 0.3 LogMAR ≤ 0.48 or > 20/40 Snellen ≤ 20/ Moderate/severe (> 0.48 LogMAR or > 20/60 Snelle Vision impairment (worse eye) None (≤ 0.3 LogMAR or ≤ 20/40 Snellen) Mild (> 0.3 LogMAR ≤ 0.48 or > 20/40 Snellen ≤ 20/ Moderate/severe (> 0.48 LogMAR or > 20/60 Snelle Marital status Single s in Phase 1 N % 13 48.1% 1 3.7% 3 11.1% 12 44.4% 11 40.7% 22 81.5% 2 7.4% 3 11.1% 2 27.4% 10 37.0% 6 22.2% 5 18.5% 4 14.8% 10 37.0% 17 63.0% 33 61.1% 18 33.3% 3 5.6% 9 33.3% 5 18.5% 2 7.4% 8 29.6% 3 11.1% 4 14.8% 7 25.9% 10 37.0% 6 22.2% 26 96.3% 9 33.3% 11 40.7% 26 96.3% 1 3.7% 0 0.0% 15 55.6% 2 7.4% 10 37.0% 3 11.1% Table 3 Sociodemographic and clinical characteristics of the 27 participants in Phase 1 Variable N % Gender Male 13 48.1% Age (Years) 40—49 1 3.7% 50—59 3 11.1% 60—69 12 44.4% 69 < 11 40.7% Ethnicity Chinese 22 81.5% Malay 2 7.4% Indian 3 11.1% Duration of glaucoma (years) 0–2 2 27.4% 3–5 10 37.0% 6–10 6 22.2% 11–15 5 18.5% > 15 4 14.8% Allergic reactions/side effects to medication Yes 10 37.0% No 17 63.0% Glaucoma type (per eye) POAG/ NTG 33 61.1% PACG 18 33.3% None 3 5.6% Glaucoma severity (better eye) None 9 33.3% Mild 5 18.5% Moderate 2 7.4% Severe 8 29.6% Advanced/End-stage 3 11.1% Glaucoma severity (worse eye) Mild 4 14.8% Moderate 7 25.9% Severe 10 37.0% Advanced/End-stage 6 22.2% Glaucoma treatments (in at least one eye) Topical medication 26 96.3% Laser 9 33.3% Surgery 11 40.7% Vision impairment (better eye) None (≤ 0.3 LogMAR or ≤ 20/40 Snellen) 26 96.3% Mild (> 0.3 LogMAR ≤ 0.48 or > 20/40 Snellen ≤ 20/60) 1 3.7% Moderate/severe (> 0.48 LogMAR or > 20/60 Snellen) 0 0.0% Vision impairment (worse eye) None (≤ 0.3 LogMAR or ≤ 20/40 Snellen) 15 55.6% Mild (> 0.3 LogMAR ≤ 0.48 or > 20/40 Snellen ≤ 20/60) 2 7.4% Moderate/severe (>0 48 LogMAR or>20/60 Snellen) 10 37 0% Page 7 of 13 Fenwick et al. Comparison of GlauCAT™‑Asian and other PROMs used to measure HRQoL in glaucoma While Ocular Comfort Symptoms, Activity Limita- tion, Lighting, and Mobility were present in both the GlauCAT™-Asian and GlauCAT™-Western instruments [17, 18], the remaining domain structure differed (Fig. 1). For example, rather than having three separate domains for Emotional, Concerns and Social as in GlauCAT™- Western, GlauCAT™-Asian consolidated these items under a single ‘Psychosocial’ domain in an effort to streamline the instrument. Similarly, there is no Driving domain in GlauCAT™-Asian, reflecting the fact that few elderly people in Singapore drive.h The domain and item content of nine existing paper– pencil questionnaires used to measure HRQoL in glau- coma [10, 11, 36–42] and the glaucoma IB developed by Matsuura and colleagues[16] was also compared with our new GlauCAT™-Asian instrument (see Additional file 4). Overall, Activity Limitation, Lighting, Mobility, and Psy- chosocial were reasonably well represented, although the number of items with which to measure the domains in Our focus group discussions revealed many issues with ocular comfort, especially relating to treatment side effects, such as dry, red and tired eyes, a ‘sunken’ eye appearance, and stickiness and stains around eye- lids and lashes, some (but not all) of which have been reported in other studies [43]. While some instruments, such as the Glaucoma Symptom Scale (GSS) [36], and the Table 5 Examples of item modifications following the cognitive interview process Quality of life domain Item Type of change Reason for change Ocular comfort symptoms ‘How often have you experienced sunken eyes, or your eyes feeling sunken?’ Re-phrased Feedback suggested the question was hard to comprehend and the item stem was modified and rephrased for clarity Final item: ‘How much were you bothered by sunken eyes appearance?’ Activity limitation ‘How much difficulty do you have playing indoor sports, e.g. badminton, bowling, gym sessions, table tennis?’ Edited examples Participants’ suggestions on common types of indoor sports Activity limitation ‘How much difficulty do you have visually scan- ning a document for information?’ Re-phrased After several rounds of testing different wordings and phrasings, the question was modified to ‘How much difficulty do you have finding information in a document’ Lighting ‘How much difficulty do you have getting enough light to see?’ Deleted Patients found the question confusing Mobility ‘How much difficulty do you have seeing objects coming towards you, e.g. Discussion Following a robust development and refinement process, we generated 221 items across seven independent glau- coma-specific HRQoL domains. The qualitative sessions with patients were particularly productive for content generation and, while daily activity, mobility and light- ing limitations are well-known, issues relating to ocular comfort following treatment (e.g., stickiness around eye- lashes), glaucoma management (e.g. concern about hav- ing glaucoma surgery) and work (e.g. fear of job loss) are not well captured by paper–pencil glaucoma-specific HRQoL questionnaires. While four domains are com- mon to both GlauCAT™-Asian and GlauCAT™-Western instruments, the remaining domains and item content differs. Once our IBs are calibrated and operationalised via CAT, our new instrument will offer a comprehensive yet efficient measurement of glaucoma-specific HRQoL that is applicable to Asian patients, and will be of rel- evance to health professionals and researchers with an interest in value-based care. Cognitive interviews (e.g. difficulty telling the difference between similar tones and shades and difficulty with seeing haloes around lights at night) were moved to other domains, namely Activ- ity Limitation and Lighting. All remaining items were reviewed for importance, clarity and relevance after which they were either preserved, redirected to a differ- ent domain, or deleted. The number of items was even- tually reduced from 261 to 232 (Table 4, rows 3 & 4 ‘Binning & winnowing’). (e.g. difficulty telling the difference between similar tones and shades and difficulty with seeing haloes around lights at night) were moved to other domains, namely Activ- ity Limitation and Lighting. All remaining items were reviewed for importance, clarity and relevance after which they were either preserved, redirected to a differ- ent domain, or deleted. The number of items was even- tually reduced from 261 to 232 (Table 4, rows 3 & 4 ‘Binning & winnowing’). Of the 27 patients (mean ± SD age 65.8 ± 11.3; 59.3% male, 88.9% Chinese) who participated in the cogni- tive interviews, nine (33.3%), six (22.2%), six (22.2%), four (14.8%), and two (7.4%) had no, mild, moderate, severe, advanced/end-stage glaucoma in the better eye, respectively (see Additional file 3). Twenty-one patients (77.8%) had received topical medication in at least one Fenwick et al. Journal of Patient-Reported Outcomes (2022) 6:107 Fenwick et al. Journal of Patient-Reported Outcomes Page 8 of 13 eye, with seven (25.9%) and eight (29.6%) receiving laser or surgery in at least one eye, respectively. currently available PROMs was limited (median = 8.5, range 3–84). In contrast, Ocular Comfort Symptoms, Glaucoma Management and Work were largely under- represented, with the exception of the Matsuura item bank [16]. Based on the feedback from the cognitive interviews and dry-runs, the study team made several amendments (Table 5) including addition (n = 12), deletion (n = 23) and modification of items and response options (n = 10), resulting in a final 7-domain, 221-item item bank (Activ- ity Limitation, n = 72 Lighting, n = 15; Mobility, n = 19; Psychosocial, n = 55; Ocular Comfort Symptoms, n = 19; Glaucoma Management, n = 28; Work, n = 13) (Table 4, row 5 ‘Cognitive interviews’). Comparison of GlauCAT™‑Asian and other PROMs used to measure HRQoL in glaucoma A comprehensive and validated glaucoma-specific PROM is hence needed to better assess the treatment side effect-medication adher- ence relationship. in our focus groups, and which mirrors findings from other studies [53], was fear of falling. This important psy- chological burden has been linked with reduced mobility and physical activity levels and increased fall events [54, 55] in glaucoma patients and suggests that screening for, and developing interventions to minimize fear of falling, may result in important functional improvements for glaucoma patients. Unlike paper–pencil questionnaires that contain only a handful of items per domain, our HRQoL domains com- prise between 13–72 items each and, as such, are able to target the spectrum of patient ‘ability’ level. In the next stage of this multi-phase study, the items will be ranked in terms of relative difficulty in an item bank using Rasch analysis using data from a large patient sample across the spectrum of glaucoma type and severity. Items can then be administered using CAT, which applies an algorithm to administer the best-targeted items from the bank at each stage of the testing process [14], allowing precise estimates of HRQoL to be calculated with relatively few items (depending on the desired measurement precision) [56]. This results in time savings of up to 80% compared to administering equivalent paper–pencil questionnaires [57]. Each glaucoma HRQoL item bank will function independently allowing users to select relevant domains for their sample population (e.g. Glaucoma Management may be most relevant to patients on treatment). However, even if some items are not relevant (e.g., patients not on topical medication cannot answer items about eyedrops), CATs can avoid presenting these items without biasing the overall score. This is a clear advantage over paper– pencil questionnaires, where patients must answer every item regardless of applicability. While the content of the Glaucoma Management domain (GlauCAT™-Asian) and the Convenience-Treat- ment domain (GlauCAT™-Western) is similar, the Glau- coma Management domain has twice the number of items and covers a broader range of issues. For example, it contains multiple items relating to difficulty admin- istering eyedrops, an issue that has been commonly reported in the literature [29, 47], and one that has been associated with decrements in vision-related HRQoL [48] and non-adherence to medications [46]. Comparison of GlauCAT™‑Asian and other PROMs used to measure HRQoL in glaucoma cars, bikes, scooters?’ Added Patient suggestion Psychosocial ‘In the past 4 weeks, how often did you feel like you wanted to give up on your glaucoma treat- ment?’ Added A constant theme noticed in patients who have been diagnosed with glaucoma for several years Table 5 Examples of item modifications following the cognitive interview process Quality of life domain Item Type of chan Table 5 Examples of item modifications following the cognitive interview process Patient suggestion A constant theme noticed in patients who have been diagnosed with glaucoma for several years Fenwick et al. Journal of Patient-Reported Outcomes (2022) 6:107 Page 9 of 13 Fenwick et al. Journal of Patient-Reported Outcomes Ocular Comfort Symptoms OS (n=19) Mobility MB (n=19) Psychosocial PS (n=55) Work WK (n=13) Glaucoma Management GM (n=28) Mobility MB (n=20) Emotional EM (n=45) Lighting LT (n=9) Treatment Convenience CV-T (n=14) Activity Limitation AL (n=58) GlauCATTM - Asian GlauCATTM - Western Activity Limitation AL (n=72) Lighting LT (n=15) Ocular Comfort Symptoms OS (n=22) Visual Symptoms VS (n=18) Social SC (n=14) Concerns HC (n=45) Driving DV (n=13) Economic EC (n=22) General Convenience CV-T (n=23) Fig. 1 Head-to-head comparison of the domain structure of the new GlauCAT™-Asian and GlauCAT™-Western instruments. This figure shows that four domains—OS, AL, MB and LT—are the same across the two instruments, while the number and content of the remaining domains differs GlauCATTM - Asian GlauCATTM - Western Glaucoma Management GM (n=28) Treatment Convenience CV-T (n=14) General Convenience CV-T (n=23) Work WK (n=13) Fig. 1 Head-to-head comparison of the domain structure of the new GlauCAT™-Asian and GlauCAT™-Western instruments. This figure shows that four domains—OS, AL, MB and LT—are the same across the two instruments, while the number and content of the remaining domains differs Page 10 of 13 Page 10 of 13 Fenwick et al. Journal of Patient-Reported Outcomes (2022) 6:107 Fenwick et al. Journal of Patient-Reported Outcomes Comparison of Ophthalmic Medications for Tolerability (COMTOL) scale [41] contain a handful of items relat- ing to glaucoma-specific symptoms, only the GlauCAT™- Western previously developed by our group [17, 18] covers a similar breath of issues. This is important as there may be an association between worse patient- reported side effects and non-adherence to glaucoma medications; however, evidence is equivocal [29, 44–46], which may be due to the lack of an appropriate tool to adequately assess this relationship. Comparison of GlauCAT™‑Asian and other PROMs used to measure HRQoL in glaucoma This domain also comprises items relating to concerns about having to undergo glaucoma surgery or laser treatment, as well as the financial burden associated with ongoing topical medication use and/or surgery/laser; this is pertinent as inability to afford treatment is a known barrier to adher- ence [49]. An in-depth and holistic understanding of glaucoma treatment burden from the patient’s perspec- tive using a comprehensive PROM is crucial for clinicians delivering patient-centred care and to improve patient- centred and clinical outcomes. In future, the final glaucoma CAT instrument will be able to measure glaucoma-specific HRQoL cross-sec- tionally, as well as monitor changes over time, for exam- ple pre-/post-treatment interventions (surgery, changes in medication regimens) or at routine clinical appoint- ments (e.g., real-world setting). It will be relevant for clinical research studies or trials as a primary or sec- ondary endpoint to measure the magnitude of HRQoL impact in patients across the spectrum of glaucoma, with or without associated VI, and related treatment for glau- coma to support market application. The CATs will be administered on an internet-enabled digital device and will be compliant with accessibility standards for visu- ally impaired patients and the technical requirements of international data security regulatory bodies. While work-related issues were reported by some of our focus group participants, this has not been widely reported elsewhere, likely because glaucoma is an age- related condition affecting most people post-retirement. However, with many Singaporeans working well into their 60 s, 25% of our focus group participants (mean age 68 years) were still currently working either part- or full- time. Indeed, work-related issues relating to glaucoma may continue to increase as Singapore plans to raise retirement and re-employment age to 65 and 70, respec- tively by 2030 [50]. As such, we expect our Work domain to become progressively more relevant in assessing the HRQoL issues that glaucoma patients will invariably face as the workforce ages.i Our finding that glaucoma impacts on daily living activities like reading and getting out and about, espe- cially in challenging lighting conditions [51], is well sub- stantiated in the literature [52], and is reflected by the fact that both GlauCAT™ instruments contain these fun- damental HRQoL domains. Another key theme reported Our substantial qualitative component including 54 patients and four experts is a key strength of our study, as is the psychometric expertise and CAT experience of the development team [56]. Author details 1 Singapore National Eye Centre, Singapore Eye Research Institute (SERI), The Academia, 20 College Road, Level 6, Singapore 169856, Singapore. 2 Duke– NUS Medical School, National University of Singapore, Singapore, Singapore. 3 National University Health System, Singapore, Singapore. 4 The University of Melbourne, Melbourne, Australia. 5 Medical and Vision Research Foundation, Sankara Nethralaya, Chennai, India. Comparison of GlauCAT™‑Asian and other PROMs used to measure HRQoL in glaucoma We used a systematic and accepted item reduction process [25], and were guided by Fenwick et al. Journal of Patient-Reported Outcomes (2022) 6:107 Page 11 of 13 Page 11 of 13 empirical evidence [27] when generating item stems and response options. Our thorough pre-testing process via cognitive interviewing is also a strength [58]. However, as it was not feasible to conduct in-depth interviews on all 221 items, most were only pre-tested eight times; as such, it is possible that potential issues were missed. Our pur- posive sampling technique may have introduced selection bias; however, our aim was to obtain detailed information from sub-groups of interest rather from a representa- tive population-based sample. We had fewer Indian and Malay participants compared to Chinese and therefore may have missed culturally-specific content; however, we will raise recruitment numbers for these minority groups in subsequent phases. While our new instru- ment has been developed in Asian patients, these were limited to one country, Singapore; as such, the content may not be applicable to patients residing in other parts of Asia. Further work to test the cultural appropriateness of GlauCAT™-Asian is required. Finally, we did not gain qualitative feedback from carers who may have provided valuable information on the burden of glaucoma. Acknowledgementsi The first draft of the manuscript was written by Eva Fenwick and Belicia Lim and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Author contributions Study conception and design was performed by EF, BL, RM, and EL. Material preparation, data collection and analysis were performed by EF, BL and RM, MB, MN, JVI, JRL, and EL. All authors read and approved the final manuscript. Availability of data and materials Availability of data and materials Data reported in this study are available upon reasonable request. y Data reported in this study are available upon reasonable request Ethics approval and consent to participate This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by SingHealth Centralised Institutional Review Board (CIRB #2018/2459). Written informed consent from participants was obtained prior to study commencement. Supplementary Information Received: 13 April 2022 Accepted: 16 September 2022 Received: 13 April 2022 Accepted: 16 September 2022 The online version contains supplementary material available at https://doi. org/10.1186/s41687-022-00513-3. The online version contains supplementary material available at https://doi. org/10.1186/s41687-022-00513-3. Additional file 1. Moderator’s guide for the focus groups. Open-ended questions were asked about how glaucoma, vision loss, and associated glaucoma treatments affected various areas of patients’ health-related quality of life, including activity limitation, symptoms, emotional well- being, social life, and work. Consent for publicationfi We generated 221 items across seven independent glau- coma-specific HRQoL domains. Once operationalised by CAT, our GlauCAT™-Asian instrument will be useful for clinicians to better understand the impact of glaucoma on patients’ HRQoL, especially once it is fully imple- mented into routine clinical care and integrated with patients’ electronic medical records; and for researchers to assess the patient-centred impact of novel glaucoma treatment therapies or models of care. The authors affirm that human research participants provided informed con- sent for publication of the data in Table 3 and Additional file 3. The authors have no relevant financial or non-financial interests to disclose. The authors have no relevant financial or non-financial interests to disclose. Declarations Ethics approval and consent to participate Funding This study was funded by a National Medical Research Council Health Services Research Grant (PI Lamoureux, and Co-Is Fenwick, Man, Baskaran, and Aung, #0070/2016). 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https://openalex.org/W4206522674	https://vuzbiochemi.elpub.ru/jour/article/download/716/407	Russian	null	Influence of mechanochemical activation on dissolving model corrosion films formed on ion-exchange resins using Trilon B	Izvestiâ vuzov. Prikladnaâ himiâ i biotehnologiâ	2,022	cc-by	7,457	1© Паламарчук М. С., Шлык Д. Х., Братская С. Ю., 2021 https://vuzbiochemi.elpub.ru/jour ИЗВЕСТИЯ ВУЗОВ. ПРИКЛАДНАЯ ХИМИЯ И БИОТЕХНОЛОГИЯ 2021 Том 11 N 4 PROCEEDINGS OF UNIVERSITIES. APPLIED CHEMISTRY AND BIOTECHNOLOGY 2021 Vol. 11 No. 4 ИЗВЕСТИЯ ВУЗОВ. ПРИКЛАДНАЯ ХИМИЯ И БИОТЕХНОЛОГИЯ 2021 Том 11 N 4 PROCEEDINGS OF UNIVERSITIES. APPLIED CHEMISTRY AND BIOTECHNOLOGY 2021 Vol. 11 No. 4 ХИМИЧЕСКАЯ ТЕХНОЛОГИЯ Для цитирования: Паламарчук М. С., Шлык Д. Х., Братская С. Ю. Влияние механохимической акти- вации на растворение трилоном Б модельных отложений продуктов коррозии, образованных на ионо- обменных смолах // Известия вузов. Прикладная химия и биотехнология. 2021. Т. 11. N 4. С. 663–672. https://doi.org/10.21285/2227-2925-2021-11-4-663-672. Влияние механохимической активации на растворение трилоном Б модельных отложений продуктов коррозии, образованных на ионообменных смолах Марина Сергеевна Паламарчук, Дарья Хамитовна Шлык, 1 Светлана Юрьевна Братская Институт химии ДВО РАН, г. Владивосток, Российская федерация Автор, ответственный за переписку: Паламарчук Марина Сергеевна, marina_p@ich.dvo.ru Финансирование. Работа выполнена при финансовой поддержке Российского научного фонда (проект 18-73-10066, https://rscf.ru/project/18-73-10066/). Influence of mechanochemical activation on dissolving model corrosion films formed on ion-exchange resins using Trilon B Marina S. Palamarchuk, Darya Kh. Shlyk, Svetlana Yu. Bratskaya Institute of Chemistry FEB RAS, Vladivostok, Russian Federation Corresponding author: Marina S. Palamarchuk, marina_p@ich.dvo.ru Abstract. Inorganic deposits formed during operation and intermediate storage contain radionuclides, whose removal during the chemical decontamination of spent ion-exchange resins used in filters for special water purification at nuclear power plants has proved to be a challenge. In such deposits, radionuclides of the cor- rosion group (58.60Co, 54Mn, 51Cr) are typically located in the crystal lattice of poorly soluble iron oxides. The present work discusses the possibility of using mechanochemical activation in the decontamination of spent ion-exchange resins polluted with deposits of activated corrosion products from structural materials. Samples of natural and synthesised on the surface of the KU-2-8 cation exchanger in the presence of the 57Co label magnetites were used as model deposits. It was shown that an increase in the duration of mechanochemical activation leads to an increase in the dissolution rate of magnetite in model decontamina- tion solutions based on еthylenediaminetetraacetic acid disodium salt (Trilon B) and nitric acid. It was shown that, when using Trilon B, magnetite dissolves more efficiently, which is explained by the interaction between the oxide surface and organic complexing agents. It can be assumed that solid-phase reactions occur during the mechanochemical activation of magnetite in the presence of dry reagents (Trilon B, oxalic, ascorbic and citric acids). Therefore, a poorly soluble shell formed on the oxide surface hinders the dissolution at a low magnetite/solution ratio. Unlike the reagent-free activation, for magnetite activated in the presence of oxalic acid, an increase in the solution/magnetite ratio promotes the dissolution of iron oxides. Using the example of a model cation exchanger, it was shown that the rate and efficiency of decontamination of spent ion- exchange resins polluted with deposits containing activated corrosion products increase significantly after mechanochemical activation in the presence of oxalic acid. eywords: spent ion-exchange resins, cobalt radionuclides, corrosion products, iron oxides, decon exchange resins, cobalt radionuclides, corrosion products, iron oxides, decontamination cknowledgements. XRD investigations and atomic absorption analysis were conducted using Far East Center of Structural Studies (Institute of Chemistry, FEB RAS, Vladivostok, Russia). Funding. The study was financially supported by the Russian Science Foundation (project 18-73-10066P, https://rscf.ru/project/18-73-10066/). Funding. The study was financially supported by the Russian Science Foundation (project https://rscf.ru/project/18-73-10066/). Funding. The study was financially supported by the Russian Science Foundation (project 18-73-10066P, https://rscf.ru/project/18-73-10066/). Марина Сергеевна Паламарчук, Дарья Хамитовна Шлык, 1 Светлана Юрьевна Братская Институт химии ДВО РАН, г. Владивосток, Российская федерация Автор, ответственный за переписку: Паламарчук Марина Сергеевна, marina_p@ich.dvo.ru Марина Сергеевна Паламарчук, Дарья Хамитовна Шлык, 1 Светлана Юрьевна Братская Институт химии ДВО РАН, г. Владивосток, Российская федерация Автор, ответственный за переписку: Паламарчук Марина Сергеевна, marina Аннотация. При химической дезактивации отработанных ионообменных смол фильтров спецво- доочистки атомных электростанций возникает проблема удаления из них радионуклидов, входя- щих в состав неорганических отложений, образованных в процессе эксплуатации и промежуточ- ного хранения. Как правило, в таких отложениях радионуклиды коррозионной группы (58,60Co, 54Mn, 51Сr) зафиксированы в кристаллической решетке труднорастворимых оксидов железа. Цель рабо- ты – исследование возможности применения механохимической активации при дезактивации от- работанных ионообменных смол, загрязненных отложениями активированных продуктов коррозии конструкционных материалов. В качестве модельных отложений использован образец природного магнетита и магнетит, синтезированный на поверхности катионита КУ-2-8 в присутствии метки 57Со. Показано, что увеличение времени механохимической активации приводит к увеличе- нию скорости растворения магнетита в модельных дезактивирующих растворах на основе дина- триевой соли этилендиаминтетрауксусной кислоты (трилон Б) и азотной кислоты. Показано, что при использовании трилона Б магнетит растворяется более эффективно, что объясняется особенностями взаимодействия поверхности оксида с органическими комплексообразующими агентами. Предположено, что при механохимической активации магнетита в присутствии сухих реагентов (трилона Б, щавелевой, аскорбиновой и лимонной кислот) протекают твердофазные реакции, приводящие к образованию на поверхности оксида труднорастворимой оболочки, что затрудняет растворение при невысоких соотношениях магнетит/раствор. Для магнетита, ак- тивированного в присутствии щавелевой кислоты, показано, что увеличение отношения рас- твор/магнетит способствует растворению оксидов железа по сравнению с безреагентной акти- вацией. На примере модельного катионита показано, что скорость и эффективность дезактива- ции отработанных ионообменных смол, загрязненных отложениями активированных продуктов коррозии, существенно возрастает после механохимической активации в присутствии щавелевой кислоты. Ключевые слова: отработанные ионообменные смолы, радионуклиды кобальта, продукты корро- зии, оксиды железа, дезактивация Благодарности. Рентгенофазовый анализ и определение содержания железа методом атомно- абсорбционной спектроскопии выполнены на оборудовании ЦКП «Дальневосточный центр струк- турных исследований» (Институт химии ДВО РАН, Владивосток, Россия). Финансирование. Работа выполнена при финансовой поддержке Российского научного фонда (проект 18-73-10066, https://rscf.ru/project/18-73-10066/). Финансирование. Работа выполнена при финансовой (проект 18-73-10066, https://rscf.ru/project/18-73-10066/). Для цитирования: Паламарчук М. С., Шлык Д. Х., Братская С. Ю. Влияние механохимической акти- вации на растворение трилоном Б модельных отложений продуктов коррозии, образованных на ионо- обменных смолах // Известия вузов. Прикладная химия и биотехнология. 2021. Т. 11. N 4. С. 663–672. https://doi.org/10.21285/2227-2925-2021-11-4-663-672. 663 https://vuzbiochemi.elpub.ru/jour CHEMICAL TECHNOLOGY Influence of mechanochemical activation on dissolving model corrosion films formed on ion-exchange resins using Trilon B For citation: Palamarchuk M. S., Shlyk D. Kh., Bratskaya S. Yu. Influence of mechanochemical activation on dissolving model corrosion films formed on ion-exchange resins using Trilon B. Izvestiya Vuzov. Priklad- naya Khimiya i Biotekhnologiya = Proceedings of Universities. Applied Chemistry and Biotechnology. 2021;11(4):663-672. (In Russian). https://doi.org/10.21285/2227-2925-2021-11-4-663-672. https://vuzbiochemi.elpub.ru/jour Паламарчук М. С., Шлык Д. Х., Братская С. Ю. Влияние механохимической активации Palamarchuk M. S., Shlyk D. Kh., Bratskaya S. Yu. Influence of mechanochemical activation ВВЕДЕНИЕ ально подобранными растворами с переводом ОИОС в нерадиоактивные отходы [7]. Однако при дезактивации ОИОС возникает проблема удаления из них радионуклидов, входящих в со- став неорганических отложений, образованных в процессе эксплуатации и промежуточного хра- нения смол [8, 9]. Значительную проблему пред- ставляет загрязнение смол активированными продуктами коррозии (ПК), вынесенными из ак- тивной зоны в виде частиц, в которых радио- нуклиды коррозионной группы (58,60Co, 54Mn, 51Сr) зафиксированы в кристаллической решетке труднорастворимых оксидов железа [10, 11]. ально подобранными растворами с переводом ОИОС в нерадиоактивные отходы [7]. Однако при дезактивации ОИОС возникает проблема удаления из них радионуклидов, входящих в со- став неорганических отложений, образованных в процессе эксплуатации и промежуточного хра- нения смол [8, 9]. Значительную проблему пред- ставляет загрязнение смол активированными продуктами коррозии (ПК), вынесенными из ак- тивной зоны в виде частиц, в которых радио- нуклиды коррозионной группы (58,60Co, 54Mn, 51Сr) зафиксированы в кристаллической решетке труднорастворимых оксидов железа [10, 11]. Выведенные из эксплуатации отработанные ионообменные смолы (ОИОС) из фильтров спецводоочистки относятся к радиоактивным отходам и нуждаются в кондиционировании, то есть мероприятиях, направленных на иммобили- зацию радионуклидов при долговременном хра- нении (захоронении) [1]. В настоящее время предложен ряд методов кондиционирования ОИОС от прямого отверждения с включением в неорганические и органические компаунды (це- мент, битум, пластмассы) [2, 3] до переработки с полной деструкцией органической матрицы смол (сжигание, пиролиз, жидкофазное окисление) [4–6]. Весьма перспективным является подход, вклю- чающий глубокую дезактивацию смол, то есть отмывку органической матрицы ОИОС специ- Этилендиаминтетрауксусная кислота (ЭДТА) является аминополикарбоксилатным комплексо- ном, образующим устойчивые комплексы с ра- дионуклидами коррозионной группы и ионами https://vuzbiochemi.elpub.ru/jour 664 Паламарчук М. С., Шлык Д. Х., Братская С. Ю. Влияние механохимической активации Palamarchuk M. S., Shlyk D. Kh., Bratskaya S. Yu. Influence of mechanochemical activation раствора (Aк+р). После измерений раствор воз- вращали в емкость и продолжали эксперимент. Активность катионита, Aк, определяли по формуле раствора (Aк+р). После измерений раствор воз- вращали в емкость и продолжали эксперимент. Активность катионита, Aк, определяли по формуле железа. Так, логарифм константы устойчивости комплексов Со-ЭДТА достигает 16 и 41 для Со(II) и Co(III), а комплексов Fe-ЭДТА – 14 и 25,7 для Fe(II) и Fe(III) соответственно. Благодаря этому ЭДТА и ее соли (главным образом, трилон Б) широко используются в атомной энергетике в со- ставе дезактивирующих растворов для удаления продуктов коррозии с конструкционных материалов и механически загрязненных катионитов. к р+к р. ВВЕДЕНИЕ A A A   (1) (1) Степень дезактивации катионита рассчиты- вали по формуле В последнее время для переработки отходов все чаще предлагаются методы, включающие механохимическую активацию (МХА) [12–16]. В процессе МХА может происходить снижение размера частиц, образование новых поверхно- стей, точечных дефектов, изменение кристалли- ческой структуры [14, 15]. В настоящей работе исследовано влияние МХА на растворимость модельных железооксидных отложений в мо- дельных дезактивирующих растворах, содержа- щих трилон Б. к 0 % (1 ( / )) 100, S A A    (2) (2) где Aк – активность образца, Бк; A0 – исходная активность образца, Бк. Перед отбором проб для анализа проводили центрифугирование при скорости 2000 об./мин в течение 2 мин с использованием центрифуги МТ-45 (Hangzhou MIU Instruments Co Ltd., КНР). Содержание железа в растворах определяли атомно-абсорбционным методом на спектромет- ре Solaar M6 (Thermo Scientific, США). Актив- ность радионуклидов 57Со определяли с исполь- зованием гамма-бета-спектрометра МКС-АТ1315 (УП «Атомтех», Беларусь). Рентгенограммы об- разцов записывали на дифрактометре D8 ADVANCE (Bruker, Германия), идентификацию кристаллических фаз проводили с использова- нием программы Qualx2.0 [17] и открытой кри- сталлографической базы (COD, SQLITE3). Рас- пределение по размеру и медианное значение размера частиц (d 0,5) после ультразвуковой об- работки определяли методом лазерной дифрак- ции на анализаторе Mastersizer 2000 с модулем Hydro 2000S (Malvern, Великобритания). ЭКСПЕРИМЕНТАЛЬНАЯ ЧАСТЬ В работе использовали концентрат природ- ного магнетита и синтетический магнетит, нане- сенный на поверхность катионита КУ-2-8. Синтез магнетита на поверхности катионита проводили в присутствии метки 57Со по методике, описан- ной в работе [9]. Затем катионит перенесли в колонку и обработали раствором 4 М азотной кислоты и раствором, содержащим нитрат натрия и гидроксид натрия (2,25 и 0,75 М соот- ветственно) до удаления всех обменно- связанных с матрицей катионита радионуклидов с установлением постоянной удельной активно- сти, не снижаемой последующими обработками. После отмывки дистиллированной водой катио- нит обезвоживали в течение 5 ч в сушильном шкафу при 120 ºС. Удельная активность полу- ченного катионита составила 154 Бк/г. РЕЗУЛЬТАТЫ И ИХ ОБСУЖДЕНИЕ Наибольшая разница в относительном содержа- нии железа в растворах трилона Б и азотной кислоты наблюдалась на третьи сутки экспери- мента: в 155 раз для 10 мин МХА с плавным снижением до разницы в 80 раз для 90 мин МХА. Такое различие в относительном содержании железа объясняется различием механизмов рас- творения оксида железа в зависимости от соста- ва раствора. В присутствии минеральных кислот (азотной, серной) растворение происходит глав- ным образом при участии протонов, в то время как в присутствии органических лигандов на рас- творение в основном влияют процессы комплек- сообразования и переноса заряда [19]. факторов: с одной стороны, снижением размера частиц, с другой – усилением процессов агрега- ции мелкодисперсной фракции магнетита. При использовании 3 М азотной кислоты происходит постепенное возрастание концентрации железа в растворе, изломов на кинетических кривых не наблюдается. После 60 суток контакта в азотно- кислый раствор переходит в 3–4 раза больше ионов железа, чем в раствор трилона Б, однако, относительное содержание железа, отнесенное к количеству молей кислоты, значительно ниже, чем в эксперименте с трилоном Б (рис. 3, b). Наибольшая разница в относительном содержа- нии железа в растворах трилона Б и азотной кислоты наблюдалась на третьи сутки экспери- мента: в 155 раз для 10 мин МХА с плавным снижением до разницы в 80 раз для 90 мин МХА. Такое различие в относительном содержании железа объясняется различием механизмов рас- творения оксида железа в зависимости от соста- ва раствора. В присутствии минеральных кислот (азотной, серной) растворение происходит глав- ным образом при участии протонов, в то время как в присутствии органических лигандов на рас- творение в основном влияют процессы комплек- сообразования и переноса заряда [19]. рение реального размера частиц, однако плав- ное снижение медианного размера частиц с уве- личением времени МХА показывает, что процесс измельчения магнетита продолжается. Рис. 1. Рентгенограммы магнетита: исходный образец (1); образцы после МХА в течение 10 (2), 20 (3), 30 (4) и 60 (5) мин Fig. 1. X-ray diffraction patterns of magnetite: initial sample (1); samples after MCA for 10 (2), 20 (3), 30 (4), and 60 (5) minutes Рис. 1. Рентгенограммы магнетита: исходный образец (1); образцы после МХА в течение 10 (2), 20 (3), 30 (4) и 60 (5) мин Fig. 1. X-ray diffraction patterns of magnetite: initial sample (1); samples after MCA for 10 (2), 20 (3), 30 (4), and 60 (5) minutes Согласно литературным данным, процесс растворения оксидов железа в присутствии ор- ганических лигандов включает комплексообра- зование в растворе и на поверхности. РЕЗУЛЬТАТЫ И ИХ ОБСУЖДЕНИЕ В качестве модельных отложений, вынесенных на ОИОС из активной зоны, был использован об- разец природного магнетита Fe3O4. Рентгенограм- ма образца приведена на рис. 1 (спектр 1). Можно увидеть, что магнетит содержит примеси гемати- та Fe2O3, обедненных по титану ильменитов состава Fe1+xTi1-xO3 и шпинелоидов состава Fe3-xSixO4. Уже после 10 мин МХА наблюдается снижение рефлексов примесей (спектр 2), а при увеличении времени обработки до 30 мин проис- ходит полная аморфизация примесей, о чем свидетельствует изменение соотношения пиков в спектрах: после 30–60 мин МХА наблюдаются дифракционная картина, характерная для чи- стых фаз магнетита и гематита. МХА образцов оксида и катионита проводили в вертикальной планетарной мельнице (Сhangsha Tianchuang Powder Technology Co Ltd., КНР) при скорости 800 об./мин, материал стаканов и ша- ров – карбид вольфрама, диаметр шара – 10 мм, масса шара – 7 г. Эксперименты по растворению и дезактива- ции проводили в полипропиленовых емкостях с герметичной крышкой в статических условиях при перемешивании на шейкере. Растворение оксида проводили при соотношении Ж:Т, равном 100, 250, 320 и 500 мл/г (для образцов, получен- ных после МХА с реагентами, объем раствора относили к исходной массе оксида в смеси с реа- гентом). Для определения содержания железа в растворе отбирали аликвоты объемом 0,2 мл. Дезактивацию катионита приводили при соотно- шении раствор/катионит – 50 мл/г. Для опреде- ления активности отбирали пипеткой половину объема раствора и измеряли активность раство- ра (Aр), а также оставшейся смеси катионита и Анализ распределения частиц магнетита в зависимости от продолжительности МХА пока- зал, что в первые 10 мин происходит наиболее резкое снижение медианного размера (d 0,5), в дальнейшем наряду с увеличением доли частиц с размером менее 10 мкм, появляются крупные вторичные агрегаты (рис. 2). Такое поведение, характерное для мелкодисперсного магнетита и описанное в литературе [18], затрудняет изме- https://vuzbiochemi.elpub.ru/jour 665 Паламарчук М. С., Шлык Д. Х., Братская С. Ю. Влияние механохимической активации Palamarchuk M. S., Shlyk D. Kh., Bratskaya S. Yu. Influence of mechanochemical activation факторов: с одной стороны, снижением размера частиц, с другой – усилением процессов агрега- ции мелкодисперсной фракции магнетита. При использовании 3 М азотной кислоты происходит постепенное возрастание концентрации железа в растворе, изломов на кинетических кривых не наблюдается. После 60 суток контакта в азотно- кислый раствор переходит в 3–4 раза больше ионов железа, чем в раствор трилона Б, однако, относительное содержание железа, отнесенное к количеству молей кислоты, значительно ниже, чем в эксперименте с трилоном Б (рис. 3, b). РЕЗУЛЬТАТЫ И ИХ ОБСУЖДЕНИЕ Kh., Bratskaya S. Yu. Influence of mechanochemical activation … Паламарчук М. С., Шлык Д. Х., Братская С. Ю. Влияние механохимической активации … Palamarchuk M. S., Shlyk D. Kh., Bratskaya S. Yu. Influence of mechanochemical activation … a b Рис. 3. Относительное содержание железа в 0,02 М раствора трилона Б (а) и 3 М раствора HNO3 (b) в зависимости от времени растворения образцов: исходного магнетита (1) и магнетита после МХА в течение 10 (2), 20 (3), 30 (4), 60 (5) и 90 (6) минут. Ж:Т – 250 мл/г b a a Рис. 3. Относительное содержание железа в 0,02 М раствора трилона Б (а) и 3 М раствора HNO3 (b) в зависимости от времени растворения образцов: исходного магнетита (1) и магнетита после МХА в течение 10 (2), 20 (3), 30 (4), 60 (5) и 90 (6) минут. Ж:Т – 250 мл/г Fig. 3. Relative iron content in 0.02 M solution of Trilon B (a) and 3 M solution of HNO3 (b) depending on the dissolution time of the samples: initial magnetite (1) and magnetite after MCA Таблица 1. Относительная концентрация железа, перешедшего в 0,05 М растворы кислот при растворении магнетита, активированного в течение 30 мин (Ж:Т – 100 мл/г) Таблица 1. Относительная концентрация железа, перешедшего в 0,05 М растворы кислот при растворении магнетита, активированного в течение 30 мин (Ж:Т – 100 мл/г) Table 1. Relative concentration of iron transferred to 0.05 M acids solutions at dissolution of magnetite activated for 30 minutes (L:S – 100 ml/g) Table 1. Relative concentration of iron transferred to 0.05 M acids solutions at dissolution of magnetite activated for 30 minutes (L:S – 100 ml/g) Кислота рНисх Железо/кислота, моль/моль t, сут. 1 3 7 10 20 30 Щавелевая 1,55 0,584 0,610 0,641 0,650 0,603 0,599 Аскорбиновая 2,78 0,148 0,340 0,455 0,510 0,524 0,529 Лимонная 2,22 0,033 0,063 0,097 0,131 0,165 0,227 повышению эффективности растворения оксидов в присутствии трилона Б. В качестве активирую- щих добавок использовали сухие реагенты – ща- велевую, аскорбиновую, лимонную кислоты, а так- же трилон Б (табл. 2). Исходя из результатов эксперимента по рас- творению оксида кислотами было сделано пред- положение, что при МХА в присутствии органиче- ских кислот, добавленных в виде сухих реагентов, возможны твердофазные реакции, приводящие к Таблица 2. Относительная концентрация железа, перешедшего в 0,02 М раствор трилона Б при растворении магнетита, активированного в течение 30 мин Таблица 2. РЕЗУЛЬТАТЫ И ИХ ОБСУЖДЕНИЕ Адсорб- ция, в результате которой образуется поверх- ностный комплекс, ослабляет связь металл– кислород на поверхности кристаллической ре- шетки. Кроме того, комплексообразование с по- верхностным Fe(III) облегчает перенос электро- нов между растворенным Fe(II) и поверхностным Fe(III) и способствует растворению поверхност- ного Fe(III) [20]. Эксперимент по растворению активированного магнетита органическими кис- лотами показывает, что эффективность раство- рения снижается в ряду щавелевая кислота > аскорбиновая > лимонная (табл. 1). Константы устойчивости комплексов железа со щавелевой и лимонной кислотами (log K1) различаются не- значительно для Fe(II)/Fe(III) и составляют 4,7/9,4 и 3,2/11,85 соответственно. Существен- ное различие в растворении магнетита объясня- ется не только химией растворов этих кислот (диссоциация и комплексообразование), но и влиянием их структурных различий на поверх- ностное комплексообразование: щавелевая кис- лота занимает меньшую площадь на поверхно- сти оксида, поэтому концентрация поверхност- ных комплексов выше [22], а в отношении аскор- биновой кислоты преобладает механизм восста- новительного растворения (для щавелевой он дополняет поверхностное комплексообразова- ние). Поэтому в щавелевой и аскорбиновой кис- лотах растворение протекает с большей эффек- тивностью [23]. Рис. 2. Распределение и медианное значение размера частиц (врезка) магнетита: исходный образец (1); образцы после МХА в течение 10 (2), 20 (3), 30 (4), 60 (5) и 90 (6) мин Fig. 2. Distribution and median values of the particle size (insert) for magnetite after MCA for 10 (2), 20 (3), 30 (4), 60 (5), and 90 (6) minutes Рис. 2. Распределение и медианное значение размера частиц (врезка) магнетита: исходный образец (1); образцы после МХА в течение 10 (2), 20 (3), 30 (4), 60 (5) и 90 (6) мин Fig. 2. Distribution and median values of the particle size (insert) for magnetite after MCA for 10 (2), 20 (3), 30 (4), 60 (5), and 90 (6) minutes На рис. 3, а представлены результаты экспе- римента по растворению магнетита трилоном Б до и после МХА. Влияние МХА выражается в резком возрастании концентрации железа в рас- творе пропорционально времени МХА в первые трое суток эксперимента. Данный эффект свя- зан, вероятнее всего, с разрушением труднорас- творимых примесных фаз, содержащих титан и кремний. Далее в наклоне кинетических кривых не обнаруживается зависимости от времени МХА для активированных образцов. Это можно объяснить разнонаправленным влиянием двух https://vuzbiochemi.elpub.ru/jour 666 Паламарчук М. С., Шлык Д. Х., Братская С. Ю. Влияние механохимической активации … Palamarchuk M. S., Shlyk D. Kh., Bratskaya S. Yu. Influence of mechanochemical activation … ламарчук М. С., Шлык Д. Х., Братская С. Ю. Влияние механохимической активации … alamarchuk M. S., Shlyk D. РЕЗУЛЬТАТЫ И ИХ ОБСУЖДЕНИЕ Однако визу- ально, несмотря на низкую десорбцию радио- нуклидов в раствор, в образце, активированном в присутствии щавелевой кислоты, наблюдалось полное растворение отложений в течение пер- вых часов. Низкая степень десорбции в данном случае объясняется вторичной сорбцией радио- нуклидов кобальта, высвобожденных при рас- творении отложений, на функциональных груп- пах катионита. Данный эффект устраняется до- бавлением в раствор конкурирующих ионов: по- сле добавления в растворы нитрата натрия до концентрации 200 г/л происходит десорбция этих радионуклидов из матрицы катионита. Как видно из данных, представленных в табл. 2, количество перешедшего в раствор железа, отне- сенное к содержанию в растворе трилона Б, уве- личивается. Однако при сопоставлении данных, представленных в табл. 1 и 2, обнаруживается, что фактически происходит снижение растворимости оксида железа по сравнению с оксидом, активиро- ванным без реагентов: суммарная концентрация комплексообразующих агентов (добавленных при МХА кислот и содержащегося в растворе трилона Б) увеличивается (см. табл. 2) по сравнению с без- реагентной МХА (см. табл. 1), а относительное со- держание железа в растворе с учетом добавлен- ных кислот снижается. Этот эффект хорошо виден на примере МХА с трилоном Б: после безреагент- ной МХА в раствор переходит больше железа, чем после МХА с трилоном Б, несмотря на то что в по- следнем случае суммарная концентрация трило- на Б выше. Данное явление мы связываем с обра- зованием труднорастворимой оболочки на поверх- ности частиц магнетита в результате твердофаз- ной реакции с комплексообразующим реагентом. Как видно из рентгенограмм образцов (рис. 4), после МХА с реагентами обнаруживает- ся новая кристаллическая фаза. Для магнетита, активированного с трилоном Б, лимонной и ас- корбиновой кислотами, она не идентифицирова- на, а в случае применения щавелевой кислоты идентифицируется оксалат железа (II) и остатки непрореагировавшей кислоты. При отмывке во- дой остатки реагента растворяются, а рефлексы оксалата железа сохраняются. Подобный случай описан в работе [24]: на поверхности частиц ме- таллического железа образовалась оболочка оксалата железа (II) в результате взаимодей- ствия сухой щавелевой кислоты и поверхностно- го гематита. Низкая растворимость образован- ных оболочек, вероятно, препятствует переходу железа в раствор, особенно при низких величи- нах Ж:Т. Увеличение отношения Ж:Т способ- ствует растворению оксида железа, что показано для образца, активированного в присутствии щавелевой кислоты: относительная концентра- ция железа существенно возрастает по сравне- нию с безреагентной МХА (см. табл. 2). Рис. 4. Рентгенограммы магнетита: исходный образец (1); образцы после МХА без реагентов (2); с трилоном Б (3); лимонной (4), аскорбиновой (5), щавелевой (6) кислотами; после МХА со щавелевой кислотой и отмывки водой (7) Рис. 4. РЕЗУЛЬТАТЫ И ИХ ОБСУЖДЕНИЕ Относительная концентрация железа, перешедшего в 0,02 М раствор трилона Б при растворении магнетита, активированного в течение 30 мин р р р , р Table 2. Relative concentration of iron transferred to 0.02 M solution of Trilon B at dissolution of magnetite activated for 30 minutes МХА Растворение Железо/трилон Б, моль/моль Реагент (кислота) Массовая доля реагента в смеси, % Ж:Т, мл/г Расчетная концентрация реагента в растворе, моль/л t, сут. 1 7 20 30 Щавелевая 50 100 0,079 0,312 0,399 0,483 0,498 Аскорбиновая 50 100 0,056 0,091 0,128 0,202 0,205 Лимонная 50 100 0,052 0,127 0,171 0,286 0,271 Трилон Б 50 100 0,027 0,023 0,055 0,095 0,107 Щавелевая 50 500 0,016 0,717 0,897 0,942 0,962 Щавелевая 20 312 0,006 0,455 0,494 0,582 0,622 Без реагентов – 100 – 0,033 0,122 0,174 0,208 Без реагентов – 500 – 0,234 0,263 0,277 0,307 Отнесено к количеству трилона Б, содержащемуся в 0,02 М растворе при указанных отношениях Ж:Т. Table 2. Relative concentration of iron transferred to 0.02 M solution of Trilon B at dissolution of magnetite activated for 30 minutes 667 Паламарчук М. С., Шлык Д. Х., Братская С. Ю. Влияние механохимической активации … Palamarchuk M. S., Shlyk D. Kh., Bratskaya S. Yu. Influence of mechanochemical activation … отложений и не удаляемыми при кислотно- щелочной регенерации. Как видно из табл. 3, МХА смолы незначительно повышает десорбцию радионуклидов 57Со из магнетитовых отложений по сравнению с неактивированным образцом. При этом для образца, активированного в при- сутствии щавелевой кислоты, после 24 ч контак- та с раствором степень дезактивации ниже, чем для неактивированного образца. Однако визу- ально, несмотря на низкую десорбцию радио- нуклидов в раствор, в образце, активированном в присутствии щавелевой кислоты, наблюдалось полное растворение отложений в течение пер- вых часов. Низкая степень десорбции в данном случае объясняется вторичной сорбцией радио- нуклидов кобальта, высвобожденных при рас- творении отложений, на функциональных груп- пах катионита. Данный эффект устраняется до- бавлением в раствор конкурирующих ионов: по- сле добавления в растворы нитрата натрия до концентрации 200 г/л происходит десорбция этих радионуклидов из матрицы катионита. отложений и не удаляемыми при кислотно- щелочной регенерации. Как видно из табл. 3, МХА смолы незначительно повышает десорбцию радионуклидов 57Со из магнетитовых отложений по сравнению с неактивированным образцом. При этом для образца, активированного в при- сутствии щавелевой кислоты, после 24 ч контак- та с раствором степень дезактивации ниже, чем для неактивированного образца. РЕЗУЛЬТАТЫ И ИХ ОБСУЖДЕНИЕ 4 приведены резуль- таты эксперимента по дезактивации модельного катионита с использованием одинакового коли- чества реагента, но в первом случае щавелевая кислота вводилась при МХА, а во втором – в дезактивирующий раствор. Видно, что наиболее эффективной дезактивации можно достичь предварительной МХА отложений в присутствии сухой щавелевой кислоты. Небольшое снижение степени дезактивации по сравнению с экспери- ментом, результаты которого приведены в табл. 3, связано, вероятно, с более низким рН раствора. чества реагента, но в первом случае щавелевая кислота вводилась при МХА, а во втором – в дезактивирующий раствор. Видно, что наиболее эффективной дезактивации можно достичь предварительной МХА отложений в присутствии сухой щавелевой кислоты. Небольшое снижение степени дезактивации по сравнению с экспери- ментом, результаты которого приведены в табл. 3, связано, вероятно, с более низким рН раствора. Таблица 4. Зависимость степени дезактивации (S, %) катионита от способа введения щавелевой кислоты при дезактивации раствором, содержащим 0,02 моль/л трилона Б и 200 г/л NaNO3 Table 4. Dependence of the degree of decontamination (S, %) of the cation-exchange resin on oxalic acid introducing mode at decontamination by solution containing 0.02 mol/L of Trilon B and 200 g/L of NaNO3 Способ введения щавелевой кислоты S, % t, ч 1 3 24 При МХА 84,5 95,7 95,9 В дезактивирующий раствор 18,5 28,9 90,9 Table 4. Dependence of the degree of decontamination (S, %) of the cation-exchange resin on oxalic acid introducing mode at decontamination by solution containing 0.02 mol/L of Trilon B a кислот приводит к образованию на поверхности частиц оксида оболочек, затрудняющих раство- рение при невысоких значениях соотношения Ж:Т. Для образцов после МХА со щавелевой кислотой наблюдали растворение оболочек при повышении Ж:Т. МХА катионита КУ-2-8, загряз- ненного модельными отложениями, содержащи- ми радионуклиды 57Со, с последующей дезакти- вацией растворами на основе трилона Б позво- лила снизить активность катионита на 95–99,5%. ВЫВОДЫ Установлено, что при МХА модельного маг- нетита происходит значительное уменьшение объема частиц и разрушение примесных фаз ильменита и шпинелита, в результате чего уско- ряется растворение оксидов железа в растворах трилона Б и азотной кислоты. Эффективность перехода железа в растворы кислот увеличива- ется пропорционально времени МХА. Предпо- ложено, что МХА в присутствии органических РЕЗУЛЬТАТЫ И ИХ ОБСУЖДЕНИЕ Рентгенограммы магнетита: исходный образец (1); образцы после МХА без реагентов (2); с трилоном Б (3); лимонной (4), аскорбиновой (5), щавелевой (6) кислотами; после МХА со щавелевой кислотой и отмывки водой (7) Fig. 4. X-ray diffraction patterns of magnetite: initial sample (1), samples after MCA without reagents (2); with Trilon B (3); with citric acid (4), with ascorbic acid (5), with oxalic acid (6); ft MCA ith li id d hi ith t (7) Для исследования влияния МХА на эффек- тивность дезактивации ОИОС был использован модельный катионит КУ-2-8, загрязненный ради- онуклидами 57Со, входящими в состав магнитных ( ); after MCA with oxalic acid and washing with water (7) мость степени дезактивации (S, %) модельного катионита от условий предварительной обработки 668 https://vuzbiochemi.elpub.ru/jour Таблица 3. Зависимость степени дезактивации (S, %) модельного катионита от условий предварительной обработки Table 3. Dependence of the decontamination degree of the model cation-exchanger on the pretreatment conditions Предварительная обработка катионита S, % Трилон Б, 0,02 М NaNO3, 200 г/л МХА Реагент t, ч t, ч 1 3 24 1 24 - - 9,08 11,8 13,9 20,5 23,2 + - 19,8 20,1 21,2 26,9 33,6 + Н2С2О4∙2Н2О 12,3 14,2 16,3 98,9 99,5 dence of the decontamination degree of the model cation-exchanger on the pretreatment conditions https://vuzbiochemi.elpub.ru/jour 668 Паламарчук М. С., Шлык Д. Х., Братская Palamarchuk M. S., Shlyk D. Kh., Bratskay Следует отметить, что добавление щаве вой кислоты в дезактивирующий раствор п дезактивации катионита, активированного в сутствии реагентов, интенсифицирует раство ние модельных отложений. Однако скоро растворения отложений при использовании щ велевой кислоты в качестве активатора зна тельно выше. Так, в табл. 4 приведены резу таты эксперимента по дезактивации модельн катионита с использованием одинакового ко Таблица 4. Зависимость степени дезактивации (S, при дезактивации раствором, содержащим 0,02 мол Table 4. Dependence of the degree of decontamination on oxalic acid introducing mode at decontamination by Способ введения щавелевой кислоты При МХА В дезактивирующий раствор Паламарчук М. С., Шлык Д. Х., Братская С. Ю. Влияние механохимической активации … Palamarchuk M. S., Shlyk D. Kh., Bratskaya S. Yu. Influence of mechanochemical activation … Следует отметить, что добавление щавеле- вой кислоты в дезактивирующий раствор при дезактивации катионита, активированного в от- сутствии реагентов, интенсифицирует растворе- ние модельных отложений. Однако скорость растворения отложений при использовании ща- велевой кислоты в качестве активатора значи- тельно выше. Так, в табл. org/10.1021/acs.iecr.0c02732. org/10.1021/acs.iecr.0c02732 org/10.1021/acs.iecr.0c02732. 1. Hussain A., Al-Othmany D. 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Владивосток, пр-т 100-летия Владивостока, 159, Российская федерация, sbratska@ich.dvo.ru https://orcid.org/0000-0003-4954-0422 Svetlana Yu. Bratskaya, Dr. Sci. (Chemistry), Corresponding member of RAS, Head of the Department of Sorption Technologies and Functional Materials, Institute of Chemistry FEB RAS, 159, 100-letya Vladivostoka Ave., Vladivostok, 690022, Russian Federation, sbratska@ich.dvo.ru https://orcid.org/0000-0003-4954-0422 С. Ю. Братская, д.х.н., член-корреспондент РАН, заведующая отделом сорбционных технологий и функциональных материалов, Институт химии ДВО РАН, 690022, г. Владивосток, пр-т 100-летия Владивостока, 159, Российская федерация, sbratska@ich.dvo.ru https://orcid.org/0000-0003-4954-0422 Darya Kh. Shlyk, Cand. Sci. (Chemistry), Researcher, Institute of Chemistry FEB RAS, 159, 100-letya Vladivostoka Ave., Vladivostok, 690022, Russian Federation, daria79@list.ru https://orcid.org/0000-0003-4247-4872 Darya Kh. Shlyk, Cand. Sci. (Chemistry), Researcher, Institute of Chemistry FEB RAS, 159, 100-letya Vladivostoka Ave., Vladivostok, 690022, Russian Federation, daria79@list.ru https://orcid.org/0000-0003-4247-4872 Д. Х. Шлык, к.х.н., научный сотрудник, Институт химии ДВО РАН, 690022, г. Владивосток, пр-т 100-летия Владивостока, 159, Российская федерация, daria79@list.ru https://orcid.org/0000-0003-4247-4872 https://vuzbiochemi.elpub.ru/jour 671 Паламарчук М. С., Шлык Д. Х., Братская С. Ю. Влияние механохимической активации Palamarchuk M. S., Shlyk D. Kh., Bratskaya S. Yu. Influence of mechanochemical activation Вклад авторов Вклад авторов Паламарчук М. С. – концептуализация; мето- дология; проведение экспериментов; анализ и визуализация экспериментальных данных; написание текста. Шлык Д. Х. – проведение экспериментов; ана- лиз и визуализация экспериментальных дан- ных. Братская С. Ю. – методология; проведение экспериментов; анализ и визуализация экспе- риментальных данных; доработка текста. Конфликт интересов Авторы заявляют об отсутствии конфликта интересов. Все авторы прочитали и одобрили оконча- тельный вариант рукописи. Информация о статье Поступила в редакцию 29.10.2021. Одобрена после рецензирования 15.11.2021. Принята к публикации 30.11.2021. Шлык Д. Х. – проведение экспериментов; ана- лиз и визуализация экспериментальных дан- ных. Братская С. Ю. – методология; проведение экспериментов; анализ и визуализация экспе- риментальных данных; доработка текста. Conflict interests The authors declare no conflict of interests re- garding the publication of this article. Конфликт интересов Авторы заявляют об отсутствии конфликта интересов. The final manuscript has been read and approved by all the co-authors. Все авторы прочитали и одобрили оконча- тельный вариант рукописи. Информация о статье Поступила в редакцию 29.10.2021. Одобрена после рецензирования 15.11.2021. Принята к публикации 30.11.2021. Д. Х. Шлык, к.х.н., научный сотрудник, Институт химии ДВО РАН, 690022, г. Владивосток, пр-т 100-летия Владивостока, 159, Российская федерация, daria79@list.ru https://orcid.org/0000-0003-4247-4872 С. Ю. Братская, д.х.н., член-корреспондент РАН, заведующая отделом сорбционных технологий и функциональных материалов, Институт химии ДВО РАН, 690022, г. Владивосток, пр-т 100-летия Владивостока, 159, Российская федерация, sbratska@ich.dvo.ru https://orcid.org/0000-0003-4954-0422 С. Ю. Братская, Svetlana Yu. Bratskaya, Dr. Sci. (Chemistry), Corresponding member of RAS, Head of the Department of Sorption Technologies and Functional Materials, Institute of Chemistry FEB RAS, 159, 100-letya Vladivostoka Ave., Vladivostok, 690022, Russian Federation, sbratska@ich.dvo.ru https://orcid.org/0000-0003-4954-0422 Институт химии ДВО РАН, 690022, г. Владивосток, 159, 100-letya Vladivostoka Ave., пр-т 100-летия Владивостока, 159, Vladivostok, 690022, Russian Federation, Contribution of the authors Palamarchuk M. S. – conceptualization; metho- dology; investigation; visualization; writing – origi- nal draft. Shlyk D. Kh. – investigation; visualization. Bratskaya S. Yu. – methodology; investigation; visualization; writing – review and editing. Information about the article Information about the article The article was submitted 29.10.2021. Approved after reviewing 15.11.2021. Accepted for publication 30.11.2021. Information about the article The article was submitted 29.10.2021. Approved after reviewing 15.11.2021. Accepted for publication 30.11.2021. 672 https://vuzbiochemi.elpub.ru/jour
https://openalex.org/W4294927840	https://www.frontiersin.org/articles/10.3389/fpls.2022.933738/pdf	English	null	Species identity and combinations differ in their overall benefits to Astragalus adsurgens plants inoculated with single or multiple endophytic fungi under drought conditions	Frontiers in plant science	2,022	cc-by	14,967	Species identity and combinations difer in their overall beneﬁts to Astragalus adsurgens plants inoculated with single or multiple endophytic fungi under drought conditions OPEN ACCESS EDITED BY Pablo Cornejo, University of La Frontera, Chile REVIEWED BY Teresa Dias, Universidade de Lisboa, Portugal Noshin Ilyas, Pir Mehr Ali Shah Arid Agriculture University, Pakistan CORRESPONDENCE Xue-Li He xlh3615@126.com SPECIALTY SECTION This article was submitted to Plant Abiotic Stress, a section of the journal Frontiers in Plant Science RECEIVED 01 May 2022 ACCEPTED 08 August 2022 PUBLISHED 07 September 2022 Yi-Ling Zuo1,2, Qian-Nan Hu1, Le Qin1, Jia-Qiang Liu1 and Xue-Li He1,2 Yi-Ling Zuo1,2, Qian-Nan Hu1, Le Qin1, Jia-Qiang Liu1 and Xue-Li He1,2* 1School of Life Sciences, Hebei University, Baoding, China, 2Key Laboratory of Microbial Diversity Research and Application of Hebei Province, Baoding, China Zuo Y-L, Hu Q-N, Qin L, Liu J-Q and He X-L (2022) Species identity and combinations difer in their overall beneﬁts to Astragalus adsurgens plants inoculated with single or multiple endophytic fungi under drought conditions. Front. Plant Sci. 13:933738. doi: 10.3389/fpls.2022.933738 Although desert plants often establish multiple simultaneous symbiotic associations with various endophytic fungi in their roots, most studies focus on single fungus inoculation. Therefore, combined inoculation of multiple fungi should be applied to simulate natural habitats with the presence of a local microbiome. Here, a pot experiment was conducted to test the synergistic efects between three extremely arid habitat-adapted root endophytes (Alternaria chlamydospora, Sarocladium kiliense, and Monosporascus sp.). For that, we compared the efects of single fungus vs. combined fungi inoculation, on plant morphology and rhizospheric soil microhabitat of desert plant Astragalus adsurgens grown under drought and non-sterile soil conditions. The results indicated that fungal inoculation mainly inﬂuenced root biomass of A. adsurgens, but did not afect the shoot biomass. Both single fungus and combined inoculation decreased plant height (7–17%), but increased stem branching numbers (13–34%). However, fungal inoculation inﬂuenced the root length and surface area depending on their species and combinations, with the greatest beneﬁts occurring on S. kiliense inoculation alone and its co- inoculation with Monosporascus sp. (109% and 61%; 54% and 42%). Although A. chlamydospora and co-inoculations with S. kiliense and Monosporascus sp. also appeared to promote root growth, these inoculations resulted in obvious soil acidiﬁcation. Despite no observed root growth promotion, Monosporascus sp. associated with its combined inoculations maximally facilitated soil organic carbon accumulation. However, noticeably, combined inoculation of the three species had no signiﬁcant efects on root length, surface area, and biomass, but promoted rhizospheric fungal diversity and abundance most, with Sordariomycetes being the dominant fungal group. TYPE Original Research PUBLISHED 07 September 2022 DOI 10.3389/fpls.2022.933738 TYPE Original Research PUBLISHED 07 September 2022 DOI 10.3389/fpls.2022.933738 TYPE Original Research PUBLISHED 07 September 2022 DOI 10.3389/fpls.2022.933738 Introduction abundance and functionality is also critical for the arid desert soil ecosystem. Fungal endophytes are ubiquitous in the roots of almost all plants, which co-evolved with hosts with their high genetic and functional diversity, endowing plants with diverse resistance and multiple evolutionary strategies (Barnes et al., 2018; Alzarhani et al., 2019; Leroy et al., 2021). Root endophytes isolated from desert habitats have been evidenced to improve plant drought tolerance and performance, enabling the establishment and survival of host plants in a stressful environment (He et al., 2021; Moghaddam et al., 2021). Rodriguez et al. (2008) suggested that the ability to confer drought tolerance to hosts may be a unique genetic resource of endophytic fungi. Under drought stress, endophytic fungi can improve the osmotic adjustment capacity of the host by increasing the production of antioxidant enzymes and active substances, thus reducing water consumption (Xu et al., 2017). Improved nutrition, phytohormones production, and acquired host resistance or immunity induction are also related to endophytic fungi stimulating plant growth and drought tolerance (Mandyam and Jumpponen, 2005; Kour et al., 2019; Fontana et al., 2021). Additionally, fungal endophytes have been found to increase soil fungal abundance and improve microbial community structure, thereby contributing to plant growth and ecological adaptability under water deﬁcit conditions (He et al., 2019). Some beneﬁcial endophytic fungi may even compensate for the low colonization of arbuscular mycorrhizal fungi (AMF) under natural conditions when plant nutrient consumption is challenged (Han et al., 2021). Compared with the well-studied AMF association, fungal endophytes are readily isolated and cultured in vitro, which are gradually regarded as promising reciprocal partners in plants (Gonçalves et al., 2021; Zhong et al., 2022). Global warming has intensiﬁed the appearance of the drought phenomenon, thus aggravating the adverse impact of drought stress, especially in arid desert areas (Zandalinas et al., 2021). As the most important threat to desert ecosystems, drought adversely inﬂuences plant growth, including its development and survival (Omer et al., 2020; Terletskaya et al., 2020; Barker et al., 2021). Plants respond to such drought environments both directly and indirectly; furthermore, indirect responses through intimate associations with endophytic fungi have recently received increased attention (Beckers et al., 2017; Trivedi et al., 2020). Therefore, identifying the most beneﬁcial combinations between plant hosts and endophytes may be of particular value for improving the plant growth in desert habitats and mitigating the negative eﬀects on soil ecosystems. Species identity and combinations difer in their overall beneﬁts to Astragalus adsurgens plants inoculated with single or multiple endophytic fungi under drought conditions This indicates the response of plant growth to fungal inoculation may be diferent from that of the rhizospheric fungal community. Structural equation modeling also demonstrated that fungal inoculation signiﬁcantly inﬂuenced the interactions © 2022 Zuo, Hu, Qin, Liu and He. 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. Frontiers in Plant Science Frontiers in Plant Science 01 frontiersin.org Zuo et al. Zuo et al. 10.3389/fpls.2022.933738 10.3389/fpls.2022.933738 among the growth of A. adsurgens, soil factors, and rhizospheric fungal groups. Our ﬁndings suggest that, based on species-speciﬁc and combinatorial efects, endophytic fungi enhanced the plant root growth, altered soil nutrients, and facilitated rhizospheric fungal community, possibly contributing to desert plant performance and ecological adaptability. These results will provide the basis for evaluating the potential application of fungal inoculants for developing sustainable management for desert ecosystems. root endophytes, combination inoculation, promoting efects, soil fungal community, drought stress Frontiers in Plant Science frontiersin.org Introduction The rhizosphere is known as the soil region surrounding the plant roots, as well as the reservoir of soil microorganisms (Li et al., 2021). Changes in microorganisms signiﬁcantly inﬂuence plant productivity, survival, and stress resistance by acting on geochemical characteristics of subsurface soils, such as material cycling, decomposition rate, and pathogenicity (Bardgett and van der Putten, 2014; Zhang et al., 2020). The rhizospheric fungal community, in particular, plays a central role in improving soil nutrient recycling and availability, resulting in beneﬁcial eﬀects on plant growth and adaptation (Bardgett and van der Putten, 2014). Recent studies have shown that utilizing beneﬁcial endophytic fungi can alter the composition of soil fungal communities and promote the abundance and beneﬁcial interactions of soil fungi (Azcón et al., 2013; Nanjundappa et al., 2019; Li et al., 2020). Furthermore, endophytic fungi could inﬂuence plant– rhizosphere interactions to alleviate abiotic stress (Dimkpa et al., 2009). For example, increased production of fungal exopolysaccharides and microbial activity under water- deﬁcit conditions can impact soil water retention and ﬁeld performance of tomatoes (Le Gall et al., 2021). In this light, optimizing plant–fungal partnership to increase soil microbial Most studies have focused on assessing the eﬀects of single strains on plants under drought stress in essentially sterile conditions (González-Teuber et al., 2018; Li et al., 2019a; Hereme et al., 2020). In natural habitats, plant roots are often colonized by multiple fungi species, which together perform complicated ecological functions, rather than a single fungus Frontiers in Plant Science 02 frontiersin.org frontiersin.org 10.3389/fpls.2022.933738 Zuo et al. alone (Durán et al., 2018; Liu et al., 2020a). Additionally, natural habitats are non-sterile environments due to the presence of local microbiota. Therefore, combined inoculation of multiple endophytic fungi simulates natural conditions better than single inoculations, and is thought to be more competitive and eﬀective in a non-sterile natural environment (Baez-Rogelio et al., 2017; Li et al., 2021). Chen et al. (2017) evaluated the eﬀects of combined inoculation of diﬀerent AMF species on the growth, nutrient absorption, and photosynthesis of cucumber seedlings, which indicated that the AMF composition consists of distant AMF species showing a better synergistic eﬀect than a single or closely related AMF spp. Nanjundappa et al. (2019) reported that under ﬁeld conditions, the synergistic eﬀect of AMF and Bacillus spp. on crop growth and soil nutrient uptake was much greater than that of inoculation with either AMF or Bacillus alone. He et al. Experimental design A pot experiment was used to carry out single and multiple cross-inoculations of three root endophytic fungi on seedlings of desert plant A. adsurgens. Considering the threat of drought in the natural environment, we conducted this pot experiment under drought stress to test the response of the A. adsurgens plant to fungal inoculation under conditions of drought stress. The A. adsurgens seedlings were inoculated under 8 inoculation treatments, which included a non-inoculated control (C), inoculation with Alternaria chlamydospora (A), Sarocladium kiliense (S), Monosporascus sp. (M), co-inoculation of A. chlamydospora and S. kiliense (AS), co-inoculation of A. chlamydospora and Monosporascus sp. (AM), co-inoculation of S. kiliense and Monosporascus sp. (SM), and the combination of the three species (ASM). There were 5 replicates for each treatment (two plants/pot/replicate), and a total of 40 pots were set up for the experiment. The experiment lasted for 4 months and was performed in a growth chamber with a 14/10 h photoperiod, day/night temperatures of 27/22◦C, and 60% mean relative humidity. Astragalus adsurgens Pall. (Leguminosae) is a native perennial herbaceous mainly distributed in the desert region of Northwest China (Liu et al., 2018). As typical desert herbage and ﬁne forage, this species is a preferred plant for desert ecological restoration and desert grassland planting because it is strong drought and sandstorm resisting plant (Chen et al., 2013). Therefore, considering the concept of sustainability and the need to enhance the growth status and drought resistance of grassland plants, understanding the interaction between plants and beneﬁcial microbes is crucial to receive beneﬁt from the symbiotic mechanisms. Frontiers in Plant Science Introduction (2022) suggested that the dual inoculation of dark septate endophytes and Trichoderma viride enriched beneﬁcial microbiota, altered soil nutrient status, and might contribute to enhancing the cultivation of medicinal plants in dryland. Hence, it is essential to identify the combinations of diﬀerent fungal species and determine their subsequent synergistic eﬀects in non-sterile environments, especially with the inclusion of drought stress. height, leaf number, root length, surface area, and diameter), (3) soil physicochemical properties, and (4) the rhizospheric fungal community composition. Such data would display endophytic fungal groups that could withstand the drought conditions that impacted host growth, and their potential for improving the stress tolerance and symbiotic performance of A. adsurgens in drought-aﬀected arid lands. Inoculation assay and drought stress treatment Approximately 1,000 g of culture substrate containing 500 g of soil mixed with 500 g of river sand (<2 mm) were placed in sterile plastic pots (11.5 cm height, 13.6 cm diameter at the top, and 9.5 cm diameter at the base) and prepared for inoculation assay. One week old A. adsurgens seedlings were transplanted into sterile plastic pots for subsequent growth. The mixture substrates contained 9.98 mg/g of organic matter, 63.85 µg/g of nitrate nitrogen, 94.89 µg/g of ammonia nitrogen, and 16.37 µg/g of available phosphorus. After 1 month of seedlings’ acclimation to the pot, fungal endophytes (alone or in combination) were inoculated. The fungal inoculums were produced by sterile culture of fungal strains in Petri dishes using a PDA culture medium and inoculated in the form of liquid suspensions. Speciﬁcally, six mycelia discs (9 mm in diameter) from the active mycelium of 10-day-old PDA colonies were transferred to a container of 200 ml sterile water and then were crushed with a homogenizer in intervals of 30 s for 3 min (Del Barrio-Duque et al., 2020). Subsequently, the prepared fungal suspensions were irrigated uniformly in the soil substrates where A. adsurgens seedlings grew. The viability of the broken fungal fragments has been conﬁrmed by culturing on a PDA culture medium. Three mycelia discs of each fungus were, respectively, used in the pairwise fungal inoculation treatments, and two discs were, respectively, used in the combination of inoculation with three fungi. For the non-inoculated treatments, the plugs excised from the sterile medium without any fungi were used instead. Plant biomass and morphological parameters were cultured at 27◦C in darkness and subcultured every 2 weeks. Mature seeds of A. adsurgens were provided by Linquan Ecological Seed Corporation in Minqin, Gansu Province, China, and stored at 4◦C. The seeds were surface-sterilized in a solution of 70% ethanol for 3 min and 2.5% sodium hypochlorite for 10 min with agitation. The sterilized seeds were gently washed several times with sterile water and then aseptically planted onto water–agar medium (containing 10 g/L agar) in petri dishes for germination at 27◦C in the dark. At the end of the experiment, plant height and stem branching number were recorded for each replicate with two plants in each pot. Shoots and roots from each replicate were separately harvested, and roots were gently washed with deionized water to remove the sand. The root sections for each replicate were ﬂoated in water at ∼1 cm depth in a plexiglass tray and scanned using a desktop scanner. Morphological characteristics of roots, such as total root length, average root diameter, and root surface area, were determined using the WinRHIZO image analysis system (Chen et al., 2012). The remaining roots and fresh shoots were dried at 80◦C for at least 48 h prior to calculating the plant biomass. The total biomass production of plants was the sum of the dry weights of both shoots and roots. Fungal isolates and plant materials In a previous study, we investigated the inﬂuences of several endophytic fungal strains alone from the roots of desert shrubs on the performance of Hedysarum scoparium under diﬀerent soil water conditions with sterile substrates. These strains established a positive symbiosis with the host plant depending on fungal species and water availability (Li et al., 2019b). In the current study, the eﬀects of single and mixed inoculation of three desert endophytic fungi on A. adsurgens plants were studied in a greenhouse pot experiment with non-sterile substrates. Since A. adsurgens is a desert plant, our pot experiment was conducted under drought stress which simulated the natural conditions for the plant host and the root endophytes. We hypothesize that mixed inoculations of desert endophytic fungi could either promote plant growth or change the rhizospheric fungal community of A. adsurgens, and the synergistic eﬀects between mixed inoculations would depend on the combination of diﬀerent fungal species. Based on our conjecture, we investigated the eﬀects of mixed inoculations on (1) plant biomass (shoot and root biomass), (2) the morphological parameters (plant Endophytic fungal strains of A. chlamydospora, S. kiliense, and Monosporascus sp. used in this experiment were isolated from the roots of xerophytic shrubs in an extremely arid desert of Northwest China, such as Sympegma regelii, Salsola passerine, and Ephedra przewalskii. In our previous studies, these three endophytic fungi have been shown to be drought- tolerant in vitro, and are therefore considered to confer adaptive beneﬁts to host plants (Zuo et al., 2021, 2022). The identiﬁcation of the three fungal species was performed based on the similarity alignment of internal transcribed spacer (ITS) sequences in GenBank with the BLASTN program (Supplementary Figure 1). The ITS sequences for the fungi are available in the GenBank database under accession numbers OM304622 (A. chlamydospora), OM304621 (S. kiliense), and OM304623 (Monosporascus sp.). These fungal strains are deposited in the culture collection of the Laboratory of Mycorrhizal Biology, Hebei University, China. All fungal strains Frontiers in Plant Science 03 frontiersin.org Zuo et al. 10.3389/fpls.2022.933738 Soil enzyme activities and physico-chemical properties Rhizospheric soils from each replicate were collected when harvesting plants. Part of the soil samples was stored at 4◦C for enzyme analyses, the subsamples were air-dried at room temperature to determine soil physicochemical properties, while the remaining soils were frozen at −80◦C for further analysis of fungal community composition. Soil pH was determined with a digital pH meter in a (1:2.5, soil: water) suspension. The activities of soil urease (U) and acid phosphatase were determined, respectively, using the method of Hoﬀmann and Teicher (1961) and Tarafdar and Marschner (1994). Soil organic carbon (SOC) was estimated according to the potassium dichromate oxidation method (Rowell, 1994). Soil nitrate and ammonia were examined by a continuous ﬂow analyzer (Smart Chem 200, Alliance, France) via extraction in a 2 mol/L of KCl solution (1:5 w/v), during which samples were shaken for 1 h at 250 r min−1 and then ﬁltered. Soil available phosphorus (P) was extracted for 30 min by using 0.5 M sodium bicarbonate and examined by the chlorostannus-reduced molybdophosphoric blue color method (Olsen et al., 1954). One month after inoculation with fungi, pots were kept at low water content (30% ﬁeld capacity) in accordance with the median value (approximately 28% ﬁeld capacity) in the natural habitat of A. adsurgens in Northwest China to simulate drought stress (Bai et al., 2009). The drought stress experiment lasted for 2 months, and the signiﬁcant eﬀect of this level of drought has been demonstrated in our previous experiments (Li et al., 2019a,b). Water loss was determined by regular weighing and was supplemented with sterile distilled water to meet the requirements of water capacity. Frontiers in Plant Science Soil fungal community composition The genomic DNA of soil samples was extracted using a Powersoil R⃝DNA Extraction kit. The DNA concentration and purity were determined using a NanoDrop 2000 UV- vis spectrophotometer, and DNA quality was checked with 1% agarose gel electrophoresis. The universal primers of ITS1F (5′-CTTGGTCATTTAGGAAGTAA-3′) and ITS2R (5′-GCTGCGTTCTTCATCATGATGC-3′) were employed to Frontiers in Plant Science 04 frontiersin.org Zuo et al. 10.3389/fpls.2022.933738 10.3389/fpls.2022.933738 among treatments, and Duncan’s multiple-range tests were performed (p < 0.05). For the sequenced rhizospheric fungal community, comparisons of the fungal alpha diversity were examined by Student’s tests for paired comparisons between samples (De Winter, 2013). A Venn diagram was used to visualize the numbers of fungal OTUs that were shared among treatments. Non-metric multidimensional scaling (NMDS) was used to visualize the community composition dissimilarities of soil fungi based on the Bray–Curtis, and the analysis of similarity (ANOSIM) was used to examine signiﬁcant diﬀerences based on 999 permutations (Clarke et al., 2006). The relative abundances of the top 50 abundant fungal OTUs were depicted using the “pheatmap” function to display the clustering of fungal community composition among diﬀerent treatment groups. Linear discriminant analysis (LDA) eﬀect size (LEfSe), which can take into account the statistical signiﬁcance and biological relevance, was conducted to identify OTUs diﬀerentially represented between diﬀerent inoculation treatments by using the non-parametric factorial Kruskal–Wallis (KW) sum-rank test and LDA (Segata et al., 2011; Guerrero-Preston et al., 2016). High LDA scores reﬂect a signiﬁcantly higher abundance of certain taxa. The fungal groups that were signiﬁcantly correlated with soil factors and plant growth parameters were identiﬁed based on the coeﬃcient of Pearson’s correlation, and the p- value was adjusted based on the false discovery rate (FDR) (Yao et al., 2019; He et al., 2022). The interrelationships among soil factors, the fungal community, and plant growth parameters were evaluated by analysis of Pearson’s correlation and structural equation modeling (SEM) via AMOS 21.0. The SEM procedure was started with an a priori model on the basis of our predictions and related literature. The chi-square (χ2) test, p- value, comparative ﬁt index (CFI), and root mean square error of approximation (RMSEA) were applied to assess model ﬁt. All the statistical analysis and plotting of graphs were performed by SPSS 19.0 and R package vegan. generate the ampliﬁcation of fungal internal transcribed spacer 1 (ITS1) regions with a thermocycler PCR system (GeneAmp 9700, Applied Biosystems ABI, Foster City, CA, USA). Plant shoot and root biomass Despite the drought stress imposed, all inoculated seedlings were green, alive, and healthy after 4 months of growth, including the control seedlings. Compared with the non- inoculated plants, fungal inoculation did not inﬂuence the shoot biomass of A. adsurgens seedlings but obviously inﬂuence the root biomass (Figure 1). Under the treatment of single fungus inoculation, the root biomass showed the maximum value when inoculated with S alone. However, in terms of 2-species inoculations, the shoot biomass under AM and SM were higher than that of M inoculated alone, and inoculated with AS and AM induced an increase in root biomass than that of M and non-inoculated plants. Overall, the total biomass of A. adsurgens Frontiers in Plant Science Soil fungal community composition The PCR reactions were performed in 20 µl mixtures containing 4 µl of 5 × FastPfu Buﬀer, 2 µl of 2.5 mM dNTPs, 0.8 µl of each primer (5 µM), 0.4 µl of FastPfu Polymerase, and 10 ng of template DNA. The PCRs were conducted using the following program: 3 min of denaturation at 95◦C, 27 cycles of 30 s at 95◦C, 30 s for annealing at 55◦C, 45 s for elongation at 72◦C, and a ﬁnal extension at 72◦C for 10 min. The resulting PCR products were extracted from a 2% agarose gel and further puriﬁed using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) and quantiﬁed using a QuantiFluorTM -ST system (Promega, Madison, WI, USA) according to the manufacturer’s protocol. The puriﬁed amplicons were pooled in equimolar amounts and paired-end sequenced [2 × 300 base pairs (bp)] using the Illumina MiSeq platform (PE300, Illumina, San Diego, CA, USA) with standard protocols by Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China). The Raw FASTQ sequences were quality-ﬁltered using Trimmomatic software and merged using FLASH software to obtain valid and high-quality sequences. All reads were assembled according to their overlapping sequences longer than 10 bp, and sequences that could not be assembled were discarded. The maximum mismatch ratio of overlapping regions was 0.2. Samples were distinguished according to the barcodes. Operational taxonomic units (OTUs) were clustered by UPARSE (version 7.0.1090, http://www.drive5.com/uparse/) according to 97% similarity with a novel “greedy” algorithm that performs chimera ﬁltering and OTU clustering simultaneously after dereplication and discarding all singletons (Edgar, 2013). The taxonomy of each representative sequence was analyzed with the RDP Classiﬁer algorithm (http://rdp.cme.msu.edu/) against the UNITE version 8.0 database (https://unite.ut.ee/) using a conﬁdence threshold of 70% (Kõljalg et al., 2013). To eliminate the eﬀects of the diﬀerent numbers of sequences among the samples on the identiﬁed fungal community, the number of sequences per sample was normalized to the smallest sample size using the subsample command in MOTHUR. Subsequently, rarefaction curves were assembled, and the alpha diversity indices of OUT richness, Simpson index, Shannon diversity, and evenness were calculated. The relative abundance of speciﬁc fungal taxa was deﬁned as the number of reads of a particular taxon, which is a percentage of the number of all reads in a sample. Plant growth and root morphology traits Fungal combination inoculation inﬂuenced the growth of A. adsurgens seedlings (Figure 2). Regardless of the single or combined inoculation, all fungal inoculation decreased the plant height of A. adsurgens seedlings, but the 2-species inoculation of SM and 3-species inoculation of ASM slightly promoted plant height growth (Figure 2A). Compared with the control plant, all fungal inoculation increased the stem branching numbers of A. adsurgens except M inoculation; and the AM inoculation had the greatest eﬀect on plant height, which was higher than that of A and M inoculation alone (Figure 2B). Statistical analysis The Shapiro–Wilk test and Bartlett’s test were employed to check the normality and homoscedasticity of all the data, respectively. One-way analysis of variance (ANOVA) was applied for plant growth and rhizospheric soil parameters Frontiers in Plant Science frontiersin.org 05 Zuo et al. 10.3389/fpls.2022.933738 FIGURE 1 Efects of combined inoculation efects of root endophytic fungi on plant biomass of A. adsurgens seedlings. Diferent lowercase letters indicate signiﬁcant diferences (p < 0.05). C, non-inoculated control; A, inoculation with Alternaria chlamydospora; S, inoculation with Sarocladium kiliense; M, inoculation with Monosporascus sp.; AS, co-inoculation of Alternaria chlamydospora and Sarocladium kiliense; AM, co-inoculation of Alternaria chlamydospora and Monosporascus sp.; SM, co-inoculation of Sarocladium kiliense and Monosporascus sp.; ASM, combination inoculation of the three species. FIGURE 1 Efects of combined inoculation efects of root endophytic fungi on plant biomass of A. adsurgens seedlings. Diferent lowercase letters indicate signiﬁcant diferences (p < 0.05). C, non-inoculated control; A, inoculation with Alternaria chlamydospora; S, inoculation with Sarocladium kiliense; M, inoculation with Monosporascus sp.; AS, co-inoculation of Alternaria chlamydospora and Sarocladium kiliense; AM, co-inoculation of Alternaria chlamydospora and Monosporascus sp.; SM, co-inoculation of Sarocladium kiliense and Monosporascus sp.; ASM, combination inoculation of the three species FIGURE 1 Efects of combined inoculation efects of root endophytic fungi on plant biomass of A. adsurgens seedlings. Diferent lowercase letters indicate signiﬁcant diferences (p < 0.05). C, non-inoculated control; A, inoculation with Alternaria chlamydospora; S, inoculation with Sarocladium kiliense; M, inoculation with Monosporascus sp.; AS, co-inoculation of Alternaria chlamydospora and Sarocladium kiliense; AM, co-inoculation of Alternaria chlamydospora and Monosporascus sp.; SM, co-inoculation of Sarocladium kiliense and Monosporascus sp.; ASM, combination inoculation of the three species. plants under S alone and SM inoculations were signiﬁcantly higher than that of control plants. every single inoculation compared with the control treatment (Figures 2C,D). The inoculation of M showed no signiﬁcant diﬀerence with the control plant, but the 2-species inoculations of AM and SM existed promoting eﬀects on the root growth of A. adsurgens seedlings. However, there was no diﬀerence between inoculated A. adsurgens and control plants when the three fungal species were combined inoculated. In contrast, all fungal inoculation had a negative impact on root diameter, except M and the 3-species inoculation of ASM (Figure 2E). Frontiers in Plant Science Soil enzymes and physicochemical properties The activity of soil urease and acid phosphatase was not inﬂuenced by fungal inoculations, but only inoculations with AS and ASM were associated with higher soil urease activity (Figures 3A,B). In terms of soil physicochemical properties, inoculations of A, AS, and AM all resulted in a decrease Inoculated with A and S alone increased the total root length and root surface area of A. adsurgens to the greatest extent, whereas AS showed a lower promotion eﬀect than 06 frontiersin.org Zuo et al. 10.3389/fpls.2022.933738 FIGURE 2 Efects of combined inoculation efects of root endophytic fungi on the morphological parameters of A. adsurgens seedlings. (A) Plant height, (B) stem branching, (C) root length, (D) root surface area, and (E) root diameter. Diferent lowercase letters indicate signiﬁcant diferences (p < 0.05). C, non-inoculated control; A, inoculation with Alternaria chlamydospora; S, inoculation with Sarocladium kiliense; M, inoculation with Monosporascus sp.; AS, co-inoculation of Alternaria chlamydospora and Sarocladium kiliense; AM, co-inoculation of Alternaria chlamydospora and Monosporascus sp.; SM, co-inoculation of Sarocladium kiliense and Monosporascus sp.; ASM, combination inoculation of the three species. FIGURE 2 Efects of combined inoculation efects of root endophytic fungi on the morphological parameters of A. adsurgens seedlings. (A) Plant height, (B) stem branching, (C) root length, (D) root surface area, and (E) root diameter. Diferent lowercase letters indicate signiﬁcant diferences (p < 0.05). C, non-inoculated control; A, inoculation with Alternaria chlamydospora; S, inoculation with Sarocladium kiliense; M, inoculation with FIGURE 2 GU Efects of combined inoculation efects of root endophytic fungi on the morphological parameters of A. adsurgens seedlings. (A) Plant height, (B) stem branching, (C) root length, (D) root surface area, and (E) root diameter. Diferent lowercase letters indicate signiﬁcant diferences (p < 0.05). C, non-inoculated control; A, inoculation with Alternaria chlamydospora; S, inoculation with Sarocladium kiliense; M, inoculation with Monosporascus sp.; AS, co-inoculation of Alternaria chlamydospora and Sarocladium kiliense; AM, co-inoculation of Alternaria chlamydospora and Monosporascus sp.; SM, co-inoculation of Sarocladium kiliense and Monosporascus sp.; ASM, combination inoculation of the three species. of ASM signiﬁcantly increased the availability of soil available phosphorus (Figure 3G). in soil pH (Figure 3C). Although there was no eﬀect on the morphological growth of A. adsurgens seedlings, M inoculation alone increased soil organic carbon content to the greatest extent (Figure 3D). Soil enzymes and physicochemical properties Fungal inoculation treatments did not have an eﬀect on soil nitrate and seemed to reduce the content of soil ammonia, with the remarkably reduced soil ammonia under M and AS inoculations (Figures 3E,F). Compared with the control treatment, inoculation with A, S, and M alone resulted in an insigniﬁcant decrease in soil available phosphorus, but 2- species inoculations of AS, AM, SM, and 3-species inoculation Soil fungal community composition A total of 1,183,752 non-chimeric eﬀective fungal sequences were obtained after removing sequences of low quality, potential chimeras, and singletons. Subsequently, the number of sequences per sample was normalized to the smallest sample Frontiers in Plant Science 07 frontiersin.org Zuo et al. 10.3389/fpls.2022.933738 FIGURE 3 Efects of combined inoculation efects of root endophytic fungi on soil enzymes and physicochemical properties of A. adsurgens seedlings. (A) Soil urease, (B) soil acid phosphatase, (C) soil pH, (D) soil organic carbon, (E) soil nitrate, (F) soil ammonia, and (G) soil available phosphorus. Diferent lowercase letters indicate signiﬁcant diferences (p < 0.05). C, non-inoculated control; A, inoculation with Alternaria chlamydospora; S, inoculation with Sarocladium kiliense; M, inoculation with Monosporascus sp.; AS, co-inoculation of Alternaria chlamydospora and Sarocladium kiliense; AM, co-inoculation of Alternaria chlamydospora and Monosporascus sp.; SM, co-inoculation of Sarocladium kiliense and Monosporascus sp.; ASM, combination inoculation of the three species. FIGURE 3 Efects of combined inoculation efects of root endophytic fungi on soil enzymes and physicochemical properties of A. adsurgens seedlings. (A) Soil urease, (B) soil acid phosphatase, (C) soil pH, (D) soil organic carbon, (E) soil nitrate, (F) soil ammonia, and (G) soil available phosphorus. Diferent lowercase letters indicate signiﬁcant diferences (p < 0.05). C, non-inoculated control; A, inoculation with Alternaria chlamydospora; S, FIGURE 3 FIGURE 3 Efects of combined inoculation efects of root endophytic fungi on soil enzymes and physicochemical properties of A. adsurgens seedlings. (A) Soil urease, (B) soil acid phosphatase, (C) soil pH, (D) soil organic carbon, (E) soil nitrate, (F) soil ammonia, and (G) soil available phosphorus. Diferent lowercase letters indicate signiﬁcant diferences (p < 0.05). C, non-inoculated control; A, inoculation with Alternaria chlamydospora; S, inoculation with Sarocladium kiliense; M, inoculation with Monosporascus sp.; AS, co-inoculation of Alternaria chlamydospora and Sarocladium kiliense; AM, co-inoculation of Alternaria chlamydospora and Monosporascus sp.; SM, co-inoculation of Sarocladium kiliense and Monosporascus sp.; ASM, combination inoculation of the three species. 08 Frontiers in Plant Science frontiersin.org Zuo et al. 10.3389/fpls.2022.933738 the A inoculation alone. There were 23, 24, 25, 24, 25, and 33 OTUs present only in the non-inoculated control, A inoculation, M inoculation, and 2-species inoculations of AS, AM, and SM, respectively. Rarefaction curves for the Sobs index at the OTU level across all samples approached an asymptote and showed that the overall fungal diversity was well represented (Supplementary Figure 2). Soil fungal community composition size (49,323 reads), and the remaining eﬀective fungal sequences were ﬁnally clustered into 1,275 operational taxonomic units (OTUs) at 97% sequence similarity level. The identiﬁed fungal OTUs were dominated by Ascomycota (94.36%) and represented by 21 classes, 68 orders, 176 families, 396 genera, and 689 species. Of 1,275 fungal OTUs, 312 were shared by all the fungal inoculation treatments (Figure 4). The inoculation of ASM represented the highest number of unique OTUs (59), while the lowest number of unique OTUs (10) occurred under The member of Sordariomycetes was the dominant class, with a relative abundance range of 60.94–83.10% across FIGURE 4 Venn diagram displaying the overlap in fungal OUTs composition of A. adsurgens under diferent fungal inoculation treatments. C, non-inoculated control; A, inoculation with Alternaria chlamydospora; S, inoculation with Sarocladium kiliense; M, inoculation with Monosporascus sp.; AS, co-inoculation of Alternaria chlamydospora and Sarocladium kiliense; AM, co-inoculation of Alternaria chlamydospora and Monosporascus sp.; SM, co-inoculation of Sarocladium kiliense and Monosporascus sp.; ASM, combination inoculation of the three species. FIGURE 4 Venn diagram displaying the overlap in fungal OUTs composition of A. adsurgens under diferent fungal inoculation treatments. C, non-inoculated control; A, inoculation with Alternaria chlamydospora; S, inoculation with Sarocladium kiliense; M, inoculation with Monosporascus sp.; AS, co-inoculation of Alternaria chlamydospora and Sarocladium kiliense; AM, co-inoculation of Alternaria chlamydosp and Monosporascus sp.; SM, co-inoculation of Sarocladium kiliense and Monosporascus sp.; ASM, combination inoculation of the three spe 09 Frontiers in Plant Science frontiersin.org Zuo et al. 10.3389/fpls.2022.933738 the various treatments (Figure 5A). However, the member of Agaricomycetes (27.40%) was the most abundant under A inoculated alone when compared with other inoculation treatments. At the level of order classiﬁcation, Hypocreales and Sordariales were the dominant fungal taxon, with relative abundance ranging between 38.53–50.87% and 18.72–33.60%, respectively (Figure 5B). The members of Agaricales (27.36 %) and Coniochaetales (2.63 %) were mainly distributed under A Zuo et al. 10.3389/fpls.2022.933738 the various treatments (Figure 5A). However, the member of Agaricomycetes (27.40%) was the most abundant under A inoculated alone when compared with other inoculation treatments. At the level of order classiﬁcation, Hypocreales and Sordariales were the dominant fungal taxon, with relative abundance ranging between 38.53–50.87% and 18.72–33.60%, respectively (Figure 5B). The members of Agaricales (27.36 %) and Coniochaetales (2.63 %) were mainly distributed under A FIGURE 5 Relative abundances of soil fungi on class (A) and order (B) levels of A. adsurgens. Soil fungal alpha and beta diversities Sobs index values were calculated to describe the observed OTU richness. Alpha diversity, including the Simpson index, Shannon diversity, and evenness index, were analyzed based on OTU richness (Figure 6). The OTU richness of soil fungi under the inoculation of SM was signiﬁcantly higher than that in non-inoculated control, and inoculations of M and AS (Figure 6A). Inoculation with A alone revealed the lowest soil fungal diversity, while SM slightly promoted soil fungal diversity (Figures 6B,C). The inoculation of A alone also caused a decrease in the evenness of the soil fungal community (Figure 6D). In terms of plant growth parameters, Cladorrhinum bulbillosum, unclassiﬁed Coniochaetales, unclassiﬁed Hypocreales fam Incertae sedis, unclassiﬁed Lasiosphaeriaceae, and Phialophora geniculate highly corresponded to plant height and stem branching numbers. Several fungal species were signiﬁcantly correlated with the root growth and biomass, for example, unclassiﬁed Pleosporaceae, as well as unclassiﬁed Lasiosphaeriaceae, were corresponded to root length, surface area, and diameter, and unclassiﬁed Aspergillus and Humicola nigrescens were correlated with root biomass. Nonmetric multidimensional scaling (NMDS) showed that inoculation treatment had no signiﬁcant eﬀect on the composition of the soil fungal community (Supplementary Figure 3). However, the clustering heatmap based on the top 50 most abundant fungal OTUs indicated that the abundance of soil fungi under diﬀerent inoculation treatments changed. Moreover, soil fungal composition associated with A and SM inoculations was far away from other inoculation treatments and revealed certain diﬀerences (Supplementary Figure 4). Frontiers in Plant Science Soil fungal community composition The fungal class and order represents <0.01% of the total reads and were all assigned to “Others.” C, non-inoculated control; A, inoculation with Alternaria chlamydospora; S, inoculation with Sarocladium kiliense; M, inoculation with Monosporascus sp.; AS, co-inoculation of Alternaria chlamydospora and Sarocladium kiliense; AM, co-inoculation of Alternaria chlamydospora and Monosporascus sp.; SM, co-inoculation of Sarocladium kiliense and Monosporascus sp.; ASM, combination inoculation of the three species. FIGURE 5 Relative abundances of soil fungi on class (A) and order (B) levels of A. adsurgens. The fungal class and order represents <0.01% of the total reads and were all assigned to “Others.” C, non-inoculated control; A, inoculation with Alternaria chlamydospora; S, inoculation with Sarocladium kiliense; M, inoculation with Monosporascus sp.; AS, co-inoculation of Alternaria chlamydospora and Sarocladium kiliense; AM, co-inoculation of Alternaria chlamydospora and Monosporascus sp.; SM, co-inoculation of Sarocladium kiliense and Monosporascus sp.; ASM, combination inoculation of the three species. Relative abundances of soil fungi on class (A) and order (B) levels of A. adsurgens. The fungal class and order represents <0.01% of the total reads and were all assigned to “Others.” C, non-inoculated control; A, inoculation with Alternaria chlamydospora; S, inoculation with Sarocladium kiliense; M, inoculation with Monosporascus sp.; AS, co-inoculation of Alternaria chlamydospora and Sarocladium kiliense; AM, co-inoculation of Alternaria chlamydospora and Monosporascus sp.; SM, co-inoculation of Sarocladium kiliense and Monosporascus sp.; ASM, combination inoculation of the three species. 10 Frontiers in Plant Science frontiersin.org Zuo et al. 10.3389/fpls.2022.933738 inoculated alone, while a member of Glomerellales (4.09%) was mainly distributed in the inoculation of SM. sp., unclassiﬁed Penicillium, unclassiﬁed Aspergillus, and Clonostachys sp. were highly correlated with soil urease; Gibellulopsis nigrescens and Cephaliophora sp. had a positive eﬀect on soil acid phosphatase and available phosphorus, and Volvopluteus earlei and Stachybotrys chlorohalonata were related to soil nitrate. However, most fungal species revealed a close relationship with soil organic carbon (14 species) and soil ammonia (9 species), such as unclassiﬁed Sordariomycetes, Chaetomium piluliferum, unclassiﬁed Pleosporales, and unclassiﬁed Sordariales. sp., unclassiﬁed Penicillium, unclassiﬁed Aspergillus, and Clonostachys sp. were highly correlated with soil urease; Gibellulopsis nigrescens and Cephaliophora sp. had a positive eﬀect on soil acid phosphatase and available phosphorus, and Volvopluteus earlei and Stachybotrys chlorohalonata were related to soil nitrate. However, most fungal species revealed a close relationship with soil organic carbon (14 species) and soil ammonia (9 species), such as unclassiﬁed Sordariomycetes, Chaetomium piluliferum, unclassiﬁed Pleosporales, and unclassiﬁed Sordariales. Interrelationship among soil factors, fungi community and plant growth Pearson’s correlation analyses showed signiﬁcant relationships between soil factors and fungal microbial community, as well as the plant growth parameters (Supplementary Table 1). Referring to the correlation coeﬃcients (R values), we used structural equation modeling (SEM) to quantify the relative eﬀects of diﬀerent fungal inoculation treatments on the relationship between the growth of A. adsurgens and soil factors and fungal community (χ2 = 64.206, df = 57, p = 0.239, CFI = 0.945, RMSEA = 0.074; Figure 9). Inoculation treatment inﬂuenced soil pH and nitrate by acting on the abundance of soil fungal species thereby indirectly inﬂuencing the stem branching number, root diameter, and total biomass. The fungal diversity had a signiﬁcant direct eﬀect on soil available phosphorus, which then further inﬂuenced the total biomass. Moreover, the soil fungal community impacted soil nitrate by acting on soil ammonia and ﬁnally inﬂuenced the root diameter architecture. Subsequently, the change in root diameter signiﬁcantly corresponded to the growth of root length and stem branching numbers, thus inﬂuencing the total biomass of host plants. Linear discriminant analysis of efect size We used linear discriminant analysis (LDA) of eﬀect size (LEfSe) to determine the taxa that most likely explain the diﬀerences of soil fungi among inoculation treatments. Indicator fungal species with LDA scores of 2 or greater in the fungal community were conﬁrmed to predict the best biomarker for each category (Figure 7). The signiﬁcant inoculation eﬀect observed among diﬀerent treatment levels was explained by 5, 1, 8, 2, 4, 11, 8, and 6 indicator fungi species, respectively. For example, the taxon of Pluteaceae was signiﬁcantly enriched under A inoculation alone; Scleroramularia, Gaeumannomyces radicicola, Gaeumannomyces, Scleroramularia musae, and an unclassiﬁed Pleosporaceae were signiﬁcantly enriched under the inoculation of AM; and two unclassiﬁed Pleosporaceae and Auxarthron were signiﬁcantly enriched under the inoculation of SM. The inoculation of SM presented the most abundant species with signiﬁcantly diﬀerent eﬀects (11 indicator taxa). Synergistic efect of combined fungal inoculation depending on fungal species The relationship between fungal taxa with the abundance of top 50 and soil factors, as well as plant growth parameters, was presented in heatmap (Figure 8). Members of Cephaliophora In this study, we determined the eﬀects of single or combined inoculation of three root endophytic fungi on 11 frontiersin.org Zuo et al. 10.3389/fpls.2022.933738 FIGURE 6 Species richness and diversity index of soil fungi of A. adsurgens. (A) OTU richness estimated by sobs index; (B) Shannon diversity; (C) Simpson diversity; (D) Shannon’s evenness. C, non-inoculated control; A, inoculation with Alternaria chlamydospora; S, inoculation with Sarocladium kiliense; M, inoculation with Monosporascus sp.; AS, co-inoculation of Alternaria chlamydospora and Sarocladium kiliense; AM, co-inoculation of Alternaria chlamydospora and Monosporascus sp.; SM, co-inoculation of Sarocladium kiliense and Monosporascus sp.; ASM, combination inoculation of the three species. FIGURE 6 Species richness and diversity index of soil fungi of A. adsurgens. (A) OTU richness estimated by sobs index; (B) Shannon diversity; (C) Simpson diversity; (D) Shannon’s evenness. C, non-inoculated control; A, inoculation with Alternaria chlamydospora; S, inoculation with Sarocladium kiliense; M, inoculation with Monosporascus sp.; AS, co-inoculation of Alternaria chlamydospora and Sarocladium kiliense; AM, co-inoculation of Alternaria chlamydospora and Monosporascus sp.; SM, co-inoculation of Sarocladium kiliense and Monosporascus sp.; ASM, combination inoculation of the three species. promoting the growth of desert plant A. adsurgens under a non-sterile substrate simulating natural drought conditions. Meanwhile, the parallel experiment of well-watered treatment was carried out when the fungus was inoculated alone, and the reduced plant height of A. adsurgens seedlings under drought conditions evidenced the eﬀectiveness of imposed drought stress in our experiment (Supplementary Figure 5). Under the drought stress, we demonstrated the synergistic eﬀects between diﬀerent fungal combinations, which are consistent with that of He et al. (2022), who documented the increased dual inoculation eﬀects of beneﬁcial endophytic fungi between Paraboeremia putaminum and Trichoderma viride on the growth of medicinal plant Astragalus mongholicus under water stress conditions. However, not all fungal inoculations participated in plant growth promotion. Regardless of the single or combined inoculations, the eﬀects mainly depended on diﬀerent fungal species and combinations, which approved the previous reports that fungal species may be one of the factors inﬂuencing symbiotic relationships and plant performance (Wazny et al., 2018; Geisen et al., 2021). Liu et al. (2020b) found that the synergistic eﬀect of arbuscular mycorrhizal fungi and endophytic fungi on tall fescue depended on the species of arbuscular mycorrhizal fungi. Frontiers in Plant Science Synergistic efect of combined fungal inoculation depending on fungal species C, non-inoculated control; A, inoculation with Alternaria chlamydospora; S, inoculation with Sarocladium kiliense; M, inoculation with Monosporascus sp.; AS, co-inoculation of Alternaria chlamydospora and Sarocladium kiliense; AM, co-inoculation of Alternaria chlamydospora and Monosporascus sp.; SM, co-inoculation of Sarocladium kiliense and Monosporascus sp.; ASM, combination inoculation of the three species. the root biomass, but did not inﬂuence the shoot biomass, which may be related to the fungi-mediated morphological growth of roots (Li et al., 2019a; Jabborova et al., 2021; Toppo et al., 2022). In this study, inoculation of all fungi alone decreased the plant height of A. adsurgens seedlings, but signiﬁcantly increased the total root length and root surface area. This may be related to endophytic fungal symbiosis cost or resource allocation. Wäli et al. (2006) have documented that seedlings of Festuca ovina containing Epichloë endophytes grew smaller than those without endophytes. Alternatively, the theory of the optimal allocation of plant resources states that plants allocate biomass preferentially to obtain the most growth-limiting resources (Puglielli et al., 2021). Thus, plants tend to invest in root growth at the expense of reduced aboveground growth in an arid environment, resulting in a decrease in aboveground plant height and biomass (Schall et al., 2012; Azizi et al., 2021). However, the decreased root diameter indicated that the fungal symbionts promote the growth and development of ﬁne roots of A. adsurgens seedlings. A well-developed root system and architecture are beneﬁcial for desert plants to improve water and nutrient absorption in deep dry soil (Likar and Regvar, 2013; Li et al., 2019a). This contributes to the ecological adaptation and resistance of host plants to adverse environments in desert habitats. also increase or decrease certain microbial populations to alter the rhizospheric microbial community composition (Achouak et al., 2019). However, the high diversity of fungal species also means that more plant resources are needed to sustain microbial growth and reproduction, as well as more complex interspeciﬁc interactions (Voges et al., 2019; Wagg et al., 2019). Thus, the allocation of plant resources to microorganisms may be responsible for our failure to observe the fungal promoting plant growth under the combined inoculation of ASM. Additionally, the interactions between fungal species may inﬂuence plant performance by altering the way host nutrient acquisition, uptake, and metabolism (He et al., 2017; Liu et al., 2020a). Synergistic efect of combined fungal inoculation depending on fungal species Therefore, various interactions of diﬀerent fungal species in plant symbiosis and competition may have important implications for host plant growth and health. Synergistic efect of combined fungal inoculation depending on fungal species Yang et al. (2021) also showed that root endophytes represented an important role in promoting the biological process of the host relying on the type of host and the species of endophytes. Therefore, the use of suitable fungal endophytes, either singly or in combination, remains a major challenge as diﬀerent fungal species may play diﬀerent roles in plant symbiosis. It is worth noting that in our study, the response of plant growth to fungal inoculation is diﬀerent from that of the soil fungal community. The combined inoculation of ASM did not have positive eﬀects on the growth of A. adsurgens, but most signiﬁcantly promoted the diversity and abundance of soil fungal community. This suggests that combined fungal inoculation mediates the composition and ecological adaptation of the soil microbial community (Pang et al., 2020; He et al., 2022). Studies have shown that fungal inoculation may impact competition for niches in the fungal community, resulting in changes in species abundance (Wagg et al., 2015; Van Nuland and Peay, 2020). Due to the dependence of endophytes on plant habitat and nutrition, host plants may face tradeoﬀs in resource allocation among symbionts (Niwa et al., 2018). Under drought conditions, plants may allow more fungal species to coexist by controlling the abundance of dominant taxa; it may Frontiers in Plant Science 12 frontiersin.org Zuo et al. 10.3389/fpls.2022.933738 FIGURE 7 Indicator fungal species with LDA scores of 2 or greater in the rhizosphere of A. adsurgens under diferent inoculation treatments. LDA Efect Size (LEfSe) algorithm was used on OTUs level to determine taxa that were diferentially represented among inoculation treatments. C, non-inoculated control; A, inoculation with Alternaria chlamydospora; S, inoculation with Sarocladium kiliense; M, inoculation with Monosporascus sp.; AS, co-inoculation of Alternaria chlamydospora and Sarocladium kiliense; AM, co-inoculation of Alternaria chlamydospora and Monosporascus sp.; SM, co-inoculation of Sarocladium kiliense and Monosporascus sp.; ASM, combination inoculation of the three species. FIGURE 7 Indicator fungal species with LDA scores of 2 or greater in the rhizosphere of A. adsurgens under diferent inoculation treatments. LDA Efect Size (LEfSe) algorithm was used on OTUs level to determine taxa that were diferentially represented among inoculation treatments. Fungal inoculation-mediated morphological growth of A. adsurgens plants Using biomass quantiﬁcation as an indicator of fungal inoculation performance, we found that fungi mainly promoted frontiersin.org 13 Frontiers in Plant Science Zuo et al. 10.3389/fpls.2022.933738 FIGURE 8 Correlation analysis results showing fungal species associated with soil physicochemical properties and plant growth parameters. U, soil urease; ACP, soil acid phosphatase; P, soil available phosphorus; SOC, soil organic carbon; NO3, soil nitrate; NH4, soil ammonia; HEI, plant height; STB, stem branching numbers; RLE, root length; RSA, root surface area; RDA, root diameter; SBI, shoot biomass; RBI, root biomass; TBI, total biomass. Signiﬁcance level is shown at: p < 0.05; p < 0.01; *p < 0.001. Correlation analysis results showing fungal species associated with soil physicochemical properties and plant growth parameters. U, soil urease; ACP, soil acid phosphatase; P, soil available phosphorus; SOC, soil organic carbon; NO3, soil nitrate; NH4, soil ammonia; HEI, plant height; STB, stem branching numbers; RLE, root length; RSA, root surface area; RDA, root diameter; SBI, shoot biomass; RBI, root biomass; TBI, total biomass. Signiﬁcance level is shown at: p < 0.05; p < 0.01; p < 0.001. Frontiers in Plant Science Efects of fungal inoculation on soil properties and rhizospheric fungal community of plant nutrient uptake (Yadav et al., 2015; Koshila Ravi et al., 2019). In our study, the decreased soil pH under inoculations of A. chlamydospora may be related to the enrichment of the acidophilic fungus Pluteaceae. The members of Pluteaceae have been reported to mainly adapted to grow in acidic soil with pH 4.5–4.99 with a strong ability to decompose organic matter, thus Soil fungi interact in complex ways and play important roles in soil nutrient cycling, transformation, and promotion Frontiers in Plant Science 14 frontiersin.org Zuo et al. 10.3389/fpls.2022.933738 FIGURE 9 Structural equation model (SEM) showing the causal relationships among soil fungal community, soil factors, and plant growth parameters of A. adsurgens associated with diferent inoculation treatments. The solid lines and dashed lines indicate signiﬁcant and non-signiﬁcant pathways, respectively. The width of the solid lines indicates the strength of the causal efect, and the numbers near the arrows indicate the standardized path coefcients. Signiﬁcant values are indicated by (p < 0.05), (p < 0.01), and (p < 0.001). The numbers in the upper-right corner of the box indicate the R2 values and represent the proportion of variance explained for each variable. P, soil available phosphorus; SOC, soil organic carbon; NO3, soil nitrate; NH4, soil ammonia; STB, stem branching numbers; RLE, root length; RDA, root diameter; TBI, total biomass. FIGURE 9 Structural equation model (SEM) showing the causal relationships among soil fungal community, soil factors, and plant growth parameters of A. adsurgens associated with diferent inoculation treatments. The solid lines and dashed lines indicate signiﬁcant and non-signiﬁcant pathways, respectively The width of the solid lines indicates the strength of the causal efect and the numbers near the arrows indicate the standardized FIGURE 9 Structural equation model (SEM) showing the causal relationships among soil fungal community, soil factors, and plant growth parameters of A. adsurgens associated with diferent inoculation treatments. The solid lines and dashed lines indicate signiﬁcant and non-signiﬁcant pathways, respectively. The width of the solid lines indicates the strength of the causal efect, and the numbers near the arrows indicate the standardized path coefcients. Signiﬁcant values are indicated by (p < 0.05), (p < 0.01), and *(p < 0.001). The numbers in the upper-right corner of the box indicate the R2 values and represent the proportion of variance explained for each variable. Frontiers in Plant Science Efects of fungal inoculation on soil properties and rhizospheric fungal community Moreover, the most enriched number of diﬀerential fungal species appeared in the inoculation of SM further indicating that fungal cross-inoculation plays a dominant role in the process of plant–soil ecological adaptation by adjusting the abundance and quantity of soil fungal taxa. The synergistic eﬀect indicated that fungal combination inoculation can improve the rhizospheric ecological environment in natural habitat or the presence of a local microbiome, which is more than that of single strain inoculation (Vorholt et al., 2017). Potential application of fungal assemblages in desert area Promoting better growth of desert plants, improving the soil environment, and the maintenance of soil microbial diversity, are critical for the stability of arid ecosystems. In the present study, the enhanced stem branching and root growth A. adsurgens under S. kiliense and SM inoculations will help desert plants to grow better and adapt to the desert environment to promote wind prevention and sand ﬁxation (Li et al., 2018; Zuo et al., 2020). However, soil acidiﬁcation under A. chlamydospora and co-inoculations of AM and AS indicates that this species may be potentially applied to neutralize soil pH in alkaline areas. In contrast, Monosporascus sp. alone and its combined inoculation of AM and SM markedly promoted the accumulation of soil organic carbon, which is speculated to be important for the nutrient and fertility health of desert soil (Hammad et al., 2020). Most importantly, the synergistic eﬀects of combined inoculation on the increased diversity and abundance of rhizospheric fungal community suggest that more fungal species than fewer might be conducive to regulating the composition and structure of microbial community (Mawarda et al., 2020; Cheng et al., 2021). This may be beneﬁcial to the subsequent restoration of soil microbial functions in the Efects of fungal inoculation on soil properties and rhizospheric fungal community Acremonium and Diatrypaceae have been widely reported to be isolated from desert halophytes and showed the ability to improve host resistance to biotic stress (Jalili et al., 2020; Ameen et al., 2021). However, Scleroramularia and Gaeumannomyces have been reported as pathogens of crops (Coombs et al., 2004; Li et al., 2011), which may be related to the transformation of endophytic fungal lifestyle. Studies have shown that the phase of endosymbiosis represents a balanced interaction between fungal virulence and host defense factors (Kuo et al., 2015). In the presence of appropriate environmental factors, saprophytic and pathogenic bacteria may transform into endophytes. Nevertheless, some beneﬁcial fungal species, such as Aspergillus, Psilocybe, and Strophariaceae taxa, were enriched under the inoculation of ASM. Aspergillus has been reported to be the most common genus of desert medicinal plants and can stimulate the growth of desert medicinal plants (Ntemafack et al., 2021). Psilocybe was considered to be an important biological source for medical compounds and displays important research value in phytochemistry, antioxidant and cellular immunity (Nkadimeng et al., 2020). Strophariaceae were found as basidiomycetes, and their mycelia biomass had a signiﬁcant eﬀect on garlic scale and cucumber seed germination and seedling growth (Regeda et al., 2021). We speculate that the combined inoculation of the three fungi in this study may have promoted the increase of Basidiomycetes in the rhizospheric soil of A. adsurgens. This may also suggest that when inoculated with ASM, plants may reduce resource allocation for growth in order to recruit more beneﬁcial microorganisms (Vandenkoornhuyse et al., 2015). According to previous studies, due to the diﬀerent growth rates of fungal species, the symbiotic eﬀects of slow-growing fungi may be delayed (Getachew et al., 2019; Vrabl et al., 2019). Consequently, considering the short duration of our pot experiment, the beneﬁcial eﬀects of the rhizospheric fungal microbes recruited by ASM mixed inoculation on plant growth will likely take a longer time to manifest. Moreover, the most enriched number of diﬀerential fungal species appeared in the inoculation of SM further indicating that fungal cross-inoculation plays a dominant role in the process of plant–soil ecological adaptation by adjusting the abundance and quantity of soil fungal taxa. The synergistic eﬀect indicated that fungal combination inoculation can improve the rhizospheric ecological environment in natural habitat or the presence of a local microbiome, which is more than that of single strain inoculation (Vorholt et al., 2017). Efects of fungal inoculation on soil properties and rhizospheric fungal community P, soil available phosphorus; SOC, soil organic carbon; NO3, soil nitrate; NH4, soil ammonia; STB, stem branching numbers; RLE, root length; RDA, root diameter; TBI, total biomass. playing an important role in soil bioremediation (Mohammadi- Sichani et al., 2017; Kunca and Pavlik, 2019). However, the increase of soil organic carbon, especially under Monosporascus sp. inoculation, may be related to the carbon conversion process mediated by the increased abundance of soil fungi (Kohout et al., 2018). Alternatively, the dominant group Sordariomycetes may also be associated with the increased organic carbon, which has been reported to favor the mineralization of soil aggregate organic matter, thereby promoting the content of soil organic matter (Xu et al., 2021). Nevertheless, the reduced soil ammonia under fungal inoculations probably corresponded to the nitrogen ﬁxation by rhizobia. In our experiment, due to the identity of the leguminous plant, nodules were indeed observed in the roots of A. adsurgens plants, especially under the fungal inoculations. Therefore, the presence of adequate nitrogen-ﬁxing bacteria may reduce the dependence of plants on microbial nitrogen mineralization (Kakraliya et al., 2018). Meanwhile, the symbiosis of nitrogen-ﬁxing bacteria may also be related to plant growth promotion, which in turn triggers the higher demands of the host plant for other nutrients, especially phosphorus demand in N-ﬁxing legumes (Png et al., 2017). In our study, the increases in soil available P under fungal combined inoculation were indeed observed, which was inferred to be relevant to the secretion of various hydrolases by fungi to promote the transformation of insoluble phosphorus (Fabia´nska et al., 2019). Thus, we speculate that symbiotic nitrogen-ﬁxing bacteria and endophytic fungi may exhibit complementary beneﬁts in terms of nutrient access to their common host (Tang et al., 2019; Primieri et al., 2021). Such tri-partite symbiosis of plant-endophytic fungi-N ﬁxing bacteria should be further explored in future studies. In the current study, the enrichment of soil fungal communities was diﬀerent under the combined inoculation treatments. Despite the acidiﬁed soil pH, inoculations of AM and AS mitigated the loss of fungal diversity and enrichment of acidophilic fungi caused by acid soils. In contrast, members of Acremonium, Entoloma cremeoalbum, Scolecobasidium, and Diatrypaceae were enriched under AS inoculation; while Frontiers in Plant Science 15 frontiersin.org Zuo et al. Zuo et al. 10.3389/fpls.2022.933738 Scleroramularia and Gaeumannomyces were enriched under AM inoculation. Efects of fungal inoculation on soil properties and rhizospheric fungal community rhizospheric microorganisms (Trivedi et al., 2020). In our study, we found that fungal inoculation mainly inﬂuenced soil nutrients by changing soil fungal species diversity and richness, thereby indirectly impacting the number of stem branches, root diameter, and total biomass of A. adsurgens seedlings. Certain fungal species, such as Sordariomycetes, Chaetomium piluliferum, and Pleosporales, were directly linked to soil nutrient cycling. However, in addition to the indirect eﬀects, the fungal community themselves could also directly inﬂuence plant growth. For example, Aspergillus, Humicola nigrescens, Pleosporaceae, and Lasiosphaeriaceae were highly correlated with root growth of A. adsurgens. This may be due to the formation of a stable symbiotic relationship between root fungi and host plants (Bouasria et al., 2012; Yeh et al., 2019). Plants can recruit target microbial communities through signaling molecules, and subsequently exert selective pressure through the immune system, speciﬁc nutrient supply or habitat type, etc., allowing the successful colonization of beneﬁcial microorganisms (Foster et al., 2017; Martin et al., 2017; Cordovez et al., 2019). The interaction or synergistic eﬀect among root endophytic fungi responds to plant growth by creating favorable microﬂora in the rhizospheric environment of A. adsurgens plants. Therefore, the enrichment of beneﬁcial microbial communities in the rhizospheric soil is crucial for plant survival and development and can confer abiotic and biotic tolerance in plants to improve ﬁtness. rhizospheric microorganisms (Trivedi et al., 2020). In our study, we found that fungal inoculation mainly inﬂuenced soil nutrients by changing soil fungal species diversity and richness, thereby indirectly impacting the number of stem branches, root diameter, and total biomass of A. adsurgens seedlings. Certain fungal species, such as Sordariomycetes, Chaetomium piluliferum, and Pleosporales, were directly linked to soil nutrient cycling. However, in addition to the indirect eﬀects, the fungal community themselves could also directly inﬂuence plant growth. For example, Aspergillus, Humicola nigrescens, Pleosporaceae, and Lasiosphaeriaceae were highly correlated with root growth of A. adsurgens. This may be due to the formation of a stable symbiotic relationship between root fungi and host plants (Bouasria et al., 2012; Yeh et al., 2019). Plants can recruit target microbial communities through signaling molecules, and subsequently exert selective pressure through the immune system, speciﬁc nutrient supply or habitat type, etc., allowing the successful colonization of beneﬁcial microorganisms (Foster et al., 2017; Martin et al., 2017; Cordovez et al., 2019). Frontiers in Plant Science Efects of fungal inoculation on soil properties and rhizospheric fungal community The interaction or synergistic eﬀect among root endophytic fungi responds to plant growth by creating favorable microﬂora in the rhizospheric environment of A. adsurgens plants. Therefore, the enrichment of beneﬁcial microbial communities in the rhizospheric soil is crucial for plant survival and development and can confer abiotic and biotic tolerance in plants to improve ﬁtness. Scleroramularia and Gaeumannomyces were enriched under AM inoculation. Acremonium and Diatrypaceae have been widely reported to be isolated from desert halophytes and showed the ability to improve host resistance to biotic stress (Jalili et al., 2020; Ameen et al., 2021). However, Scleroramularia and Gaeumannomyces have been reported as pathogens of crops (Coombs et al., 2004; Li et al., 2011), which may be related to the transformation of endophytic fungal lifestyle. Studies have shown that the phase of endosymbiosis represents a balanced interaction between fungal virulence and host defense factors (Kuo et al., 2015). In the presence of appropriate environmental factors, saprophytic and pathogenic bacteria may transform into endophytes. Nevertheless, some beneﬁcial fungal species, such as Aspergillus, Psilocybe, and Strophariaceae taxa, were enriched under the inoculation of ASM. Aspergillus has been reported to be the most common genus of desert medicinal plants and can stimulate the growth of desert medicinal plants (Ntemafack et al., 2021). Psilocybe was considered to be an important biological source for medical compounds and displays important research value in phytochemistry, antioxidant and cellular immunity (Nkadimeng et al., 2020). Strophariaceae were found as basidiomycetes, and their mycelia biomass had a signiﬁcant eﬀect on garlic scale and cucumber seed germination and seedling growth (Regeda et al., 2021). We speculate that the combined inoculation of the three fungi in this study may have promoted the increase of Basidiomycetes in the rhizospheric soil of A. adsurgens. This may also suggest that when inoculated with ASM, plants may reduce resource allocation for growth in order to recruit more beneﬁcial microorganisms (Vandenkoornhuyse et al., 2015). According to previous studies, due to the diﬀerent growth rates of fungal species, the symbiotic eﬀects of slow-growing fungi may be delayed (Getachew et al., 2019; Vrabl et al., 2019). Consequently, considering the short duration of our pot experiment, the beneﬁcial eﬀects of the rhizospheric fungal microbes recruited by ASM mixed inoculation on plant growth will likely take a longer time to manifest. Interactions among plants growth, soil nutrients, and fungal community In natural ecosystems, endophytic fungi have complex interactions with host plants and are closely related to Frontiers in Plant Science 16 frontiersin.org Zuo et al. 10.3389/fpls.2022.933738 10.3389/fpls.2022.933738 Funding This study was ﬁnancially supported by the National Natural Science Foundation of China (Project No. 31770561). 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. SUPPLEMENTARY FIGURE 2 Rarefaction curves for the observed soil fungal operational taxonomic units (OTUs). C, non-inoculated control; A, inoculation with Alternaria chlamydospora; S, inoculation with Sarocladium kiliense; M, inoculation with Monosporascus sp.; AS, co-inoculation of Alternaria chlamydospora and Sarocladium kiliense; AM, co-inoculation of Alternaria chlamydospora and Monosporascus sp.; SM, co-inoculation of Sarocladium kiliense and Monosporascus sp.; ASM, combination inoculation of the three species. Conclusion 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. This study explored the synergistic eﬀects of three extremely arid habitat-adapted root endophytes, on the growth of non- host desert plant A. adsurgens, under drought and non- sterile conditions. Fungal inoculation markedly improved the root growth and biomass of A. adsurgens, and this favorable eﬀect was dependent on speciﬁc fungal species and their combinations. However, the synergistic eﬀect of fungal inoculation on the rhizospheric soil fungi community was inconsistent with that of plant growth. The three species combined inoculation that promoted the diversity of rhizospheric fungal community most had no eﬀect on plant root growth when compared to the control treatments. This insinuates the diﬀered responses between plant growth and rhizospheric fungal community to the multiple fungal interactions. Thus, the fungal species and the combined consortium used should be carefully evaluated owing to their diﬀerential eﬀects on plant growth and soil microhabitat, as well as their complementary functional roles. Further investigation of interactions between host plants and rhizospheric soil microhabitat mediated by fungal combined inoculations are still needed to be elucidated. Author contributions desert. However, this needs to be interpreted with caution, as fungal communities do not independently in line with the plant growth-promoting eﬀects. In desert habitats, the microbial community still includes bacteria, archaea, nematodes, and other groups, which may have a very distinct response to the endophytic fungi inoculation (Trivedi et al., 2020). Therefore, the changes in the entire microbial community may be in line with the plant growth-promoting eﬀects and fully reﬂect the soil microbial functions. Based on our experimental results, inoculations of S. kiliense alone and SM were recommended for use in the desert plant A. adsurgens due to their promoted plant performance, improved soil fungal diversity, and soil organic carbon without the acidiﬁcation of soil pH. Our research hints at the combined consortium of multiple fungi as a possible solution for promoting sustainable desert conservation and restoration. However, the optimal combination of strains and the complementary eﬀects between diﬀerent species should be emphasized when constructing a synthetic community (Li et al., 2021). Y-LZ and X-LH conceived and designed the experiments and wrote the manuscript. Y-LZ and Q-NH performed the experiments. Y-LZ, LQ, and J-QL analyzed the data. All authors contributed to the article and approved the submitted version. 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Cluster analysis was performed based on Bray–Curtis similarities. C, non-inoculated control; A, inoculation with Alternaria chlamydospora; S, inoculation with Sarocladium kiliense; M, inoculation with Monosporascus sp.; AS, co-inoculation of Alternaria chlamydospora and Sarocladium kiliense; AM, co-inoculation of Alternaria chlamydospora and Monosporascus sp.; SM, co-inoculation SUPPLEMENTARY FIGURE 5 Comparison of plant height under well-irrigated water and drought treatment when fungi were inoculated alone. Diferent lowercase letters indicate signiﬁcant diferences (p < 0.05). C, non-inoculated control; A, inoculation with Alternaria chlamydospora; S, inoculation with Sarocladium kiliense; M, inoculation with Monosporascus sp.; WW, well-irrigated water; DS, drought stress. SUPPLEMENTARY FIGURE 3 The datasets presented in this study can be found in online repositories. The raw data sequences were deposited in the NCBI Sequence Read Archive (SRA) under the Bioproject number PRJNA798873 and Bio Sample accession numbers SAMN25131707 to SAMN25131730. Non-metric multidimensional scaling (NMDS) ordination of soil fungal community composition of A. adsurgens. The dissimilarities of fungi were based on the Bray–Curtis method and the non-parametric ANOSIM test was used to examine the signiﬁcant diference based on 999 permutations. Ellipses in the plots represent the grouping interval of fungi in diferent inoculation treatments. C, non-inoculated control; A, inoculation with Alternaria chlamydospora; S, inoculation with Sarocladium kiliense; M, inoculation with Monosporascus sp.; AS, Frontiers in Plant Science 17 frontiersin.org Zuo et al. 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https://openalex.org/W2800004384	https://www.repository.cam.ac.uk/bitstream/1810/279263/3/1-s2.0-S0006899318302312-main.pdf	English	null	The physiological and pathological biophysics of phase separation and gelation of RNA binding proteins in amyotrophic lateral sclerosis and fronto-temporal lobar degeneration	Brain research	2,018	cc-by	13,007	a r t i c l e i n f o Article history: Received 20 February 2018 Received in revised form 27 April 2018 Accepted 28 April 2018 Available online 30 April 2018 Keywords: Amyotrophic lateral sclerosis Arginine methylation Cation-pi interactions Frontotemporal dementia Phase separation RNA binding proteins Article history: Received 20 February 2018 Received in revised form 27 April 2018 Accepted 28 April 2018 Available online 30 April 2018 Many RNA binding proteins, including FUS, contain moderately repetitive, low complexity, intrinsically disordered domains. These sequence motifs have recently been found to underpin reversible liquid: liq- uid phase separation and gelation of these proteins, permitting them to reversibly transition from a monodispersed state to liquid droplet- or hydrogel-like states. This function allows the proteins to serve as scaffolds for the formation of reversible membraneless intracellular organelles such as nucleoli, stress granules and neuronal transport granules. Using FUS as an example, this review examines the biophysics of this physiological process, and reports on how mutations and changes in post-translational state alter phase behaviour, and lead to neurodegenerative diseases such as amyotrophic lateral sclerosis and fron- totemporal lobar degeneration. Keywords: Amyotrophic lateral sclerosis Arginine methylation Cation-pi interactions Frontotemporal dementia Phase separation RNA binding proteins 2018 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/). The discovery of missense mutations in multiple RNA binding proteins, such as fused in sarcoma (FUS) (Kwiatkowski et al., 2009; Vance et al., 2009), transactive response DNA binding pro- tein 43 (TDP-43) (Kabashi et al., 2008; Sreedharan et al., 2008), heterogeneous nuclear ribonucleoprotein (hnRNP) A1 and A2/B1 (Kim et al., 2013), in patients with familial forms of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), has opened up a new and highly productive avenue of research into the pathobiology of ALS and FTD. It has also shed light on a previously poorly recognised ﬁeld of cell biology, namely the role of intrinsically disordered proteins in the formation and function of membraneless intracellular organelles such as nucleoli, stress granules, ribonucleoprotein (RNP) granules, P-bodies and neuronal transport granules. A common neuropathological feature of ALS and FTLD associ- ated with mutations in RNA binding proteins is the deposition of visible aggregates of the corresponding proteins in the nucleus and/or cytoplasm of neurons and glia (Mackenzie and Neumann, 2016; Neumann et al., 2006, 2009; Rademakers et al., 2012). Peter St George-Hyslop a,b,⇑, Julie Qiaojin Lin c, Akinori Miyashita b, Emma C. Phil Suzanne J. Randle a, GuoZhen Wang a a Cambridge Institute for Medical Research, Department of Clinical Neurosciences, University of Cambridge, Cambridge CB2 0XY, UK b Tanz Centre for Research in Neurodegenerative Diseases, and Departments of Medicine, Medical Biophysics and Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario M5S 3H2, Canada c Department of Physiology, Development, and Neuroscience, University of Cambridge, Cambridge CB2 3DY, UK https://doi.org/10.1016/j.brainres.2018.04.036 0006-8993/ 2018 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/). Brain Research 1693 (2018) 11–23 Brain Research 1693 (2018) 11–23 Abbreviations: ADMA FUS, asymmetrically di-methylated arginine FUS; CHOP, C/EBP homologous protein gene; DDX4, DEAD box helicase 4; EWS, Ewing sarcoma protein; fALS, familial amyotrophic lateral sclerosis; FTLD, frontotemporal lobar degeneration; FUS, fused in sarcoma protein; hnRNP, heterogeneous nuclear ribonucleoprotein; PTM, post-translational modiﬁcation; PY-NLS, proline tyrosine nuclear localisation signal; QGSY, glutamine glycine serine and tyrosine repeats motif; RGG, arginine glycine glycine repeat motif; RRM, RNA recognition Motif; SMN, survival motor neuron; TAF15, TATA box binding protein 15; TDP-43, transactive response DNA binding protein 43; TNPO1, transportin 1/karyopherin b2. ⇑Corresponding author. E-mail address: p.hyslop@utoronto.ca (P. St George-Hyslop). The physiological and pathological biophysics of phase separation and gelation of RNA binding proteins in amyotrophic lateral sclerosis and fronto-temporal lobar degeneration The physiological and pathological biophysics of phase separation and gelation of RNA binding proteins in amyotrophic lateral sclerosis and fronto-temporal lobar degeneration Peter St George-Hyslop a,b,⇑, Julie Qiaojin Lin c, Akinori Miyashita b, Emma C. Phillips a, Seema Qamar a, Suzanne J. Randle a, GuoZhen Wang a E-mail address: p.hyslop@utoronto.ca (P. St George-Hyslop). a r t i c l e i n f o These protein aggregates display subtle differences in their staining and biochemical characteristics compared to conventional amyloid b-sheet rich aggregates associated with other neurodegenerative diseases such as Ab, tau and a-synuclein. For instance, they stain poorly with amyloidophyllic dyes such as thioﬂavin T, they contain little b-sheet content on circular dichroism, and they are partially soluble in urea (Mann and Snowden, 2017; Neumann et al., 2006, 2009; Rademakers et al., 2012). Because these assemblies are dis- tinguishable from classical amyloids, it has become of intense interest to determine the underlying mechanisms of the neurotox- icity of these aggregates (Bowden and Dormann, 2016; Coady and Manley, 2015; Kim and Taylor, 2017; Lagier-Tourenne et al., 2010). P. St George-Hyslop et al. / Brain Research 1693 (2018) 11–23 12 This review focuses on recent work suggesting that many of these soluble RNA binding proteins possess the unique biophysical property of being able to reversibly transition between a dispersed (mixed) state; a phase-separated state as liquid protein droplets suspended within a liquid; or in a gelled state as soft hydrogels somewhat similar to jelly dessert. This review will also describe work that has shown that disease associated mutations and disease associated changes in the post-translational modiﬁcations (PTM) in some of these proteins dramatically alter this process, resulting in formation of stable condensed gelled states that are not easily reversible by the cell. The physics of separation of polymers into droplets and gels are well known in materials science, but were not widely thought about in biological science until very recently. As a result, the terminology used in this ﬁeld is in a state of ﬂux, although efforts are being made to assemble a uniform nomencla- ture for these processes. Currently, the biophysical condensation process of polymer phase separation from a dispersed state into liquid droplet suspended in a liquid has been termed ‘‘liquid: liquid phase separation” or ‘‘coacervation”. The phase separation from a dispersed or liquid droplet state into a gelled state has been termed ‘‘gelation”. A more general term that is often applied to these alter- nate biophysical states is ‘‘condensate” (Shin and Brangwynne, 2017). However, the reader is alerted to the fact that many other terms, such as ‘‘de-mixing” are also frequently used. rium states. Instead, they are ‘‘metastable” assemblies whose bio- physical state is fragile, and is easily perturbed by external modulators. a r t i c l e i n f o Such external modulators include post-translational modiﬁcations and remodelling by ATPase-driven chaperones and disaggregases (see below). Conceptually, the simplest membraneless intracellular orga- nelle would be a ‘‘homotypic” droplet composed of a single phase separating polymer. However, in practical terms, most cellular membraneless organelles are more complex. As an example of morphological complexity, stress granules have been described as having a semisolid gel-like cores (Jain et al., 2016) or multiple, distributed small nanocores (Niewidok et al., 2018), a difference which might reﬂect differences between ﬁxed and unﬁxed samples and/or in cell type of origin. Other membraneless organelles such as nucleoli, are also composed of multiple distinct liquid droplets within a larger encompassing droplet (Feric et al., 2016), which can be re-created experimentally (Schmidt and Rohatgi, 2016). Membraneless organelles are also biochemically complex, often containing hundreds or even thousands of RNA and protein com- ponents (Fritzsche et al., 2013; Jain et al., 2016; Markmiller et al., 2018; Youn et al., 2018). Moreover, the component proteins are generally not speciﬁc to a single type of membraneless organelle, with many proteins being components of several nosologically dis- tinct membraneless organelles (Fritzsche et al., 2013; Jain et al., 2016; Markmiller et al., 2018; Youn et al., 2018). The composition of some complexes can differ under different contexts (normal ver- sus disease) or cell type (Markmiller et al., 2018; Youn et al., 2018). Nevertheless, despite this morphological and biochemical com- plexity each of these condensates are still governed by, and pre- dicted by, the same general biophysical rules that govern the assembly of simpler complexes, which are discussed below. 1. Liquid droplets and hydrogels as the building block of membraneless organelles The unique biophysical property of reversible condensation of RNA binding proteins associated with ALS and FTD (and also of a growing list of other proteins) turns out to be crucial for the forma- tion of a subset of intracellular organelles which lack limiting membranes. These membranous organelles include nucleoli, stress granules, neuronal transport granules, and postsynaptic densities to name a few. Most intracellular organelles possess a limiting membrane which, because of their very different biophysical prop- erties and ability to bind histological dyes, facilitated their visual- isation by early investigators using conventional light microscopy. In contrast, intracellular organelles that lack limiting membranes, and that have biophysical properties very similar to those of the surrounding cytoplasm, were difﬁcult to visualise by conventional light and electron microscopy. As a result, their authenticity was initially questioned. However, recent advances in live cell imaging methods including the use of ﬂuorescent tags and tools such as optical tweezers, have made it clear that membraneless organelles such as nucleoli, stress granules and RNP granules are indeed authentic physiological structures (Banani et al., 2017; Brangwynne et al., 2009; Hyman et al., 2014). 2. Functional implications of physiological phase separation and gelation The biophysical properties of membraneless organelles, which allow reversible transition between dispersed, liquid droplet and gel-like states, confer the ability of these organelles to perform a wide range of biological functions such as transport, storage and physical colocalization of components of intracellular signalling or metabolic machinery. For instance, stress granules allow sequestration of cargo (i.e. key translationally-stalled mRNA tran- scripts in the case of stress granules) during cellular stress (Protter and Parker, 2016). Neuronal transport granules sequester and transport key cargo elements involved in regulated local pro- tein translation in axon terminals and dendrites (Holt and Bullock, 2009; Holt and Schuman, 2013; Shigeoka et al., 2016). Other membraneless organelles, such as the post-synaptic density in neuronal dendritic spines allow the assembly of speciﬁc reaction components necessary for signalling downstream of the post- synaptic membrane (Zeng et al., 2016). The diversity of the cell types harbouring membraneless organelles, and the diversity of their functions, emphasizes the critical role played in cell biology by membraneless organelles and by the components which under- pin their ability to phase separate. Generally, but with some exceptions, membraneless organelles have the biophysical characteristics expected of liquid droplets suspended in an immiscible liquid. They are often spherical. They fuse into larger droplets when they contact each other (Fig. 1). They display viscosity similar to that of water. The component polymers that make the 3D structure of the condensate (‘‘scaf- folds”) are generally not bound to each other by strong covalent forces. As a result, the component polymers of these membraneless organelles often display rapid exchange with the surrounding solute. Other molecules in the solute which are capable of binding to the scaffold proteins can also rapidly partition between the solute and the droplet/gel phase by diffusion. The efﬁciency of the partitioning of these ‘‘client” or ‘‘cargo” molecules reﬂects a variety of factors including the afﬁnity of the scaffold protein for the client (e.g. afﬁnity for binding of speciﬁc RNAs to the RNA Recognition Motifs of RNA binding proteins). Finally, it is impor- tant to note that unlike conventional protein complexes, these membraneless organelles are not in stable, steady-state equilib- 4. Biophysics of liquid-liquid phase separating polymers group of soluble proteins that can exist in a soluble dispersed state, but that can also transition into a separate physical state in which the protein exists in liquid droplets of concentrated protein sus- pended within the liquid solvent (i.e. the cytoplasm/nucleoplasm). Some of these proteins can also condense into jelly-like states, which are referred to as ‘‘hydrogels”. This ability to phase separate is also held by other biological polymers (e.g. some RNAs) (Jain and Vale, 2017) and by man-made polymers. In the last several years the biophysics of phase-separated intra- cellular structures has been probed using both theoretical and experimental approaches (Banani et al., 2017; Bergeron-Sandoval et al., 2016; Brangwynne et al., 2015; Pak et al., 2016; Shin and Brangwynne, 2017; Wei et al., 2017). The phase transition of sol- uble polymers between dispersed, liquid droplet and hydrogel- like phases, represents a well-understood thermodynamic event in which an initial uniform mixture of dispersed polymers (e.g. sin- gle proteins, single RNA, or mixtures of RNA and protein) ﬁnds a lower free energy state when the components separate into one or more distinct phases (e.g. a polymer-rich liquid droplet phase and a separate polymer-depleted solute phase) (Fig. 1). This situa- tion typically occurs when each of the component molecules in the mixture ‘‘prefers” interactions with itself over interactions with the solute molecules. In the simplest instance, this might result in the partitioning of the protein or RNA polymer into a polymer-rich droplet (where its concentration is then much higher than in the initial dispersed liquid state) suspended in a polymer-depleted liq- uid solvent (Fig. 1). However, other more complicated phase sepa- rations can be imagined. For instance, two protein molecules, or protein plus RNA molecules, might cooperatively interact with each other to form a liquid droplet enriched in both components, A common feature of these phase-separating proteins is the presence of at least one domain composed of repetitive stretches of amino acids with polar side chains (glycine, glutamine, aspara- gine and serine), nonpolar side chains (proline), positive side chains (arginine, lysine), negative side chains (aspartate, gluta- mate) or aromatic side chains (phenylalanine, tryptophan and tyr- osine). Hydrophobic residues on the other hand, are typically underrepresented. These amino acids are often arranged in imper- fect, short repetitive motifs (e.g. enrichment of glutamine glycine serine tyrosine (‘‘QGSY”) repeats in the N-terminus of FUS) (Fig. 2A). 3. Composition and structure of proteins that form liquid droplets Most soluble intracellular proteins (e.g. classical enzymes) exist as uniformly mixed, dispersed solutions of either monomeric pro- tein or higher order complexes. These proteins generally fold into a limited number of well-deﬁned, well-ordered three-dimensional shapes (‘‘folds”) that confer the functional properties of these pro- teins. However, recent work has identiﬁed an increasingly large P. St George-Hyslop et al. / Brain Research 1693 (2018) 11–23 13 Fig. 1. Cartoon of phase separation. In the dispersed state, protein scaffolds (green circles) and cargo/client RNA molecules (red lines) are intermixed with solute molecules (black circles). Under appropriate conditions, protein scaffolds can phase separate to form a liquid droplet enriched in the scaffold protein and client RNA. These condensates display several features of liquid droplet including: (1) spherical shape; (2) the ability to fuse with each other; (3) viscosity that is similar to or only slightly increased relative to the viscosity of water (left red arrow denotes the FUS droplet; right red arrow denotes the solute; the ﬁgure above denotes the approximate range of viscosity difference in kilo Pascal seconds; (4) ability for both client and scaffold molecules to partition in and out; and (5) the ability to relax/disassemble back to dispersed state. They can also further condense into ﬁbrillar aggregates. The relationships between hydrogel, droplet and dispersed states can be depicted in a classical phase diagram with conversion between each of the states. Fig. 1. Cartoon of phase separation. In the dispersed state, protein scaffolds (green circles) and cargo/client RNA molecules (red lines) are intermixed with solute molecules (black circles). Under appropriate conditions, protein scaffolds can phase separate to form a liquid droplet enriched in the scaffold protein and client RNA. These condensates display several features of liquid droplet including: (1) spherical shape; (2) the ability to fuse with each other; (3) viscosity that is similar to or only slightly increased relative to the viscosity of water (left red arrow denotes the FUS droplet; right red arrow denotes the solute; the ﬁgure above denotes the approximate range of viscosity difference in kilo Pascal seconds; (4) ability for both client and scaffold molecules to partition in and out; and (5) the ability to relax/disassemble back to dispersed state. They can also further condense into ﬁbrillar aggregates. The relationships between hydrogel, droplet and dispersed states can be depicted in a classical phase diagram with conversion between each of the states. 4. Biophysics of liquid-liquid phase separating polymers Because of their reduced amino acid diversity, these domains are often referred to as ‘‘low complexity” (LC) domains. Proteins or protein domains with these features typically do not fold into a single, well-deﬁned three-dimensional structure, and are thus frequently also described as ‘‘intrinsically disordered” pro- teins or domains (See Fig. 3). 2. A. Cartoon of FUS protein topology, showing the tyrosine rich low complexity (LC) domain and the arginine rich C-terminus comprised of structured arginine g ne motifs (RGG) and RNA recognition motifs (RRM), as well as the C-terminal, atypical proline tyrosine nuclear localisation sequence (PY-NLS). B. Cartoon diagra ine: tyrosine cation- p interactions. These interactions can be enhanced by demethylation of arginines. They can be abolished by conversion of arginine to citr her stabilising intermolecular hydrogen bonding between b-sheet motifs stabilise liquid droplet and hydrogel condensates, and if excessive, the formation of irreve lar gels. C. Packing of e b-sheet motifs within the LC domain can form ﬁbrillar structures with many similarities to classical amyloid ﬁbrils (adapted from Murray 71:165, 2018. P. St George-Hyslop et al. / Brain Research 1693 (2018) 11–23 P. St George-Hyslop et al. / Brain Research 1693 (2018) 11–23 14 Fig. 2. A. Cartoon of FUS protein topology, showing the tyrosine rich low complexity (LC) domain and the arginine rich C-terminus comprised of structured arginine glycine glycine motifs (RGG) and RNA recognition motifs (RRM), as well as the C-terminal, atypical proline tyrosine nuclear localisation sequence (PY-NLS). B. Cartoon diagrams of arginine: tyrosine cation- p interactions. These interactions can be enhanced by demethylation of arginines. They can be abolished by conversion of arginine to citrulline. Further stabilising intermolecular hydrogen bonding between b-sheet motifs stabilise liquid droplet and hydrogel condensates, and if excessive, the formation of irreversible ﬁbrillar gels. C. Packing of e b-sheet motifs within the LC domain can form ﬁbrillar structures with many similarities to classical amyloid ﬁbrils (adapted from Murray et al., cell 171:165, 2018. suspended in liquid solute that is depleted in both. This latter sit- uation, as discussed below, sets up a circumstance where the rela- tive stoichiometries of the components can modulate the propensity to phase separate. (for instance cation-p interactions between the positively charged side chains of arginines or lysines with the free electrons in the aromatic rings of tyrosines, tryptophans or phenylalanines). 4. Biophysics of liquid-liquid phase separating polymers The viscosity of the condensates is depicted below the images (kPas). Adapted from Murakami et al., Neuron 88:678, 2015. Fig. 3. Phase separation and gelation can result in sequestration of client and cargo elements, such as other ribonucleoproteins and RNAs. This can be reversed upon relaxation/melting of the droplet or gel. Top row: shows single particle tracking of individual survival motor neuron protein and staufen-1 protein molecules in wild-type FUS dispersed assemblies (left column) and in liquid droplet/hydrogel condensates (middle column). The sequestration of the client/cargo element can be reversed by relaxation of the condensate a liquid droplet or dispersed state (right column). Bottom row depicts irreversible sequestration of client molecules in fALS-FUS mutant FUS condensates. In all images the length of the single molecule path is depicted and the diffusion coefﬁcient for movement in that path is colour-coded according to the heat map. The viscosity of the condensates is depicted below the images (kPas). Adapted from Murakami et al., Neuron 88:678, 2015. polymer systems (Jiang et al., 2015; Reichheld et al., 2017). Finally, weak short range intermolecular hydrogen bonding between b- sheet motifs occurs in liquid droplet, hydrogel and in denser ﬁbril- lar condensates (Hughes et al., 2018; Murray et al., 2017; Qamar et al., 2018). The denser ﬁbrillar condensates possess more exten- sive b-sheet driven intermolecular hydrogen bonding, and often result in stable pathological ﬁbrillar assemblies that cannot easily be reversed by the cell (and are often therefore referred to as ‘‘irre- versible” condensates). available interactions/nodes (e.g. by posttranslational modiﬁca- tions). Thus, as described below, the number of available interac- tions/nodes in FUS can be changed by altering the post- translational state of the key residues (e.g. arginine methylation or serine phosphorylation) or by shielding the key residues/nodes by cloaking them with interacting chaperones (e.g. transportin 1 – TNPO1) (Deng et al., 2014; Guo et al., 2018; Hofweber et al., 2018; Monahan et al., 2017; Murray et al., 2017; Qamar et al., 2018; Yoshizawa et al., 2018). Condensation into hydrogels and into stable ﬁbrillar condensates (i.e. ‘‘gelation”) likely follows somewhat similar rules, but in a time- and concentration-dependent manner in which intermolecular hydrogen bonding of b-sheet domains becomes increasingly impor- tant, especially in ﬁbrillar condensates. However, the speciﬁc details of this process are currently the topic of intense scrutiny. 4. Biophysics of liquid-liquid phase separating polymers Work on fragments of FUS using electron microscopy, solid state nuclear magnetic resonance and x-ray diffraction studies of ﬁbrillar conden- sates reveals several important differences from classical amyloids including: i) short b-sheet domains (often <5 residues); ii) a paucity of hydrophobic residues; iii) high prevalence of hydrophilic resi- dues; iv) the presence of ‘‘kinks” at glycine, proline or aromatic resi- dues which preclude long b-sheets and thus minimize the ability to make stable, steric zippers characteristic of conventional amyloids (Hughes et al., 2018; Murray et al., 2017). These motifs (e.g. residues 37-SYSGYS-42 and 54-SYSSYGQS-61 in the putative amyloid-core domain of FUS FUS) have recently been termed ‘‘low-complexity aromatic-rich kinked segments” or LARKS (Hughes et al., 2018) (Fig. 2B and C). However, while these emerging results are exciting, they should be interpreted with caution. First, the fragments studied in these experiments are not physiological. Second, there are amy- loid forming proteins (e.g. yeast prion proteins such as Sup35) that have similar amino acid content (King et al., 2012). Crucially, the interactions driving phase separation are unlike the lock-and-key interactions of well-folded proteins, which rely on precise three-dimensionally speciﬁc point-to-point interac- tions. In phase separating proteins, the interactions build upon the disordered state and the repetitive sequence motifs that are characteristic of the LC domains of these proteins. Residues within these repetitive sequences can be considered as interaction points (often referred to as ‘‘nodes”, although the use of such network ter- minology is not universally preferred). These residues/nodes per- mit the formation of networks of intra- and inter-molecular interactions within and between phase-separating polymers (Brangwynne et al., 2015; Harmon et al., 2017; Wei et al., 2017). The strength of this overall network is therefore driven by: i) the number (or ‘‘valence”) of interacting nodes (e.g. the number of arginine: tyrosine cation-p or p-p interactions); and ii) the strength of the interaction at each node. As an example of this mul- tivalency, the LC domain of FUS contains 27 tyrosines, which can form cation-p interactions with 37 arginines mostly found in the structured C-terminus (Qamar et al., 2018) (Fig. 2A). Phase separa- tion is not dependent on any individual FUS tyrosine or arginine residue (Lin et al., 2017; Qamar et al., 2018). Conversely, phase sep- aration is enhanced by selective mutagenesis which introduces additional arginine residues, and this enhancement is dependent on the number of extra arginine residues, rather than their posi- tion. 4. Biophysics of liquid-liquid phase separating polymers Other intermediate-range interactions include dipole interactions between glycine, glutamine, asparagine and serine residues, as well as p-p interactions formed by stacking of aromatic rings or between the guanidino moiety of arginines and the rings of aro- matic amino acids (Vernon et al., 2018). Most low complexity domains are relatively depleted in hydrophobic amino acids, but where they do occur, they can manifest by phase separation that occurs upon increasing temperature, rather than phase separation with decreased temperature, which is typically observed for most (for instance cation-p interactions between the positively charged side chains of arginines or lysines with the free electrons in the aromatic rings of tyrosines, tryptophans or phenylalanines). Other intermediate-range interactions include dipole interactions between glycine, glutamine, asparagine and serine residues, as well as p-p interactions formed by stacking of aromatic rings or between the guanidino moiety of arginines and the rings of aro- matic amino acids (Vernon et al., 2018). Most low complexity domains are relatively depleted in hydrophobic amino acids, but where they do occur, they can manifest by phase separation that occurs upon increasing temperature, rather than phase separation with decreased temperature, which is typically observed for most The forces (or ‘‘preferred interactions”) that drive liquid–liquid phase separation include both long-range forces that might initiate the phase separation, and intermediate-range forces that might subsequently stabilise the assembly once polymer molecules have begun to form a network with each other. The longer-range forces are typically charged interactions (e.g. glutamate – arginine), whereas the intermediate-range forces include directional interac- tions between positively charged residues and aromatic residues 15 P. St George-Hyslop et al. / Brain Research 1693 (2018) 11–23 Fig. 3. Phase separation and gelation can result in sequestration of client and cargo elements, such as other ribonucleoproteins and RNAs. This can be reversed upon relaxation/melting of the droplet or gel. Top row: shows single particle tracking of individual survival motor neuron protein and staufen-1 protein molecules in wild-type FUS dispersed assemblies (left column) and in liquid droplet/hydrogel condensates (middle column). The sequestration of the client/cargo element can be reversed by relaxation of the condensate a liquid droplet or dispersed state (right column). Bottom row depicts irreversible sequestration of client molecules in fALS-FUS mutant FUS condensates. In all images the length of the single molecule path is depicted and the diffusion coefﬁcient for movement in that path is colour-coded according to the heat map. 4. Biophysics of liquid-liquid phase separating polymers Indeed, the exact positions of the deleted or added tyrosine residues (in the LC domain) or arginine residues (in the C- terminal domain) seems irrelevant, emphasising that precise three-dimensional spatial orientation of the interactions is less critical than the valence of the interactions (Qamar et al., 2018). 6. Potential cellular consequences of pathological phase separation and gelation tion fuses the 50 part of FUS to the C/EBP HOmologous Protein Gene (CHOP). The resulting FUS/CHOP fusion protein likely phase sepa- rates in the nucleus, and then recruits chromatin remodelling or transcription factors in the same way that the EWS-FLI1 fusion protein does in Ewing sarcoma (Boulay et al., 2017). Most RNP granules are heterogeneous complexes composed of multiple RNA and protein components that serve as scaffolds or as clients/cargo. As a result, defective function of their component proteins would be anticipated to cause: i) failure of formation of the granule; ii) abnormal partitioning and binding of clients into the granule; iii) abnormal transport of condensed granules; and/ or iv) dysregulated release of clients. No examples have yet been found of mutations in clinically relevant phase separating proteins that cause defective formation or defective cargo selection. How- ever, it is now clear that disease-causing mutations and disease associated post-translational modiﬁcations of several known RNA binding scaffold proteins (e.g. FUS, TDP-43, and hnRNP A1 and A2/B1, and TIA1) can accelerate conversion of these proteins into stable, b-sheet rich, intermolecular hydrogen bonded assemblies (Conicella et al., 2016; Han et al., 2012; Kato et al., 2012; Mackenzie et al., 2017; Molliex et al., 2015; Murakami et al., 2015; Patel et al., 2015; Schmidt and Rohatgi, 2016). The rest of this review therefore focusses on: i) how disease-associated muta- tions and disease associated post-translational modiﬁcations of FUS alter its biophysical properties; and ii) how these changes might affect the function of FUS in neurons. Full-length FUS itself also has important links to neurodegener- ative diseases: namely amyotrophic lateral sclerosis (ALS-FUS); frontotemporal dementia (FTLD-FUS) and essential tremor type 4 (Merner et al., 2012). The latter illness appears to be due to frame- shift mutations causing nonsense-mediated decay and a loss-of- function effect, which differs considerably from the effects of mis- sense mutations and altered post-translational modiﬁcation of FUS that lead to ALS and FTLD, which are the focus of the present review. A small proportion (approximately 1–4%) of familial ALS (fALS) cases arise from missense or frameshift mutations in FUS (>50 mutations have been described to date). Most of these missense mutations and virtually all of the frameshift mutations cluster in the C-terminal region of FUS between residues 495 and 526, near the atypical nuclear localisation sequence (NLS) domain. 5. Mathematical modelling and complex membraneless organelles The fundamental biophysical processes underlying these phase separations can be reasonably well modelled mathematically based upon Flory Huggins models (which consider a network of the molecules interacting on a lattice) (Das et al., 2015; Flory, 1942; Huggins, 1942), or more general ternary (three phase) solu- tion models (which can be applied to understanding more complex Because liquid: liquid phase separation is primarily driven by multiple inter- and intra-polymer interactions that are not highly restricted to speciﬁc three-dimensional orientations, this allows phase separation to be tuned simply by manipulating number of P. St George-Hyslop et al. / Brain Research 1693 (2018) 11–23 16 multicomponent systems) that are probably the norm for most biological membraneless organelles (Lee et al., 2013). These math- ematical models are well reviewed (Brangwynne et al., 2015; Shin and Brangwynne, 2017). region 1 (EWS/EWSR1) and TATA box binding protein 15 (TAF15) (FET) family of RNA binding proteins (Ratti and Buratti, 2016; Schwartz et al., 2015) (Fig. 2A). It is composed of an N-terminal intrinsically disordered region (residues 1–214), which has reduced amino acid content diversity (i.e. is a low complexity (LC) domain), and which contains multiple glutamine, glycine, ser- ine and tyrosine (QGSY) repeats. In its middle and C-terminal domains, FUS has a well-conserved RNA recognition motif (RRM), a zinc ﬁnger domain, and two domains that are enriched in argi- nine, glycine, glycine (RGG) motifs (Ratti and Buratti, 2016; Schwartz et al., 2015). Finally, FUS contains an atypical proline tyr- osine nuclear localisation sequence (PY-NLS) (Dormann et al., 2010, 2012; Suarez-Calvet et al., 2016). In these more physiological complex coacervation systems, liq- uid: liquid phase separation is probably driven both by homotypic interactions between a single type of polymer, and by heterotypic interactions (e.g. protein #1: protein #2 or protein: RNA interac- tions). The existence of such complex systems, allows for: i) the cooperative interaction of the different polymers in forming the phase separation at lower polymer concentrations; and ii) the exis- tence of membraneless organelles with internal structures com- prised of shells, cores, or multiple sub-droplets. 6.2. FUS in disease FUS has an important role in the formation of myxoid/round cell liposarcoma cancers. In these cancers, a chromosomal transloca- tion fuses the 50 part of FUS to the C/EBP HOmologous Protein Gene (CHOP). The resulting FUS/CHOP fusion protein likely phase sepa- rates in the nucleus, and then recruits chromatin remodelling or transcription factors in the same way that the EWS-FLI1 fusion protein does in Ewing sarcoma (Boulay et al., 2017). FUS has an important role in the formation of myxoid/round cell liposarcoma cancers. In these cancers, a chromosomal transloca- 5. Mathematical modelling and complex membraneless organelles As discussed below, a simple and intuitive example of coopera- tive interaction is where the introduction of small quantities of RNA can bind multiple FUS proteins through their RNA binding motifs, thereby bringing the protein molecules together at much higher effective/local concentrations than would occur in the absence of the ‘‘nucleating” RNA (Berry et al., 2015; Li et al., 2012; Lin et al., 2015; Saha et al., 2016; Zhang et al., 2015). FUS is predominantly located in the nucleus, where it plays a crucial role in both DNA and RNA biology, being involved in both DNA repair as well as RNA transcription and processing (Ratti and Buratti, 2016; Schwartz et al., 2015). FUS is also present at much lower abundance in RNP granules in the cytoplasm in axons and dendrites, where it supports regulated local new protein syn- thesis (Figs. 4 and 5). Nevertheless, even here it plays a crucial role in mRNA and micro-RNA transport and processing. In neurons, many of these cytoplasmic FUS-related RNAs are involved in synaptic biology and neuronal plasticity (Sephton and Yu, 2015). These aspects of FUS biology have been extensively discussed in the literature, and readers are referred to the many recent and excellent reviews on these topics for further details (Dormann and Haass, 2013; Ratti and Buratti, 2016; Sephton and Yu, 2015). FUS is normally post-translationally modiﬁed both by asymmetric dimethylation of arginine residues by protein arginine methyl transferases (Rappsilber et al., 2003), and by serine phosphoryla- tion by DNA protein kinase (Deng et al., 2014). The molecular diversity of membraneless organelles also allows for the co-existence of multiple phase states in the same conden- sate. The content of each sub-phase (i.e. shell, core, sub-droplet) is then driven by: i) the relative differences in the component poly- mers to partition into the different sub-phases based on their rel- ative propensities to form homotypic or heterotypic interactions with each other; and ii) the resulting differences in surface ten- sions of the component droplets. Clearly these purely biophysical considerations underpin the structure and function of a diverse group of highly dynamic intra- cellular organelles. However, as described below using FUS as an example, the precarious balancing act between i) mixed/dispersed; ii) reversible liquid droplet; and iii) stable ﬁbrillar states that may be irreversible in biological systems, and thus places them at risk of causing disease states. 6. Potential cellular consequences of pathological phase separation and gelation A much smaller proportion of ALS associated mutations have been identi- ﬁed in the central and N-terminal region, with two mutations (Ser96Del and Gly156Asp) occurring within the LC domain itself (Kapeli et al., 2017; Rainero et al., 2017). this review therefore focusses on: i) how disease-associated muta- tions and disease associated post-translational modiﬁcations of FUS alter its biophysical properties; and ii) how these changes might affect the function of FUS in neurons. The clinicopathological characteristics of these fALS-FUS cases have been summarised in many recent reviews and will therefore not be discussed further here beyond noting that the pathog- nomonic neuropathological features of fALS-FUS are the presence of: profound cytoplasmic FUS aggregates; signiﬁcant depletion of 6.1. FUS The mechanism by which the FUS aggregates induce either fALS-FUS or FTLD-FUS has been extensively discussed in recent reviews, with three non-mutually exclusive ideas currently being considered, namely: 1) impairment of the nuclear transcriptional and splicing roles of FUS due to either its presence in the nucleus as pathological aggregates or due to its depletion from the nucleus; 2) loss of the normal cytoplasmic functions of FUS due to its sequestration in pathological assemblies; and 3) toxic gain of func- tion effects arising from the pathological effects of the FUS assem- blies such as retention of RNA templates inside pathological aggregates. Recent work using a variety of approaches has begun to allow these possibilities to be examined in ﬁner detail. Thus, recent studies comparing the phenotype of conditional FUS knock- out mice with that of mice expressing fALS-FUS mutant FUS sug- gest that a gain of toxic function is more probable (Sharma et al., 2016). Similarly, recent studies examining the effect of forced ecto- pic expression of aggregating proteins have shown that toxicity can be mitigated by using strong nuclear localization signals to force expression out of the cytoplasm and into the nucleus, clearly suggest that defects in nucleo-cytoplasmic translocation play a key role (Kim and Taylor, 2017; Matsukawa et al., 2016; Woerner et al., 2016). Tentative support for the possibility that the toxic effect is primarily a gain of toxic function effect in the cytoplasmic com- partment, rather than the nuclear compartment, comes from stud- ies of the earliest pathological changes in mice expressing mutant human FUS. In these mice, the earliest changes, seen by electron microscopy, occur in the structure of presynaptic terminals (So et al., 2018). However, this review will focus on the recently emerging insights into the biophysics of FUS and its propensity to undergo phase separation, and the impact of FUS mutations and post-translational modiﬁcation of FUS on this process. Extensive cytoplasmic and some nuclear deposits of FUS are also present in neurons and glia in a signiﬁcant proportion (approximately 10%) of apparently sporadic FTLD cases (Hortobagyi and Cairns, 2017; Mackenzie and Neumann, 2016). In these FTLD-FUS cases, there is usually no family history, and sequencing of the FUS gene has usually failed to detect missense or truncation mutations, although 2 FUS mutations (P106L and M254V) have been described in individual patients with FTLD- FUS. 6.1. FUS FUS is a 526 amino acid heterologous nuclear ribonucleoprotein (hnRNP), and a member of the FUS, Ewing sarcoma Breakpoint 17 P. St George-Hyslop et al. / Brain Research 1693 (2018) 11–23 17 Fig. 4. Pathological fALS-FUS mutant FUS condensates have impaired function. By sequestering the machinery of RNA metabolism and translation, the mutant ﬁbrilla condensates cause signiﬁcant reductions FUS RNP granule function, as measured by reductions in new protein synthesis in axon terminals. New proteins are identiﬁed by a assay in which puromycin incorporated into new proteins can be detected using an anti-puromycin antibody. Adapted from Murakami et al., neuron 88: 678, 2015. Fig. 4. Pathological fALS-FUS mutant FUS condensates have impaired function. By sequestering the machinery of RNA metabolism and translation, the mutant ﬁbrillar condensates cause signiﬁcant reductions FUS RNP granule function, as measured by reductions in new protein synthesis in axon terminals. New proteins are identiﬁed by an assay in which puromycin incorporated into new proteins can be detected using an anti-puromycin antibody. Adapted from Murakami et al., neuron 88: 678, 2015. Calvet et al., 2016). Finally, FUS deposits in FTLD-FUS are occasion- ally ubiquitinated, whereas in fALS-FUS, FUS is not ubiquitinated. FUS from the nucleus; and occasional intranuclear FUS aggregates. Crucially, these pathological FUS assemblies typically contain nor- mally methylated (i.e. arginine residues are normally asymmetri- cally dimethylated) FUS, and the aggregates do not contain other FET proteins or transportin (TNPO1) (Hortobagyi and Cairns, 2017; Mackenzie and Neumann, 2016; Neumann et al., 2012; Troakes et al., 2013). FUS from the nucleus; and occasional intranuclear FUS aggregates. Crucially, these pathological FUS assemblies typically contain nor- mally methylated (i.e. arginine residues are normally asymmetri- cally dimethylated) FUS, and the aggregates do not contain other FET proteins or transportin (TNPO1) (Hortobagyi and Cairns, 2017; Mackenzie and Neumann, 2016; Neumann et al., 2012; Troakes et al., 2013). Calvet et al., 2016). Finally, FUS deposits in FTLD-FUS are occasion- ally ubiquitinated, whereas in fALS-FUS, FUS is not ubiquitinated. 6.1. FUS FUS RNP granules in axon terminals colocalise th markers of RNA (Cy5). C. FUS and TNPO1 colocalise in axons and axon terminals expressing endogenous FUS and Cherry tagged TNPO1. Adapted from Murakami et al., uron 88: 678, 2015 and Qamar et al. Cell, in press, 2018. FUS C C TNPO1 Merge 2μm Fig. 5. A. FUS RNP granules are detectable in axon terminals and axons using GFP labelled anti-FUS antibody is (arrowheads). B. FUS RNP granules in axon terminals colocalise with markers of RNA (Cy5). C. FUS and TNPO1 colocalise in axons and axon terminals expressing endogenous FUS and Cherry tagged TNPO1. Adapted from Murakami et al., neuron 88: 678, 2015 and Qamar et al. Cell, in press, 2018. As described below, these emerging experimental results provide compelling molecular insights into how FUS aggregates occur and how they might injure neurons. 2018). This cooperativity arises because the positively charged guanidino side chains on arginine residues form cation-p interac- tions with free electrons in the aromatic ring of tyrosine residues that are predominantly located at the N-terminus of FUS (including the LC domain) (Figs. . 2A and 6). Similar cation-p interactions are known to occur with Tudor domain containing proteins like SMN (Tripsianes et al., 2011; Zhang et al., 2017), and with other phase separating proteins like DDX4 (Nott et al., 2015) (Fig. 7A). These cation-p interaction-forming proteins contain lysine or arginine as the cation donor, and tyrosine, phenylalanine or tryptophan as the electron donor. There are four biologically important aspects of this cation-p interaction that are worth further inspection. 6.1. FUS However, these mutations have not been shown to co- segregate with the disease, and their association with FTLD-FUS is therefore not fully proven. Two distinct neuropathological FTLD-FUS sub-phenotypes have been described, namely: neuronal intermediate ﬁlament inclusion body disease (NIFID) and basophi- lic inclusion body disease (BIBD). Despite the distinctive neu- ropathology, these two subtypes of FTLD-FUS do not have clinical phenotypes that are distinguishable. However, the biochemical signature of FUS inclusions in both subtypes of FTLD-FUS is strik- ingly different from those of FUS inclusions in fALS-FUS. As men- tioned above, in fALS-FUS, FUS inclusions do not contain other FET RNA binding proteins, or transportin, and FUS is typically phys- iologically asymmetrically di-methylated (Neumann et al., 2012; Troakes et al., 2013). In contrast, in FTLD-FUS, the pathological FUS deposits contain EWS, TAF15 and transportin. Furthermore, arginine residues in FTLD-FUS are signiﬁcantly demethylated, being predominantly comprised of unmethylated FUS or monomethylated argininines (Dormann et al., 2010; Suarez- 2016). Tentative support for the possibility that the toxic effect is primarily a gain of toxic function effect in the cytoplasmic com- partment, rather than the nuclear compartment, comes from stud- ies of the earliest pathological changes in mice expressing mutant human FUS. In these mice, the earliest changes, seen by electron microscopy, occur in the structure of presynaptic terminals (So et al., 2018). However, this review will focus on the recently emerging insights into the biophysics of FUS and its propensity to undergo phase separation, and the impact of FUS mutations and post-translational modiﬁcation of FUS on this process. 18 P. St George-Hyslop et al. / Brain Research 1693 (2018) 11–23 FUS FUS FUS Cy5 Merge Merge TNPO1 A B C 2μm 2μm 5μm 5μm S RNP granules are detectable in axon terminals and axons using GFP labelled anti-FUS antibody is (arrowheads). B. FUS RNP granules in axon terminals colocalise s of RNA (Cy5). C. FUS and TNPO1 colocalise in axons and axon terminals expressing endogenous FUS and Cherry tagged TNPO1. Adapted from Murakami et al., 678, 2015 and Qamar et al. Cell, in press, 2018. P. St George-Hyslop et al. / Brain Research 1693 (2018) 11–23 FUS A 5μm 5μm A FUS Cy5 Merge B 2μm B FUS Merge TNPO1 C 2μm g. 5. A. FUS RNP granules are detectable in axon terminals and axons using GFP labelled anti-FUS antibody is (arrowheads). B. 7. Physiological FUS phase separation At physiological concentration range of FUS and salt, and at physiological temperature, FUS undergoes phase separation and gelation in a FUS protein concentration-dependent and salt concentration-dependent manner (Han et al., 2012; Kato et al., 2012; Mann and Snowden, 2017; Murakami et al., 2015; Murray et al., 2017; Patel et al., 2015; Qamar et al., 2018). These condensa- tion processes require the presence of the LC domain, and the LC domain can itself phase separate. However, recent work suggests that for FUS, and likely for many other phase separating proteins, non-LC domains also play a critical role. In FUS, binding of RNA to the RRM and zinc ﬁnger domains in the C-terminus of FUS dra- matically facilitates phase separation, such that FUS phase separa- tion occurs at lower FUS concentrations than in the absence of RNA (Schwartz et al., 2013). More recently, the C-terminus of FUS itself has also been found to cooperate with the LC domain to induce phase separation at lower FUS concentrations (Qamar et al., The ﬁrst biologically important feature is the presence of multi- ple positively charged residues (37 arginines, 14 lysines) and mul- tiple aromatic residues (36 tyrosines, 13 phenylalanines, and 3 tryptophans) that could participate in cation-p interactions. This feature creates the basis for scalable, multivalent, location- independent interactions that drive phase separation, as discussed earlier. The current model for the role of these electrostatic cation- p interactions is that they initiate the phase separation process by promoting FUS: FUS interactions that recruit multiple FUS mole- cules into restricted volumes wherein there is a much higher local P. St George-Hyslop et al. / Brain Research 1693 (2018) 11–23 19 Fig. 6. Cartoon depictions of arginine: tyrosine cation-p interactions between the guanidino group in the side-chain of arginine residues, and the aroma These cation-p interactions can be modulated by arginine methylation by protein arginine methyl transferase enzymes (PRMT), or by conversion to c arginine deiminase (PAD). g y p / ( ) Fig. 6. Cartoon depictions of arginine: tyrosine cation-p interactions between the guanidino group in the side-chain of arginine residues, and the aromatic ring of tyrosines. These cation-p interactions can be modulated by arginine methylation by protein arginine methyl transferase enzymes (PRMT), or by conversion to citrulline by protein arginine deiminase (PAD). Fig. 6. 7. Physiological FUS phase separation However, recent work has uncovered a role for TNPO1 in maintaining FUS in a dispersed state and melting pre-existing FUS droplets and gels. TNPO1 can both block FUS phase separation and melt preassem- bled FUS condensates (Monahan et al., 2017; Qamar et al., 2018). Crucially, TNPO1 is not only expressed at the nuclear pore, but is also expressed in axons and dendrites (Chang and Tarn, 2009; Twyffels et al., 2014), where it is able to modulate the assembly and relaxation of FUS condensates (Qamar et al., 2018) (Fig. 8). By altering the biophysical state of FUS in RNP granules, and thus sequestration/release of FUS-bound mRNAs in the granule, TNPO1 can modulate local RNA translation in axon terminals (Qamar et al., 2018). FUS concentrations than in the overall solute (Qamar et al., 2018). This process permits the subsequent formation of stabilising short- range interactions as discussed below. The cooperative effect of RNA on FUS phase separation likely has a similar basis (Schwartz et al., 2013), namely recruitment of multiple FUS proteins into a small volume. The second biologically important feature is that both arginine and lysine residues are post-translationally modiﬁable by methyla- tion. Arginine can also be enzymatically deiminated to form citrul- line (Fig. 6). These post-translational modiﬁcations affect the interaction strength, and thus allow the cation-p interaction to be ‘‘tuned”. Citrulllination ablates the cation-p interaction by changing the ketimine moiety into a ketone (Qamar et al., 2018). Methylation weakens the cation-p interactions (Qamar et al., 2018), and in other proteins such as Tudor domain proteins, argi- nine dimethylation may make the arginine residues more selective only for speciﬁc tyrosine residues that exist in three-dimensional pockets, termed ‘‘aromatic cages”, comprised by the clustering of aromatic side chains of two or more tyrosine, tryptophan and/or phenylalanine residues (Tripsianes et al., 2011; Zhang et al., 2017) (Fig. 7A). It is unlikely that well-formed aromatic cages are created by tyrosine residues in the intrinsically disordered N- terminus of FUS. However additional studies will be required to determine whether a similar, but transient three-dimensional clus- tering of speciﬁc tyrosines occur in FUS during liquid: liquid phase separation. 7. Physiological FUS phase separation Cartoon depictions of arginine: tyrosine cation-p interactions between the guanidino group in the side-chain of arginine residues, and the aromatic ring of tyrosines. These cation-p interactions can be modulated by arginine methylation by protein arginine methyl transferase enzymes (PRMT), or by conversion to citrulline by protein arginine deiminase (PAD). Fig. 7. A. Cartoon of an aromatic cage. In some proteins using cation-p interactions, the arginine side-chain ﬁts inside an aromatic cage composed of multiple tyrosine residues in the receptor protein. These aromatic cages are often tuned to preferentially interact with asymmetrically di-methylated arginine (ADMA) residues. Adapted from Tripsianes, Nature Structural and Molecular Biology 18:1414, 2011. B. The terminal helix of FUS interacts closely with the side chains of residues in TNPO1. Many of the C- terminal fALS-FUS mutants (e.g. P525L, and R521G) make key interactions with TNPO1. Adapted from Zhang et al., PNAS, 109:1217, 2012. Fig. 7. A. Cartoon of an aromatic cage. In some proteins using cation-p interactions, the arginine side-chain ﬁts inside an aromatic cage composed of multiple tyrosine residues in the receptor protein. These aromatic cages are often tuned to preferentially interact with asymmetrically di-methylated arginine (ADMA) residues. Adapted from Tripsianes, Nature Structural and Molecular Biology 18:1414, 2011. B. The terminal helix of FUS interacts closely with the side chains of residues in TNPO1. Many of the C- terminal fALS-FUS mutants (e.g. P525L, and R521G) make key interactions with TNPO1. Adapted from Zhang et al., PNAS, 109:1217, 2012. Fig. 7. A. Cartoon of an aromatic cage. In some proteins using cation-p interactions, the arginine side-chain ﬁts inside an aromatic cage composed of multiple tyrosine residues in the receptor protein. These aromatic cages are often tuned to preferentially interact with asymmetrically di-methylated arginine (ADMA) residues. Adapted from Tripsianes, Nature Structural and Molecular Biology 18:1414, 2011. B. The terminal helix of FUS interacts closely with the side chains of residues in TNPO1. Many of the C- terminal fALS-FUS mutants (e.g. P525L, and R521G) make key interactions with TNPO1. Adapted from Zhang et al., PNAS, 109:1217, 2012. P. St George-Hyslop et al. / Brain Research 1693 (2018) 11–23 20 cal proline tyrosine nuclear localisation sequence (PY-NLS) (Fig. 7B) (Dormann et al., 2010, 2012; Monahan et al., 2017; Suarez-Calvet et al., 2016). Binding of FUS to TNPO1 supports the shuttling of FUS from the cytoplasm into the nucleus. 7.2. fALS-FUS 7.2. fALS-FUS The above considerations provide a partial insight into how missense and C-terminal truncating frameshift mutations in FUS might promote assembly of FUS into stable ﬁbrillar assemblies that impede FUS function in the ways described earlier (i.e. loss of nuclear functions regulating transcription and splicing, and/or loss of cytoplasmic functions in RNA transport, translation and metabolism). The third notable feature is that the initial phase separation of FUS that is induced by cation-p interactions is unstable (Qamar et al., 2018). These unstable interactions are likely to be subse- quently stabilised by the formation of b-sheet conformations in the LC domain, and by subsequent inter-molecular hydrogen bond- ing (Qamar et al., 2018). These short-range forces are also scalable. Thus, in liquid droplets, they are present, but not predominant. However, b-sheet derived hydrogen bonding forces are likely to become increasingly signiﬁcant upon further condensation into denser, hydrogel networks, and especially upon condensation into b-sheet rich, ﬁbrillar assemblies. Only a very small proportion of mutations associated with fALS- FUS occur within the FUS LC domain (FUS Ser96Del and Gly156Asp). While the precise biophysics has not yet been fully worked out, these mutations directly increase the propensity of the LC domain to phase separate, and then form hydrogels (Murakami et al., 2015; Patel et al., 2015). Thus, wild-type FUS LC domain can be cycled between dispersed and phase separated states multiple times (>5) before the process fatigues and the assemblies no longer relax back to the dispersed state after gela- tion. In contrast, the mutant LC domains have been experimentally shown to fatigue and fail to reverse back to dispersed states after only 1–2 cycles (Murakami et al., 2015). The fourth notable feature of FUS phase separation is that the arginine rich C-terminus (especially residues 495–526) bind molecular chaperones. One such chaperone is transportin 1 (TNPO1; karyopherin beta-2 (KapBeta2), which binds to the atypi- Most fALS-FUS mutations, however, occur outside the LC domain (Dormann et al., 2012; Murakami et al., 2015; Suarez- Calvet et al., 2016). Mutations affecting residues 495–526 in the Fig. 8. TNPO1 acts as a molecular chaperone for FUS. TNPO1 interacts with FUS to prevent phase separation, and to reverse pre-existing phase separation of both normally methylated ADMA FUS and also pathological hypomethylated FUS. In contrast, the Ewing sarcoma protein (EWS) has no signiﬁcant impact on FUS phase separation. Adapted from Qamar et al., Cell, in press, 2018. Fig. 8. 7.1. Pathological FUS phase separation 7.1. Pathological FUS phase separation As noted earlier, pathological deposits of FUS are prominent neuropathological features of both familial ALS (fALS FUS) and of sporadic FTLD (FTLD-FUS). 7. Physiological FUS phase separation Post-translational modiﬁcation of other residues, for instance, phosphorylation of serine by DNA protein kinase (Deng et al., 2014), and theoretically also phosphorylation of threonines and tyrosines by other enzymes, can strongly inhibit phase separa- tion, presumably by disrupting the packing of the LC domains, thereby preventing formation of hydrogen bonded b-sheets (Monahan et al., 2017; Murray et al., 2017). References Germline P granules are liquid droplets that localize by controlled dissolution/condensation Science 324 1729 1732 Brangwynne, C.P., Eckmann, C.R., Courson, D.S., Rybarska, A., Hoege, C., Gharakhani, J., Julicher, F., Hyman, A.A., 2009. Germline P granules are liquid droplets that localize by controlled dissolution/condensation. 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FUS is phosphorylated by DNA-PK and accumulates in the cytoplasm after DNA damage. J. Neurosci. 34, 7802–7813. Dormann, D., Rodde, R., Edbauer, D., Bentmann, E., Fischer, I., Hruscha, A., Than, M.E., Mackenzie, I.R., Capell, A., Schmid, B., Neumann, M., Haass, C., 2010. ALS- associated fused in sarcoma (FUS) mutations disrupt transportin-mediated nuclear import. EMBO J. 29, 2841–2857. Dormann, D., Madl, T., Valori, C.F., Bentmann, E., Tahirovic, S., Abou-Ajram, C., Kremmer, E., Ansorge, O., Mackenzie, I.R., Neumann, M., Haass, C., 2012. Arginine methylation next to the PY-NLS modulates transportin binding and nuclear import of FUS. EMBO J. 31, 4258–4275. 7.3. FTLD-FUS All authors contributed to the writing of this review manuscript. Glial and neuronal cytoplasmic and nuclear FUS aggregates are also a pathological feature of some cases of FTLD. Intriguingly, most cases, perhaps even all cases of FTLD-FUS are associated with wild-type FUS gene sequences. Moreover, in contrast to FUS inclu- sions in fALS-FUS, the FUS inclusions in FTLD-FUS coexist with EWS, TAF15 and TNPO1 (and possibly several other proteins). Importantly, while arginine residues in fALS-FUS-associated mutant FUS appears to be normally asymmetrically dimethylated (ADMA FUS), the arginine residues in FUS within the FTLD-FUS inclusions are signiﬁcantly demethylated (HYPO FUS) (Dormann et al., 2012; Hortobagyi and Cairns, 2017; Mackenzie and Neumann, 2016; Suarez-Calvet et al., 2016). Acknowledgements The mutations outside the LC domain, but not near the PY-NLS sequence have not been studied in detail. However, they probably also affect FUS phase behaviour either by changing the intrinsic phase separation propensity of FUS and/or by altering the interac- tion of FUS with nucleating ligands such as RNA and other ribonu- cleoproteins that bind to the central zinc ﬁnger and RRM domains. Supported by Canadian Institutes of Health Research, Wellcome Trust, ALS Society of Canada/Brain Canada - Canada. We apologise to the many colleagues whose prior work could not be cited due to space limitations. 7.2. fALS-FUS TNPO1 acts as a molecular chaperone for FUS. TNPO1 interacts with FUS to prevent phase separation, and to reverse pre-existing phase separation of both normally methylated ADMA FUS and also pathological hypomethylated FUS. In contrast, the Ewing sarcoma protein (EWS) has no signiﬁcant impact on FUS phase separation. Adapted from Qamar et al., Cell, in press, 2018. P. St George-Hyslop et al. / Brain Research 1693 (2018) 11–23 21 how these phase separation and gelation events occur and the effect of disease-causing mutations/post-translational modiﬁca- tions on these events is now becoming clearer. C-terminus of FUS likely have two effects. First, they directly increase the propensity of the mutant protein to phase separate and then gelate (Murakami et al., 2015). Second, the fALS-FUS mutations in this region affect residues within the 2.5 turn a- helix of FUS that makes direct contact with TNPO1 (Fig. 7B). Dis- ruption of these contacts reduces the normal binding afﬁnity (Kd = 9.5 nM) by up to 9-fold (Niu et al., 2012; Zhang and Chook, 2012). This double effect likely accounts for why fALS-FUS muta- tions in the C-terminus are associated with considerably more aggressive ALS phenotypes than mutations in the central domains and LC domains. The next phase of work will need to focus on understanding how normal assembly and relaxation/disassembly of condensates is physiologically regulated in response to cellular metabolic state. This future work may also provide some tractable molecular targets for novel treatments for diseases associated with abnormal phase separation of intrinsically disordered proteins, such as FUS, TDP-43 and other RNA binding proteins. Declarations of interest None. 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https://openalex.org/W2571563259	https://europepmc.org/articles/pmc5233428?pdf=render	English	null	A Novel Richardson-Lucy Model with Dictionary Basis and Spatial Regularization for Isolating Isotropic Signals	PloS one	2,017	cc-by	12,468	RESEARCH ARTICLE Tiantian Xu1, Yuanjing Feng1, Ye Wu1, Qingrun Zeng1, Jun Zhang1, Jianzhong He1, Qichuan Zhuge2 Tiantian Xu1, Yuanjing Feng1, Ye Wu1, Qingrun Zeng1, Jun Zhang1, Jianzhong He1, Qichuan Zhuge2 1 Institute of Information Processing and Automation, Zhejiang University of Technology, Hangzhou, Zhejiang, China, 2 Zhejiang Provincial Key Laboratory of Aging and Neurological Disorder Research, Wenzhou Medical University, Wenzhou, Zhejiang, China a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 * fyjing@zjut.edu.cn * fyjing@zjut.edu.cn a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Xu T, Feng Y, Wu Y, Zeng Q, Zhang J, He J, et al. (2017) A Novel Richardson-Lucy Model with Dictionary Basis and Spatial Regularization for Isolating Isotropic Signals. PLoS ONE 12(1): e0168864. doi:10.1371/journal.pone.0168864 Editor: Pew-Thian Yap, University of North Carolina at Chapel Hill, UNITED STATES Received: June 17, 2016 Accepted: December 7, 2016 Published: January 12, 2017 Editor: Pew-Thian Yap, University of North Carolina at Chapel Hill, UNITED STATES Editor: Pew-Thian Yap, University of North Carolina at Chapel Hill, UNITED STATES Copyright: © 2017 Xu 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: data is available in the Harvard Dataverse with the DOI 10.7910/DVN/ 3P1STN. Abstract Diffusion-weighted magnetic resonance imaging is a non-invasive imaging method that has been increasingly used in neuroscience imaging over the last decade. Partial volume effects (PVEs) exist in sampling signal for many physical and actual reasons, which lead to inaccu- rate fiber imaging. We overcome the influence of PVEs by separating isotropic signal from diffusion-weighted signal, which can provide more accurate estimation of fiber orientations. In this work, we use a novel response function (RF) and the correspondent fiber orientation distribution function (fODF) to construct different signal models, in which case the fODF is represented using dictionary basis function. We then put forward a new index Piso, which is a part of fODF to quantify white and gray matter. The classic Richardson-Lucy (RL) model is usually used in the field of digital image processing to solve the problem of spherical decon- volution caused by highly ill-posed least-squares algorithm. In this case, we propose an innovative model integrating RL model with spatial regularization to settle the suggested double-models, which improve noise resistance and accuracy of imaging. Experimental results of simulated and real data show that the proposal method, which we call iRL, can robustly reconstruct a more accurate fODF and the quantitative index Piso performs better than fractional anisotropy and general fractional anisotropy. iRL representative DTI is its inability to characterize the orientations of crossing and branching neural tracts in brain, especially fiber tracts with intersected diffusion orientations or partial volume averaged within a voxel [11–13]. Many recent high angular resolution diffusion imag- ing (HARDI) techniques have been proposed to recover the complex WM geometry [14]. Most of these methods consider water-molecule diffusion as a function of direction, such as Q-ball imaging (QBI) [15], diffusion spectrum imaging (DSI) [16] and spherical deconvolu- tion (SD) [17], which have all conquered the limitations of DTI. However, the data acquisition times for QBI and DSI are exorbitant [18] because of the high sampling numbers required to construct the full diffusion propagator. Given the linearity and sensitivity to multi-model dif- fusion [11], considerable interests have been generated with the model-free SD, which is based on convolution between fiber response function (RF) and fiber orientation distribution func- tion (fODF). Although the SD shows both good angular resolution and short computational time, the defects emerge when facing PVEs and the imaging quality is degraded by spurious directions and negative orientations caused by the truncation of high-order harmonics and ill- posed solution, even in noise-free data [19]. Competing Interests: The authors have declared that no competing interests exist. p Partial volume effects (PVEs) were put forward by Timo Roine et al. firstly [20]. It usually appears on the border of different tissues. The brain contains complex WM and non-WM tis- sues, such as gray matter (GM) and cerebrospinal fluid (CSF), which have different diffusion properties. Thus, the PVEs phenomenon is particularly obvious in human brain [12, 21, 22]. For PVEs, the SD method induces some changes on RF, but this does not solve the PVEs in essence. An informed constrained spherical deconvolution (iCSD) has been proposed to improve the estimation of fODF under non-WM PVEs by modifying RF to account for non- WM PVEs locally [23]. However, the iCSD method can’t correctly resolve fiber crossing angles of less than 60˚ under significant non-WM PVEs. Some authors have included an isotropic compartment in their signal models but these methods both require multiple b-value acquisi- tions and distinguish the signal of different tissues [24]. In other methods based on spherical deconvolution, the isotropic signal is dampened by using an iterative RL deconvolution algo- rithm [25]. Falvio et al. Introduction Magnetic resonance imaging (MRI) can offer important insights into brain disease [1]. Only diffusion-weighted MRI (DW-MRI) can provide a unique, non-invasive technique to study the microscopic structure of brain white matter (WM) in vivo [2–4]. DW-MRI provides valu- able information about the fiber architecture of tissue by measuring the diffusion of water in three-dimensional space [5, 6]. An early form of this technique, i.e., diffusion tensor imaging (DTI) [7], is widely used in clinics and provides fiber orientations of WM based on principal eigenvector of that tensor [8] and many useful quantitative indexes, including mean diffusivity (MD), fractional anisotropy (FA) [9, 10], and so on. The major shortcoming of the Funding: This work was supported in part by the National Natural Science Foundation of China (Grant No. 61379020) and by the open foundation of Wenzhou Medical University (Grant No. Funding: This work was supported in part by the National Natural Science Foundation of China (Grant No. 61379020) and by the open foundation of Wenzhou Medical University (Grant No. LKFJ014). The first funders is the corresponding author, which has a important role in study design, data collection and analysis. The second funder provide the idea and analysis the data. PLOS ONE \| DOI:10.1371/journal.pone.0168864 January 12, 2017 1 / 21 PLOS ONE \| DOI:10.1371/journal.pone.0168864 January 12, 2017 [19] infer that fODF can be represented by several discrete Dirac delta functions on unit sphere and propose a new spherical model based deconvolution approach to solve fiber crossing. They consider isotropic diffusion and anisotropic diffusion signal and combine both of two components. Dell’Acqua et al. suggest a new term, fiber orientation func- tion (FOF) to represent the weights of anisotropic and isotropic diffusion [26]. However, the FOF, as a combination of anisotropic and isotropic diffusions, can’t really take them apart. Consequently, The use of FOF is difficult. Isotropic signal existing in GM or CSF misleads the algorithms to produce spurious peaks in FOF. In this framework, Dell’Acqua et al. further combine RL spherical deconvolution algorithm with an adaptive regularization technique to yield damped Richardson-Lucy (dRL) algorithm in spherical deconvolution, aiming to attenu- ate isotropic signal while reducing spurious and non-physical fiber orientations in regions affected by PVEs [27]. dRL has its limitations. Given the different degrees of attenuation in each voxel, small FOF portions are more likely to be preserved in a low isotropic volume frac- tion, which leads to spurious fiber orientations [26]. Notably, the method based on RL has set- tled the highly ill-conditioned problem of least squares algorithm. However, in the absence of constrains of solution, even small changes in the acquired signal (e.g., MR noise) can lead to nonphysical results [17, 28]. A number of regularization algorithms have thus been developed. Yap et al. [29] develop a spatially non-negative sparse representation framework and then present an algorithm for solving l0 sparse group representation problem and apply it to tissue signal separation problem [30]. While the computational cost and intractable computation will arise when the models are more sophisticated. To make full use of spatially constraints of brain fibers, many global tractography methods considered PVEs [4, 31]have been proposed PLOS ONE \| DOI:10.1371/journal.pone.0168864 January 12, 2017 2 / 21 iRL in the last two years. But there are always many disadvantages, including computing space occupied, convergence property, sub-optimal solution and so on. There is a long way to realize global tractography perfectly. In this study, we consider a new spherical deconvolution model (hereafter denoted as iRL), which can effectively isolate isotropic signal from each DW signal. A new quantitative index is put forward to distinguish WM and non-WM of human brain, and the quantitative results of that index are better than those FA and GFA. We also propose a novel method, based on RL to efficiently reconstruct the above fiber architecture and yield high-quality fODF results. The true fODFs are gathers of delta function pointing along fiber orientations, and zero in all other orientations [32]. Thus, a dictionary basis is introduced to represent the fODF, which effec- tively helps to separate isotropic signal and renders the coefficients of fODF sparse. Finally, we integrate total variation regularization and ℓ1 norm regularization on the above framework to smooth noise and suppress spurious fiber orientations at the same time. To compare the per- formances with existing methods, the experiments are conducted on simulated and real data using the proposed method in compared with several kinds of methods, which are introduced in detail in the following sections. PLOS ONE \| DOI:10.1371/journal.pone.0168864 January 12, 2017 Spherical deconvolution Spherical deconvolution based on a relatively simple model of signal generation has been recently developed to overcome the limitations of diffusion tensor model in resolving multiple fiber orientations and to improve tractography reconstruction. The motivation of this pro- posed method is to recover fODF directly from DW signal without prior assumption or esti- mation about the number of fibers representing the information about diffusion [31]. The DW signal S can be assumed as a superposition of anisotropic and isotropic signal, which can be regard as two different diffusion models for three reasons. Firstly, the sampling voxels have a relatively large volume. On the border of WM and non-WM, the signal of each WM is affected by isotropic signal from non-WM, such as GM and CSF, which is known as PVEs phenome- non. The second is that isotropic diffusion exists in WM. Given that isotropic diffusion is weaker than anisotropic diffusion in WM, the diffusion in WM is always considered as anisot- ropy. The third one is that the complex structures of fibers such as orthogonal fibers lead to increased isotropy. Generally, the signal contributed by isotropic tissue is usually not included in spherical deconvolution models [21]. However, to facilitate calculation, researchers often try not to differentiate between the two parts and instead only make some changes in RF. The best solution is to put the two parts of DW signal segregated. In this work, we try to separate the two different parts of DW signal which would produce better imaging results especially in the DW signal existed PVEs. Let S2 be unit sphere domain and SO(3) be rotation group in R3. The anisotropic diffusion signal is modeled by convolution between a kernel R 2 L2(SO(3)) and a function f 2 L2ðS2Þ, which respectively represent the signal response function (RF) and fODF ideally composed of N Dirac delta functions for n bundles of fibers [33]. Spherical deconvolution We assume that the isotropic signal in each voxel is the same, thus the spherical deconvolution operator can be expressed as: SðgÞ ^S ¼ Z S2Rðg vÞf ðvÞdv ð1Þ ð1Þ where g are diffusion gradient orientations containing I directions and fgig I i¼1, S(g) are diffu- sion attenuation signal along g, ^S are isotropic signal, which are equal along each gradient PLOS ONE \| DOI:10.1371/journal.pone.0168864 January 12, 2017 3 / 21 iRL orientation and overlooked in most medical imaging cases, the dot stands for standard (Euclidean) dot product in R3, v is unit sphere (v also represent the discretized directions of unit sphere in the following parts), R(g v) is the RF describing DW signal intensity and f(v)dv is probability measure used to model fODF over S2 [31]. The fODF contains all desired aniso- tropic information regarding both orientations of various fiber populations that may be pre- sented and their respective volume fractions [34]. A common case is that we have N fibers in one voxel, where N is a limited natural number, and the corresponding fODF is the sum of N Dirac delta functions on sphere weighted by corresponding volume fractions. The form of fODF enables the separation of two diffusion models. Regarding the anisotropic signal, RF and fODF are defined as usual. orientation and overlooked in most medical imaging cases, the dot stands for standard (Euclidean) dot product in R3, v is unit sphere (v also represent the discretized directions of unit sphere in the following parts), R(g v) is the RF describing DW signal intensity and f(v)dv is probability measure used to model fODF over S2 [31]. The fODF contains all desired aniso- tropic information regarding both orientations of various fiber populations that may be pre- sented and their respective volume fractions [34]. A common case is that we have N fibers in one voxel, where N is a limited natural number, and the corresponding fODF is the sum of N Dirac delta functions on sphere weighted by corresponding volume fractions. The form of fODF enables the separation of two diffusion models. Regarding the anisotropic signal, RF and fODF are defined as usual. The novel fODF estimation with double models Basser et al. [7] indicate that the signal in a pulsed gradient spin echo depends on diffusion sensitive coefficient b and diffusion tensor D, the relation is: SðgiÞ ¼ etrðbgT i DgiÞ ð2Þ ð2Þ This relation relies on assumptions that the compartments have equal relaxation rates and water density, and the exchange between volumes can be neglected on the time scale of mea- surement [35]. Where S(gi) denotes the diffusion attenuation signal along i-th diffusion gradi- ent orientation gi. D is diffusion tensor, which describes the simplest model of diffusion in axon fiber bundles. The value of D is the extremum direction of diffusion, which can decide the degree of water diffusion. The RF [36–38] derived from the above signal relation has a cer- tain inaccuracy. Improving the precision of RF is of great advantage in the subsequent RL iter- ative model. Thus, we use the original Eq (2) as our RF. Our final goal is to construct the fODF which characterizes the relative likelihood of water diffusion along a given direction. Most of HARDI methods do not account for PVEs caused by non-WM tissues and orthogonal fibers. Signals contributed by GM or CSF both are actually isotropic compartments and are included in the existing model of spherical deconvolution. To accurately reconstruct brain connections from DW signal, we should properly model the dif- ferent types of water diffusion signal [39]. In order to make calculate easy, we discretize the process of spherical deconvolution (the discretized directions are still expressed as v). The reconstruction of SD method is computed as linear combination of the diffusion measure- ments [11]. The fODF can be reasonably considered as two main terms, viz. anisotropic and isotropic parts. Thus, incorporating these contributions by using double models is possible. Based on algebraic theory, we can combine the parts of anisotropy and isotropy. The novel fODF estimation with double models Thus at each voxel, the special deconvolution can be expressed as: ð3Þ SðgÞ ¼ ^Rðg vÞf ðvÞ ð3 where v are unit direction vectors which are acquired by averaging discretization of unit spherical surface along J directions and fvjg J j¼1, ^Rðg vÞ ¼ ½Raniðg vÞ Riso and where v are unit direction vectors which are acquired by averaging discretization of unit spherical surface along J directions and fvjg J j¼1, ^Rðg vÞ ¼ ½Raniðg vÞ Riso and Raniðg vÞ ðijÞ ¼ S0etrðbgT i D1giÞ, D1 is diffusion tensor of fibers (FA = 1, MD = 0.0007mm2/s), whose value is to ensure the maximum anisotropy, Rani(g v)(j) is the RF along j-th sample direction vj, which is a disc-shaped RF generated by the model presented in Eq (2) for a sin- gle fiber. There are J RFs oriented along each sampling direction. Thus, Rani(g v) is an I × J matrix, Riso = S0 e−tr(bgT D2 g) is a column vector of length I containing the signal of isotropic compartment. D2 is isotropic tensor of DW signal (FA = 0, MD = 0.0007mm2/s). Thus, the final RF ^R is an I × (J + 1) matrix. Naturally, fODF can be expressed as f(v) = [fani fiso], and i aniðg Þ 0 , 1 ( , ), whose value is to ensure the maximum anisotropy, Rani(g v)(j) is the RF along j-th sample direction vj, which is a disc-shaped RF generated by the model presented in Eq (2) for a sin- gle fiber. There are J RFs oriented along each sampling direction. Thus, Rani(g v) is an I × J matrix, Riso = S0 e−tr(bgT D2 g) is a column vector of length I containing the signal of isotropic compartment. D2 is isotropic tensor of DW signal (FA = 0, MD = 0.0007mm2/s). Thus, the final RF ^R is an I × (J + 1) matrix. Naturally, fODF can be expressed as f(v) = [fani fiso], and it PLOS ONE \| DOI:10.1371/journal.pone.0168864 January 12, 2017 4 / 21 iRL consists of two parts, the first J rows fani stand for the anisotropy. The last row fiso provides information related to isotropy. The fODF can be expressed more clearly as f = f(v) = [fani(v1), fani(v2),. . .fani(vJ), fiso]. The novel fODF estimation with double models To simplify the numerical solution, the fODF constructed by SD is originally formulated using spherical harmonics basis. Actually, the proposed method can be implemented using a number of well-characterized dictionary basis sets, which are flexible unimodal basis func- tions. This relationship can be expressed as: ð4Þ f ðvÞ ¼ Fðv; uÞcðvÞ ð4Þ where u are unit direction vectors along L (with L J) directions and fulg L l¼1, which are used to increase the accuracy of fiber directions, F(v, u) is a (J + 1) × (L + 1) matrix which will be illustrated in the next step. f(v) and c = c(v) denote (J + 1) × 1 and (L + 1) × 1 column vectors composed of estimated values of fODF and the coefficient of fODF, respectively. Notably that the diffusion measurements c also consist of two parts, the first L rows cani show the informa- tion about anisotropy; the last raw ciso represents the information related to isotropy. We can use this variable denoted as Piso to quantify the intensity of isotropy of each voxel. Piso can take place of the value of FA and GFA as well as conveys the message even better than them to some extent. Removing the isotropic part of each voxel inevitably increases the accuracy of fiber imaging. Once we have acquired the diffusion signal S(g) and ^Rðg vÞ, the unknown part fODF f can be computed using the iRL model. PLOS ONE \| DOI:10.1371/journal.pone.0168864 January 12, 2017 Dictionary basis representation Distributions of weighting estimated by solving the Least Norm method with Spherical harmonics (SH) and Dictionary basis (DB). doi:10.1371/journal.pone.0168864.g001 parameters setting are described in [41]. Thus, we can obtain a novel dictionary basis which avoids high order’s truncation of SH function and guarantees the sparsity of coefficient at a certain extent(Fig 1). To make dictionary basis be applicable to above isotropic model, we have to make some deformations on the dictionary. The final dictionary basis can be represented as F ¼ 1 1T 1 " # , where 1 represent J × 1 column vectors composed of 1. Dictionary basis representation SD has been proven to produce a good imaging result. [17] proposed to express SD directly in spherical harmonics (SH) domain, so the operation can be reduced to a simple set of matrix multiplications. Simultaneously, the presence of SH basis in the process of SD has been proven to be of great importance. From a signal processing perspective, high-order SH basis is needed if we want to represent or reconstruct crossing fibers accurately with really small separated angles. However, the higher harmonic components are more sensitive to noise. Considering numerical difficulties, typically spherical harmonic up to the order of eight is used, which lim- its their capability in reliably resolving fiber crossing of small angles [40]. An inverse relation- ship exists between high frequency term and angular resolution. Thus, we cannot obtain the highest resolution and the best resistance to noise simultaneously. On account of the above defects of SH basis, we use a new double-lobe basis function to build an over-completed dictionary basis. In this work, a set of over-completed orientation dis- tribution basis {d(v, ul)\|l = 1,. . .L} with discrete direction sets v 2 RJ and positional direction sets u 2 RL are introduced to represent fiber architecture in a voxel. The basis functions are uniformly distributed in unit sphere, thereby creating a predefined fODF field. A linear weighted combination of basis can be represented as ϕ = [d(v, u1),. . ., d(v, uL)]. By introducing an over-completed dictionary with cardinality L which is larger than unit sampling direction vectors J, we can construct a wide-ranged basis to map the fODF. Generally, fODF can be sparsely represented by the dictionary. Hence, most of the coefficients c are zero. The novel basis function quoted by [41] is proposed to establish the over-completed dictionary: dðv; ulÞ ¼ k1 sinWv;ul 1 k2 cos 22Wv;ul !t ð5Þ ð5Þ where ϑv, ul represent intersected angles between v and ul, and the other parameters, κ1, κ2 and τ are used to normalize the novel basis function. Detailed interpretation and specific PLOS ONE \| DOI:10.1371/journal.pone.0168864 January 12, 2017 5 / 21 iRL Fig 1. Weighting distributions. Distributions of weighting estimated by solving the Least Norm method with Spherical harmonics (SH) and Dictionary basis (DB). Fig 1. Weighting distributions. Distributions of weighting estimated by solving the Least Norm method with Spherical harmonics (SH) and Dictionary basis (DB). Fig 1. Weighting distributions. PLOS ONE \| DOI:10.1371/journal.pone.0168864 January 12, 2017 A new Richardson-Lucy model PðSjSÞ 1 ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 2ps2 p eðSSÞ2=2s2 ð7Þ ð7Þ Considering the premise of hypothesis that the sampling signal submitted to spherical deconvolution, we optimize the Eq (7). The RL model finds f from the observation S, knowing Considering the premise of hypothesis that the sampling signal submitted to spherical deconvolution, we optimize the Eq (7). The RL model finds f from the observation S, knowing response function ^R by maximizing the likelihood distribution. The result can be derived by minimizing the function log PðSjSÞ. We suppose that noise is independent from one voxel to another. When consider the whole brain region, the log-likelihood becomes a summation of the likelihood of all voxels. The multiplicative-type algorithm is equivalent to minimize J1(f) given by J1ðf Þ ¼ X x 1 2s2 ðSðxÞ ^Rf ðxÞÞ 2 log 1 ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 2ps2 p ð8Þ ð8Þ where x is the voxel index. Given that the function J1(f) is convex in f, looking for a minimum is equal to searching for a zero value of gradient of J1(f). We set the derivative of J1(f) with respect to f to be zero and get X x ðSðxÞ ^Rf ðxÞÞ s2 ^RT ¼ 0. There are some mathematical oper- ations which can be founded in [46]. Using the dictionary basis F to represent the fiber orien- tation f = Fc, we get: where x is the voxel index. Given that the function J1(f) is convex in f, looking for a minimum is equal to searching for a zero value of gradient of J1(f). We set the derivative of J1(f) with f f respect to f to be zero and get X x ðSðxÞ ^Rf ðxÞÞ s2 ^RT ¼ 0. There are some mathematical oper- ations which can be founded in [46]. Using the dictionary basis F to represent the fiber orien- tation f = Fc, we get: ations which can be founded in [46]. Using the dictionary basis F to represent the fiber orien- tation f = Fc, we get: ^RTS ^RT ^RðFcÞ ¼ 1 ð9Þ ð9Þ Richardson and Lucy suggested a multiplicative iterative method to solve Eq (9) ckþ1 ¼ ck ^RTS ^RT ^RðFckÞ ð10Þ ð10Þ Regularization with coefficient of fiber orientation. For obvious reasons, the operation of spherical deconvolution is a NP-hard problem. A new Richardson-Lucy model RL model is usually used in the field of astronomical imaging. This method has two advan- tages: the one is that it avoids the appearance of negative values in solutions because it satisfies non-negativity constraint of solution inherently; the other is that it well controls the instabili- ties in the process of solving and reduces the presence of noise artifacts in the solution for its robustness to noise [19]. Thus, the RL model has already been prevalent to settle the problem of fiber imaging in neurosciences field, as originally proposed by Daube-Whitherspoon and Muehllehner in [42]. Richardson-Lucy model with dictionary basis. The RL model, also known as expectation maximization (EM) algorithm, follows a statistical Bayesian approach to deconvolution prob- lem and implements an iterative estimation scheme for approximating the solutions of a maxi- mum-likelihood problem in the case of different noises [19]. Therein, to establish a necessary foundation for the presentation and development of the proposed method, a brief overview of RL model is provided firstly. Like common approach of image restoration uses a probabilistic framework: given a sampling degraded image S, we can obtain the best image S (actually is the fODF) when maximizing the probability of sampling image S. The probability PðSjSÞ obeys Bayes’ rule: PðSjSÞ ¼ PðSjSÞPðSÞ=PðSÞ. The magnitude signal of MR data is considered as Rician distribution [43], the likelihood is then: PðSjSÞ ¼ S s2 expf 1 2s2 ðS2 þ S2ÞgI0 SS s2 ð6Þ ð6Þ where I0 is the modified Bessel functions of the first kind of zero order, S denotes the true where I0 is the modified Bessel functions of the first kind of zero order, S denotes the true PLOS ONE \| DOI:10.1371/journal.pone.0168864 January 12, 2017 6 / 21 iRL magnitude signal intensity in the absence of noise, S is measured value of each voxel with noise, and σ2 is the variance of noise. When the Rician distribution is acquired with large SNR (i.e.,S=s 3), the process is better known as Gaussian approximation [19, 44, 45]. magnitude signal intensity in the absence of noise, S is measured value of each voxel with noise, and σ2 is the variance of noise. When the Rician distribution is acquired with large SNR (i.e.,S=s 3), the process is better known as Gaussian approximation [19, 44, 45]. PLOS ONE \| DOI:10.1371/journal.pone.0168864 January 12, 2017 A new Richardson-Lucy model To render the reconstruction perfectly and stably, we use regularization on the coefficient of fiber orientation, such as, total variation (TV) and sparse regularization. Putting a priori information on the coefficient of fiber orienta- tion seems reasonable. One of such information is spatial consistency. Despite many advantages of RL model, the fiber detail and noise interference are contradictory during the RL iteration process. This problem is generic for all maximum likelihood techniques because we usually want to attempt to fit the data as closely as possible. Thus, a trade-off exists between quality of image and the degree of noise interference when using RL method. In the intravoxel fODF field, voxels within a small neighbourhood usually consist of similar signals. Thus, the fODF derived from voxels ought to have a correlation in spatial structure. The advantages of using TV regulariza- tion are that it reserves the similarity of coefficient and avoids noise amplification by smooth- ing to certain extent. Here, we introduce TV constraint on the coefficient of fODF of the entire brain image to solve the above problem by adding energy function J1 reg, defined as: J1 reg ¼ lTV X x rci j j ð11Þ J1 reg ¼ lTV X x rci j j ð11Þ 7 / 21 iRL where λTV is the TV regularization parameter. Regularization is conducted in the entire field along each special gradient direction, which can be seen as I + 1 brain images. Although the images fcig I i¼1 and cI+1 have different statistical properties, regularization processing in the neighbouring voxel is not prevented. The spatial dependence introduced by TV function pro- motes smooth solutions in homogeneous regions and prevents the solution from having oscil- lations. However the process of regulation will allow the solution to have sharp discontinuities [47, 48], we need to increase another constraints. Sparse reconstruction method is broadly applied in the field of digital image processing. The sparsity constraint of the coefficient of fODF and the sparse recovery process lead us to estimate a sharp fODF from limited acquisitions. Notably, fiber orientation representation in the proposed basis is indeed sparse. The true distribution of fiber orientation can be consid- ered sparse with the assumption that only a small number of elements of fODF are non-zero physically [49]. However, the introduction of TV regularization induces excessive smoothness between neighbouring voxels. PLOS ONE \| DOI:10.1371/journal.pone.0168864 January 12, 2017 Experimental data Simulated data. Datastes are generated assuming axially symmetric diffusion tensor pro- files for each fiber population (MD = 0.7 × 10−3mm2/s) using a typical 81 directions sampling scheme [50]. To study the effect of each parameter separately in simulations, only one parame- ter at a time is varied. Details of these simulated datasets are provided in the following sections. • Simulated data1: To guarantee the impartiality of comparative methods, We build the fol- lowing simulated dataset which is the same with the data in [23], so that we can contrast iCSD method directly. Two crossing fibers are constructed, assuming the angle of crossing fiber is 70˚, with varying PVEs values ranging from 0.1 to 1 (with a step of 0.1) and with dif- ferent b-values of 1000 and 3000. The other dataset also reconstructs two crossing fibers, with varying crossing angles of fiber ranging from 40˚ to 90˚ (with a step of 10˚) and with 50% isotropic signal. Complex Gaussian noise is added to obtain noisy signals with SNR = 20. • Simulated data2: We create the synthetic data with two crossing fibers and different param- eters which determine the imaging quality. Each simulated dataset is composed by 11 times 11 voxels whose fraction of isotropy is varied from 0.1 to 1 with intervals of 0.1 along x-axis, and SNR is changed from 10 to 30 with intervals of 2 along y-axis. The dataset is used to prove the validity of iRL to solve the PVEs under the condition of PVEs and noise changed. The representative angles are 40˚ and 90˚ between fiber populations in configurations, and the diffusion weighting b = 3000s/mm2. IEEE international Symposium on Biomedical Imaging (ISBI) challenge phantom data. The second simulated dataset coming from the ISBI 2013 Reconstruction Challenge is acquired from an open-source software library (http://hardi.ep.ch/static/events/2013-ISBI/), which creates realistic phantoms in structural and diffusion MRI. The synthetic datasets con- sist of 27 simulated ground truths, including branching, kissing, and crossing structures with angles between 30˚ and 90˚. The dataset contains 64 gradient directions with b = 3000s/mm2 at SNR = 10, SNR = 20 and SNR = 30. The fODF mapping is color-coded by the standard DTI col- our scheme (red: left-right; green: front-back; and blue: up-down). In vivo human brain data. Evaluation is performed using real human data which is pub- lished on Dipy (http://nipy.org/dipy/). A new Richardson-Lucy model To ensure each fiber sparse, the sparsity constraint is often added to fODF in spherical deconvolution problem. We make full use of ℓ1 norm to ensure the sparsity of coefficient in neighboring voxels. Here, we introduce the energy function of sparsity term J2 reg, defined as J2 reg ¼ l‘1 X x ci j j ð12Þ ð12Þ where λℓ1 is the sparse regularization parameter. The two regularization terms based on maxi- mum likelihood estimation can get the derivatives of Jreg with respect to c, which can be expressed as @ @c J1 reg ¼ lTVdiv rc jrcj jX and @ @c J2 reg ¼ l‘1 rc jrcj jx, respectively, where div and r stand for divergence and differentiation, and x is voxel index indicating that regularization is conducted between voxels. The term \|rc\| is replaced by its approximate value ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ jrcj 2 þ ε q , where ε is a small positive constant [47]. The total energy function is known as J1 þ J1 reg þ J2 reg ¼ X x 1 2s2 S þ ^RFc2 log 1 ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 2ps2 p þ lTV X x rci j j þ l‘1 X x ci j j ð13Þ ð13Þ We minimize Eq (13) using multiplicative gradient-based algorithm (or equivalently using EM algorithm for penalized criterion of Eq (13) and obtain the final result defined as We minimize Eq (13) using multiplicative gradient-based algorithm (or equivalently using EM algorithm for penalized criterion of Eq (13) and obtain the final result defined as cðkþ1Þ ¼ cðkÞ ^RTS ^RT ^RðFcðkÞÞ L ðkÞ 1 TVðkÞ ð14Þ ð14Þ where c(k) is the estimated coefficient of fiber orientation, which is a ((L+1) × 1) dimension col- umn vector at iteration k at voxel x, and L ðkÞ 1 and TV(k) are the ℓ1 and TV regularization vector at iteration k. A new Richardson-Lucy model The element at different gradient positions i of ℓ1 regularized vector is computed as: L1 ð Þ ðkÞ i ¼ 1 1 l‘1 rc ðkÞ i rcðkÞ i xj ð15Þ ð15Þ PLOS ONE \| DOI:10.1371/journal.pone.0168864 January 12, 2017 8 / 21 iRL The element at different gradient position i of TV regularized vector is computed as: TV ðkÞ i ¼ 1 1 lTVdiv rc ðkÞ i rcðkÞ i xj ð16Þ ð16Þ Numerically, we notice that the regularization parameter should be neither too small nor too large. In the simulated experiments, we will discuss the selection of regularization parameters. PLOS ONE \| DOI:10.1371/journal.pone.0168864 January 12, 2017 Experimental data The whole brain is covered with contiguous 2mm slices with an in-plane resolution of 2 × 2mm2. For preprocessing of diffusion data, we use MRIcron and SPM8 toolbox. First, the DICOM images sets (.dcm) are split into NIfTI (.nii), gradient sequence (.bvecs), and sensitive coefficient (.bvals) datasets using MRIcron software, where the NIfTI dataset contains scanned sequence corresponding to the gradient sequence. DW PLOS ONE \| DOI:10.1371/journal.pone.0168864 January 12, 2017 9 / 21 iRL images are acquired along 150 uniformly distributed directions using b = 2000s/mm2 and a single b = 0s/mm2 (the size of the whole brain is 81 × 106 × 76). images are acquired along 150 uniformly distributed directions using b = 2000s/mm2 and a single b = 0s/mm2 (the size of the whole brain is 81 × 106 × 76). Comparison metrics for phantom data The performances of algorithms are quantified by comparing the obtained reconstructions with ground-truth. We adopt some of evaluation metrics widely used in the literatures [51–53]. • Average angular error (AAE): We compute the deviation between estimated fiber orienta- tion and ground truth [54]: • Average angular error (AAE): We compute the deviation between estimated fiber orienta- tion and ground truth [54]: AAE ¼ 1 O j j X x2O X np h¼1 arccos εx ~εx ð Þ j j ð17Þ ð17Þ where εx is the “ground truth” and ~εx is estimated fiber orientation, O is the local region used to compute angular error. we obtain one or more significant peaks of fODF (the num- ber of peaks defined as nP) in each voxel x 2 O, sum angular error of all peaks and finally get the average angular error. These operations are repeated about 100 times. • Average probability of false direction (APFD): APFD is used to evaluate the probability of false directions compared with real fiber number ~M x inside a voxel x. The ratio of false posi- tive (r+) and ratio of false negative (r−) are defined as rþ ¼ 1 O j j X x2O Mþ x ~Mx 100%; r ¼ 1 O j j X x2O ~Mx Mþ x 100%; ð18Þ ð18Þ In a region O, Mþ x and Mx denote the over-estimated and under-estimated number of fibers inside a voxel compared to ground truth. • Fractional anisotropy (FA): The FA characterizes the degree of “out-of-roundness” of diffu- sion ellipsoid. It measures the fraction of total magnitude of diffusion tensor that is aniso- tropic • Fractional anisotropy (FA): The FA characterizes the degree of “out-of-roundness” of diffu- sion ellipsoid. It measures the fraction of total magnitude of diffusion tensor that is aniso- tropic FA ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ l1 l 2 þ l2 l 2 þ l3 l 2 2 l2 1 þ l2 2 þ l2 3 s ð19Þ ð19Þ where λ1, λ2, λ3 are the eigenvalues provided by diffusion tensor, which is one of the most important rotationally invariant quantitative scalar parameters. l is the arithmetic mean of the three eigenvalues. where λ1, λ2, λ3 are the eigenvalues provided by diffusion tensor, which is one of the most important rotationally invariant quantitative scalar parameters. l is the arithmetic mean of the three eigenvalues. PLOS ONE \| DOI:10.1371/journal.pone.0168864 January 12, 2017 Implementation details All experiments of the proposed method are conducted on Inter(R)@2.4 GHz (48 G RAM, 64 bit). For measured signal, the obtained mask image is down-sampled to the dimensions of dMRI. Mask analysis is conducted on DSI Studio 1 (http://www.dsi-studio.labsolver.org). For the dictionary basis, the dimension of coefficients and the basis vectors are the same, represent- ing the related percentage of each dictionary basis. For the positional direction sets u of dictio- nary basis, a tessellation scheme is distributed evenly on 321 points on a hemisphere and is generated by the subdivision of the face of an icosahedron. By avoiding repeated sampling, the discrete direction sets v are made to be identical with u. To perfectly reconstruct the fODF, the reconstructed dictionary basis is designed using a symmetric sphere with 10 242 vertices from Dipy (http://dipy.org/), which is an array of 10 242 fODF values corresponding to the vertices of sphere. To ensure the applicability of in vivo data, the two RFs in vivo data are acquired according to typical value of diffusion tensor signals in the corpus callosum and cortex respec- tively [17, 23]. We choose 50 voxels with the highest FA and use the average of signals whose principal eigenvectors are aligned along z-axis to acquire the anisotropic RF. Identically, we choose 50 voxels with the lowest FA and use the average of signals to acquire the isotropic RF. We compare the proposed method iRL with the other state-of-the-art methods on simu- lated phantom and real data. The alternative approaches include Recursive calibration con- strained spherical deconvolution (RC-CSD) [55], Sparse Fascicle Model (SFM) [56], damped Richardson Lucy (dRL) [26], information constrained spherical deconvolution (iCSD) [23] and Multi-shell multi-tissue constrained spherical deconvolution(MSMT-CSD) [24]. RC-CSD is an improvement of SD, which provides an accurately calibrated RF. SFM treats each MRI voxel as two types of compartments, non-oriented tissues and oriented fascicles considering the PVEs, which is implemented using Dipy (http://nipy.org/dipy/index.html) publish library [57]. The dRL is aiming at reducing isotropic background effects in spherical deconvolution, which is implemented using a software package provided in (http://neuroimagen.es/webs/ hardi-tools/). The iCSD improves the estimation of fODF by modifying the RF to account for non-WM PVEs locally. MSMT-CSD uses CSD approach to estimate a multi-tissue ODF and implements in MRtrix (http://www.mrtrix.org/) [58]. Comparison metrics for phantom data • Generalized fractional anisotropy (GFA): Scalar measures on the fODF are useful in defin- ing tissue contrast, performing statistical analyses, or summarizing the geometric properties of fODF. We define the scalar measures GFA as • Generalized fractional anisotropy (GFA): Scalar measures on the fODF are useful in defin- ing tissue contrast, performing statistical analyses, or summarizing the geometric properties of fODF. We define the scalar measures GFA as GFA ¼ std fð Þ rms fð Þ ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ n Pn j¼1 ðf ðvjÞ fh iÞ 2 ðn 1Þ Pn j¼1 f ðvjÞ 2 v u u t ð20Þ ð20Þ where n is the number of fODF, std is the standard deviation, rms is the root-mean-square, and hf i ¼ 1n Pn j¼1 f ðvjÞ is the mean of the ODF. The GFA metric is automatically normal- ized to [0, 1]. PLOS ONE \| DOI:10.1371/journal.pone.0168864 January 12, 2017 10 / 21 iRL • Generalized relative anisotropy (GRA): The GRA scalar represents a measurement of devi- ation from the isotropic state of the fODF of each voxel: • Generalized relative anisotropy (GRA): The GRA scalar represents a measurement of devi- ation from the isotropic state of the fODF of each voxel: GRA ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ Pn j¼1 ðf ðvjÞ fh iÞ 2 n fh i s ð21Þ ð21Þ It’s worth noting that peaks in clusters that are less than half of the crossing angle (with an upper limit of 35 degrees) from the true orientations are considered correct peaks. Implementation details It’s worth mentioning that MSMT-CSD can reconstruct brain fibers using single shell data, but the function of separating different tis- sues can not work well. The number of iterations of each method is set to 200 times. The related parameters used in compared methods are set to their optimal values according to the reference documents. For dRL algorithm, η acts as a threshold parameter and controls the damped amplitude of FOF, which is set to η = 0.08. We compare the proposed method iRL with the other state-of-the-art methods on simu- lated phantom and real data. The alternative approaches include Recursive calibration con- strained spherical deconvolution (RC-CSD) [55], Sparse Fascicle Model (SFM) [56], damped Richardson Lucy (dRL) [26], information constrained spherical deconvolution (iCSD) [23] and Multi-shell multi-tissue constrained spherical deconvolution(MSMT-CSD) [24]. RC-CSD is an improvement of SD, which provides an accurately calibrated RF. SFM treats each MRI voxel as two types of compartments, non-oriented tissues and oriented fascicles considering the PVEs, which is implemented using Dipy (http://nipy.org/dipy/index.html) publish library [57]. The dRL is aiming at reducing isotropic background effects in spherical deconvolution, Results Optimal regularization parameter. The new deconvolution algorithm with TV and ℓ1 regularization has shown good imaging result with the elaborately chosen regularization PLOS ONE \| DOI:10.1371/journal.pone.0168864 January 12, 2017 11 / 21 iRL Fig 2. Simulated results of parameter selection. Average angular error using different regularization parameters in ISBI data with different SNRs. Fig 2. Simulated results of parameter selection. Average angular error using different regularization parameters in ISBI data with different SNRs. d results of parameter selection. Average angular error using different regularization parameters in ISBI data with doi:10.1371/journal.pone.0168864.g002 doi:10.1371/journal.pone.0168864.g002 parameters. The choice of good parameters value plays a crucial role in imaging results when using iRL. Thus, the first step of our experiment is to study if and how c estimation is influ- enced by setting different regularization parameters and by choosing different numbers of algorithm iterations during the process of our algorithm. To obtain the best regularization parameters and the number of iterations, we use different parameters to image the ISBI data with SNRs of 10, 20 and 30, and identify the quantitative index to evaluate image quality. To select regularization parameters, we use the AAE to be the quantitative index (Fig 2). We performe 100 repetitions with simulated data. We find that the ℓ1 regularization param- eter affects the angular resolution of imaging fiber and the TV regularization plays a vital role in resisting noise. We need only to increase the value of the TV or ℓ1 regularization parameters to improve the quality of imaging when the signals have low SNR or small angle, respectively. From Fig 2, the best regularization parameters can be set to λℓ1 = 0.01 and λTV = 0.5. The RL algorithm has certain superiority in resisting noise, but when the SNR is low, as shown in Fig 2, the imaging results are unsatisfactory and have a relatively large angular error. The RL algorithm is known to have the property of ‘semi-convergence’ [59], i.e., the solu- tion initially converges to the true value and then diverges as iterations proceed [19]. We choose 200 as the maximum iteration numbers to prevent noise amplification and generation of artifacts. Simulated data in the presence of isotropic diffusion. We use different simulated data- sets to verify the effectiveness of iRL. Comparative tests are conducted by four kinds of meth- ods. Results This experiment is used to verify the ability of imaging the signal with different volume fractions of isotropic signal (Fig 3). The other simulated datasets are generated in the same way, excepting that the diffusion weighting b is changed (Fig 4). We perform 100 repetitions with the simulated datasets that are generated randomly. Fig 3. Comparison of simulated results. AAE, False peaks and Correct peaks for different proportions of anisotropic signal (diffusion weighting 3000s/mm2, angle 70˚, and SNR 20). doi:10.1371/journal.pone.0168864.g003 Fig 3. Comparison of simulated results. AAE, False peaks and Correct peaks for different proportions of anisotropic signal (diffusion weighting 3000s/mm2, angle 70˚, and SNR 20). doi:10.1371/journal.pone.0168864.g003 PLOS ONE \| DOI:10.1371/journal.pone.0168864 January 12, 2017 12 / 21 iRL Fig 4. Comparison of simulated results. AAE, False peaks and Correct peaks for different proportions of anisotropic signal (diffusion weighting 1000s/mm2, angle 70˚, and SNR 20). doi:10 1371/journal pone 0168864 g004 Fig 4. Comparison of simulated results. AAE, False peaks and Correct peaks for different proportions of anisotropic signal (diffusion weighting 1000s/mm2, angle 70˚, and SNR 20). doi:10.1371/journal.pone.0168864.g004 Fig 4. Comparison of simulated results. AAE, False peaks and Correct peaks for different proportions of anisotropic signal (diffusion weighting 1000s/mm2, angle 70˚, and SNR 20). doi:10.1371/journal.pone.0168864.g004 doi:10.1371/journal.pone.0168864.g004 Compared with the low b value dataset, the high b value dataset shows fODF with a partial increase in angular resolution. However, no change exists in angular resolution for the signal with low proportion of anisotropy. Figs 3 and 4 show that when the signal has high proportion of anisotropy, the imagings of five kinds of methods are all accurate. The iCSD and iRL have a relative better angular resolution and less numbers of false peaks. When high isotropy exists in the simulated signal, iRL is advantageous over the other four kinds of methods in the aspect of angular resolution. Regardless of signal composition, iRL has the best and smallest angular resolution. We perform simulated experiments to investigate the simulated datasets with different fiber crossing angles. We utilize five methods to image the above simulated signals respectively. This experiment is used to verify the ability of imaging the signal with different crossing angles (the results are shown in Fig 5). We also perform 100 repetitions with the simulated datasets which are generated randomly. The five methods are all becoming more effective as the crossing angles increasing. Results Figures f1-f2 are the new index Piso. Fig 6. Comparison of the simulated results. FA and GFA with RC-CSD(a), SFM (b), dRL (c), iCSD (d) and iRL (e). Figures a1-f1 are the imagings for 40˚ cross-angle. Figures a2-f2 are the imagings for 90˚ cross-angle. Figures f1-f2 are the new index Piso. doi:10.1371/journal.pone.0168864.g006 doi:10.1371/journal.pone.0168864.g006 make Piso a reverse imaging) using different methods (Figures a1-e1) and different crossing angles (Figures a1-a2). For 40˚ cross fibers, no significant difference is observed. For 90˚ cross fibers, the quantitative index FA has an obvious deficiency in which the degree of anisotropy is lower than the normal levels. However, the quantitative indexes GFA and Piso have a correct indication. Considering both experiments, Piso has better implementation in low anisotropy. make Piso a reverse imaging) using different methods (Figures a1-e1) and different crossing angles (Figures a1-a2). For 40˚ cross fibers, no significant difference is observed. For 90˚ cross fibers, the quantitative index FA has an obvious deficiency in which the degree of anisotropy is lower than the normal levels. However, the quantitative indexes GFA and Piso have a correct indication. Considering both experiments, Piso has better implementation in low anisotropy. fODF estimation for ISBI data. We compare several different methods using the authori- tative ISBI simulated experiment data. Fig 7 compares the reconstructed fODF. We observe that the fODF estimations of each voxel are relatively independent and prone to noise. The fiber orientations reconstructed by standard RC-CSD, SFM, dRL, and MSMT-CSD methods always lack important information on fiber crossing. g p p py fODF estimation for ISBI data. We compare several different methods using the authori- tative ISBI simulated experiment data. Fig 7 compares the reconstructed fODF. We observe that the fODF estimations of each voxel are relatively independent and prone to noise. The fiber orientations reconstructed by standard RC-CSD, SFM, dRL, and MSMT-CSD methods always lack important information on fiber crossing. fODF estimation for ISBI data. We compare several different methods using the authori- tative ISBI simulated experiment data. Fig 7 compares the reconstructed fODF. We observe that the fODF estimations of each voxel are relatively independent and prone to noise. The fiber orientations reconstructed by standard RC-CSD, SFM, dRL, and MSMT-CSD methods always lack important information on fiber crossing. Fig 7. Visualization of the fODF reconstructed from ISBI dataset with HARDI data. Results In our method, the quantitative indexes of AAE and false peaks is lower for all angles and the preci- sion is improved remarkably for angles larger than 50˚ (Fig 5). It’s worth mentioning that the 40˚ crossing angle could be identified with 50% PVEs using iRL. We also perform simulated experiments to investigate simulated datasets with different PVEs and SNRs and utilize five methods to image the above simulated signals respectively (the results are shown in Fig 6). To verify the effectiveness of our method in aspect of the new iso- tropic quantitative index, we conduct the signal of simulated data2 and the imaging result is mapped to quantitative indexes, FA and GFA included. pp q In the case of anisotropy and SNR increased, the upper-left corner of each figure has the poorest simulated signal, and the lower-right corner of each figure has the best simulated sig- nal. In Fig 6, we compare FA, GFA, and our new quantitative index Piso (because the quantifi- cation of Piso is the extent of isotropy, which is contrary to FA and GFA. For comparison, we Fig 5. Comparison of simulated results. AAE, false peaks and correct peaks for different crossing angles(with 50% isotropic signal, diffusion weighting 3000s/mm2, and SNR 20). Fig 5. Comparison of simulated results. AAE, false peaks and correct peaks for different crossing angles(with 50% isotropic signal, diffusion weighting 3000s/mm2, and SNR 20). Fig 5. Comparison of simulated results. AAE, false peaks and correct peaks for different crossing angles(with 50% isotropic signal, diffusion weighting 3000s/mm2, and SNR 20). doi:10.1371/journal.pone.0168864.g005 doi:10.1371/journal.pone.0168864.g005 PLOS ONE \| DOI:10.1371/journal.pone.0168864 January 12, 2017 13 / 21 iRL Fig 6. Comparison of the simulated results. FA and GFA with RC-CSD(a), SFM (b), dRL (c), iCSD (d) and iRL (e). Figures a1-f1 are the imagings for 40˚ cross-angle. Figures a2-f2 are the imagings for 90˚ cross-angle. Figures f1-f2 are the new index Piso. Fig 6. Comparison of the simulated results. FA and GFA with RC-CSD(a), SFM (b), dRL (c), iCSD (d) and iRL (e). Figures a1-f1 are the imagings for 40˚ cross-angle. Figures a2-f2 are the imagings for 90˚ cross-angle. Figures f1-f2 are the new index Piso. Fig 6. Comparison of the simulated results. FA and GFA with RC-CSD(a), SFM (b), dRL (c), iCSD (d) and iRL (e). Figures a1-f1 are the imagings for 40˚ cross-angle. Figures a2-f2 are the imagings for 90˚ cross-angle. Results Depicted fODF profiles correspond to estimations from the RC-CSD (a), SFM (b), dRL (c), MSMT-CSD (d) and our method iRL (e). doi:10.1371/journal.pone.0168864.g007 Fig 7. Visualization of the fODF reconstructed from ISBI dataset with HARDI data. Depicted fODF profiles correspond to estimations from the RC-CSD (a), SFM (b), dRL (c), MSMT-CSD (d) and our method iRL (e). doi:10.1371/journal.pone.0168864.g007 Fig 7. Visualization of the fODF reconstructed from ISBI dataset with HARDI data. Depicted fODF profiles correspond to estimations from the RC-CSD (a), SFM (b), dRL (c), MSMT-CSD (d) and our method iRL (e). doi:10.1371/journal.pone.0168864.g007 PLOS ONE \| DOI:10.1371/journal.pone.0168864 January 12, 2017 14 / 21 iRL Fig 8. Quantification of the reconstruction accuracy. The results of RC-CSD, SFM, dRL, MSMT-CSD, and iRL in terms of AAE, n+, and n−using ISBI data. Fig 8. Quantification of the reconstruction accuracy. The results of RC-CSD, SFM, dRL, MSMT-CSD, and iRL in terms of AAE, n+, and n−using ISBI data Fig 8. Quantification of the reconstruction accuracy. The results of RC-CSD, SFM, dRL, MSMT-CSD, and iRL in terms of AAE, n+, and n−using ISBI data. Fig 8. Quantification of the reconstruction accuracy. The results of RC-CSD, SFM, dRL, MSMT-CSD, and iRL in terms of AAE, n+, and n−using ISBI data. doi:10.1371/journal.pone.0168864.g008 doi:10.1371/journal.pone.0168864.g008 In the marked regions in Fig 7, the crossing angles are very small. The iRL can separate this part of crossing, but the results are imperfect. In the crossing fiber case, performances are assessed according to two criteria: (1) the effect of miscalibration on angular resolution, and (2) the over-estimated and under-estimated number of fibers. Fig 8 shows that iRL produces fewer angular errors. About the overestimation of false peaks, iRL has a better result when compared with RC-CSD and SFM. There is a better result about underestimation of false peaks when compared with dRL and MSMT-CSD. It’s mentioning that the iRL has fewer total numbers of false peaks than the other five methods, regardless of SNR. fODF estimation for human data. Evaluation is performed using real human data acquired on public datasets (http://nipy.org/dipy/). We select two representative areas, one of the areas contains multiple functional areas of the brain, such as the cortex and CSF (i.e, con- taining possible isotropic compartment). Fig 9 compares the intravoxel fiber architecture estimated by five different methods on the human datasets. PLOS ONE \| DOI:10.1371/journal.pone.0168864 January 12, 2017 Results In the posterior thalamic radiation (refer to Human Brain in ICBM-152 Space) region (marked with a yellow box in Fig 9), the situation of fiber crossing is complex, containing single fiber and multiple fiber crossings. The iRL has a good imaging of multiple fiber crossing trends. The other methods always lack of some fiber directions. The same results can be seen in Fig 10. In addition, in the posterior thalamic radiation region, the isotropic sig- nal is stronger, and the compared results are more obvious. In particular, the fibers (red ellip- ses) in the superior temporal gyrus WM (STG-WM) and the middle temporal gyrus WM (MTG-WM) regions are well represented by iRL. Fig 9 compares the intravoxel fiber architecture estimated by five different methods on the human datasets. In the posterior thalamic radiation (refer to Human Brain in ICBM-152 Space) region (marked with a yellow box in Fig 9), the situation of fiber crossing is complex, containing single fiber and multiple fiber crossings. The iRL has a good imaging of multiple fiber crossing trends. The other methods always lack of some fiber directions. The same results can be seen in Fig 10. In addition, in the posterior thalamic radiation region, the isotropic sig- nal is stronger, and the compared results are more obvious. In particular, the fibers (red ellip- ses) in the superior temporal gyrus WM (STG-WM) and the middle temporal gyrus WM (MTG-WM) regions are well represented by iRL. Fig 9. Visualization of fODFs reconstructed from real data. Depicted fODF profiles correspond to the estimations from RC-CSD (a), SFM (b), dRL (c), MSMT-CSD (d) and iRL (e). The background images are fractional anisotropy images computed from each reconstruction. doi:10.1371/journal.pone.0168864.g009 Fig 9. Visualization of fODFs reconstructed from real data. Depicted fODF profiles correspond to the estimations from RC-CSD (a), SFM (b), dRL (c), MSMT-CSD (d) and iRL (e). The background images are fractional anisotropy images computed from each reconstruction. doi:10.1371/journal.pone.0168864.g009 15 / 21 PLOS ONE \| DOI:10.1371/journal.pone.0168864 January 12, 2017 iRL Fig 10. Visualization of fODFs reconstructed from real data. Depicted fODF profiles correspond to the estimations from RC-CSD (a), SFM (b), dRL (c), MSMT-CSD (d) and iRL (e). The background images are the fractional anisotropy images computed from each reconstruction. doi:10.1371/journal.pone.0168864.g010 Fig 10. Visualization of fODFs reconstructed from real data. Depicted fODF profiles correspond to the estimations from RC-CSD (a), SFM (b), dRL (c), MSMT-CSD (d) and iRL (e). Results The background images are the fractional anisotropy images computed from each reconstruction. doi:10.1371/journal.pone.0168864.g010 doi:10.1371/journal.pone.0168864.g010 doi:10.1371/journal.pone.0168864.g010 The quantitative indexes of GM and WM are carried out in above areas. We use Piso to quantify the difference between WM and GM in brain regions by using different indexes, including FA, GFA, and GRA. The three indexes are well-known and used in various occa- sions to describe the strength of anisotropic diffusion. The degree of diffusion anisotropy is severely underestimated using the indexes calculated by diffusion coefficients acquired in fiber orientations. Some researchers present that water diffusivity in the directions parallel to the fiber is almost 10 times higher than the average diffu- sivity in directions perpendicular to them [9]. The marked area where the fibers have vertical distribution. The anisotropy is actually very strong, whereas the figure of FA (Fig 11b1) shows a strong isotropy. The figures of Piso (Fig 11a), GFA (Fig 11b2), and GRA (Fig 11b3) show simi- lar results on anisotropy. PLOS ONE \| DOI:10.1371/journal.pone.0168864 January 12, 2017 Conclusions and Discussion We focus on PVEs in the reconstruction of fiber configuration, which rarely elicit interest of researchers. PVEs are some of the greatest obstacles in improving the accuracy of fiber imag- ing. We usually utilize the anisotropic signal to reconstruct fiber orientation, which is affected by the isotropic signal. Only by removing the isotropic signal from DW signal, can we obtain the best imaging results, as we have done in this paper. The contribution of our approach is that we initially propose a method based on the local maximum likelihood estimation to isolate the isotropic from DW signal in entire regions included in both non-WM and WM by rebuild- ing RF and fODF used to estimate the coefficients of fODF to account for tissues composition. At the same time, the separated parts can be used to quantify the degree of isotropic signal in each individual voxel. Secondly, the application of dictionary basis and RL model successfully solves the ill-posed problem and ringing effect. Finally, the spatial regularization of FOD is approximated by combining TV and ℓ1 norms that stabilize the deconvolution problem and promote sparsity in the solution. We also compare the performances of proposed method with several state-of-the-art algorithms on synthetic data and human brain datasets. Results show significant improvement over contrastive methods in its ability to reduce false positive fiber orientations and preserve angular resolution on both simulated and in vivo datasets. PLOS ONE \| DOI:10.1371/journal.pone.0168864 January 12, 2017 16 / 21 iRL Fig 11. Display of diffusion degree using four methods. (a): Piso quantifies the intensity of isotropic signal, and (b1-b3) quantify the intensity of anisotropic signal. doi:10 1371/journal pone 0168864 g011 Fig 11. Display of diffusion degree using four methods. (a): Piso quantifies the intensity of isotropic signal, and (b1-b3) quantify the intensity of anisotropic signal. Fig 11. Display of diffusion degree using four methods. (a): Piso quantifies the intensity of isotropic signal, and (b1-b3) quantify the intensity of anisotropic signal. doi:10.1371/journal.pone.0168864.g011 doi:10.1371/journal.pone.0168864.g011 Some of non-WM PVEs are due to the reduced SNR of WM compartment, which cannot be recovered, and the rest of effects are due to mostly isotropic diffusion from non-WM tissue [23]. In this paper, we extend PVEs’ influence, including the isotropic diffusion in WM and the increase in isotropy caused by complex fiber directions. By isolating the isotropic signal, the imaging results significantly improve, especially on the AAE, throughout the whole brain. PLOS ONE \| DOI:10.1371/journal.pone.0168864 January 12, 2017 Conclusions and Discussion From the Fig 8, we control the AAE within 8˚ using open ISBI data. Simulated results show that with the reduction of isotropic signal, the AAE significantly increases. As regard 50˚ of crossing fiber, although the proportion of isotropic signal is as low as 0.1, the AAE remains within 30˚. This is a complicated process because the imaging result is affected by many parameters, such as b value, regularization parameters, iterations, and so on. For different datasets, we should adjust the corresponding parameters to obtain the best imag- ing results. Notably, a lower b value leads to poorer imaging. We can find another defect, i.e., the decrease in fiber quantity is more outstanding than the overestimation of fiber in the simu- lated data. This problem is inherent in the method related to RL, which will be our concern in a future study. Real experimental results indicate that iRL efficiently improves the ability of resolving crossing fibers in regions with high PVEs, whereas in high anisotropy regions, iRL and others PLOS ONE \| DOI:10.1371/journal.pone.0168864 January 12, 2017 17 / 21 iRL produce roughly identical results. In the region of the internal capsule and the corpus callo- sum, the tracts have relatively larger amplitude, which is particularly useful in connectomics. Given the abandonment of least squares and spherical harmonic function, the spurious fODF peaks (consistent with well-known ringing artefacts) have a prominent reduction on Figs 9 and 10. The comparisons of the tract density image between iRL and others show increased tract density in the main WM tracts and decreased tract density in non-WM region, which are useful for fiber tracking. Some open areas of researches exist in iRL. Firstly, for the two different diffusion models, different choices exist for regularization parameters. Considering the different diffusion regions, the strength of regularization should be discrepant. Secondly, a calibrated RF must be used to further reduce spurious peaks. Fortunately, the methods based on RL have a low over- all sensitivity to miscalibration. Thirdly, this method has potential to considerably reduce gra- dient directions, indicating a clinically feasible acquisition time. Thus, the application of this method is significant in clinical studies in the future. Finally, the assumed unimodal Gaussian diffusion model does not apply to MRI measurements, which are completely proven to be Rician distribution model [60]. These existing problems will be studied in our future work. Acknowledgments I thank my parents for allowing me to realize my own potential. All supports they have pro- vided me over these years are the greatest gift I have ever received. Moreover, I need to thank my tutor for providing me good academic atmosphere and answering all my questions. Conceptualization: TTX YW YJF. Funding acquisition: YJF QCZG. Investigation: YJF QCZG. Methodology: TTX YW JZ. Methodology: TTX YW JZ. Project administration: TTX QRZ. Resources: TTX JZ YW. Resources: TTX JZ YW. Software: TTX YW QRZ. Software: TTX YW QRZ. Supervision: YJF QCZG. Supervision: YJF QCZG. Validation: TTX QRZ JZH. Validation: TTX QRZ JZH. Visualization: TTX YW JZH. Writing – original draft: TTX YW JZ. Writing – review & editing: TTX JZH YJF. Author Contributions Conceptualization: TTX YW YJF. Data curation: TTX YW JZ. Formal analysis: TTX QRZ YW. Funding acquisition: YJF QCZG. Investigation: YJF QCZG. Methodology: TTX YW JZ. Project administration: TTX QRZ. Resources: TTX JZ YW. Software: TTX YW QRZ. Supervision: YJF QCZG. Validation: TTX QRZ JZH. Visualization: TTX YW JZH. Writing – original draft: TTX YW JZ. Writing – review & editing: TTX JZH YJF. References 1. Ganguly D, Chakraborty S, Kim Th. A cognitive study on medical imaging. International Journal of Bio- Science and Bio-Technology. 2010; 2(3):1–18. Conceptualization: TTX YW YJF. References 1. Ganguly D, Chakraborty S, Kim Th. A cognitive study on medical imaging. International Journal of Bio- Science and Bio-Technology. 2010; 2(3):1–18. 1. 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https://openalex.org/W2792546228	http://aminbiol.com.ua/20152pdf/22.pdf	English	null	Vitamin D-status of calves at the first month of life after different routes of cholecalciferol input to cows	Bìologìâ tvarin/Biologìâ tvarin	2,015	cc-by	4,746	VITAMIN D-STATUS OF CALVES AT THE FIRST MONTH OF LIFE AFTERDIFFERENTROUTES OF CHOLECALCIFEROLINPUT TO COWS L. L.Yuskiv, V. V.Vlizlo l_yuskiv@inenbiol.com.ua Institute of animal biology NAAS, Str. V. Stus, 38, Lviv, 79034, Ukraine Vitamin D-status of early postnatal calves obtained from high-performance cows were treated with cholecalciferol orally or intramuscularly in winter-stall period was investigated.Vitamin D was added to the diet in daily dose of 30 IU per 1 kg of body weight for a month starting 7–10 days before the predicted date of calving, and then in 5–7 day after calving. Intramuscularly vitamin D was administered: the first time — at 7–10 days before the predicted date of calving and three more times starting from 5–7th day after calving every 7 days at a dose of 210 IU per 1 kg of body weight for each input.For studies in calves for took blood 5–7- and 28–30-th day after birth. In the blood examined the contents of the 25-hydroxyvitamin D, total calcium and its fractions, inorganic phosphorus, magnesium and alkaline phosphatase activity. It was established that calves derived from cows that in the last days of gestation and after calving were treated with cholecalciferol orally or intramuscularly had higher blood levels of 25-OHD3, total calcium, bounded with protein calcium and ultrafiltrated calcium, inorganic phosphorus, magnesium and lower activity of alkaline phosphatase compared to calves from cows of the control group. Blood serum of calves born by cow sthat were intramuscular injected with vitamin D3 characterized by significantly higher content of 25-OH D3, calcium and inorganic phosphorus at 5–7th and 28–30th day after birth, compared to calves of control group. Supplementation of cow diet with cholecalciferol showed the effectt on these calf blood parameters onlyon 28–30thday after birth. ff f p y y f Thus, the introduction of cholecalciferol cows in late pregnancy and early lactation is essential to ensure their calves this vitamin in the early postnatal period. Keywords: COWS, CALVES, VITAMIN D, METABOLISM, BLOOD, 25- HYDROXYCHOLECALCIFEROL, CALCIUM, PHOSPHORUS, MAGNESIUM, ALKALINE PHOSPHATASE Біологія тварин, 2015, т. 17, № 2 Біологія тварин, 2015, т. 17, № 2 63.2:577.115.16:546.41.18 VITAMIN D-STATUS OF CALVES AT THE FIRST MONTH OF LIFE AFTERDIFFERENTROUTES OF CHOLECALCIFEROLINPUT TO COWS UDC 363.2:577.115.16:546.41.18 D-ВІТАМІННИЙ СТАТУС ТЕЛЯТ У ПЕРШИЙ МІСЯЦЬ ЖИТТЯ ЗА РІЗНИХ СПОСОБІВ ВВЕДЕННЯ ХОЛЕКАЛЬЦИФЕРОЛУ КОРОВАМ Л. Л. Юськів, В. В. Влізло l_yuskiv@inenbiol.com.ua Л. Л. Юськів, В. В. Влізло l_yuskiv@inenbiol.com.ua Інститут біології тварин НААН, вул. В. Стуса, 38, Львів, 79034, Україна У зимово-стійловий період досліджували D-вітамінний статус телят раннього постнатального періоду розвитку, які були отримані від високопродуктивних корів, котрим перорально і парентерально вводили холекальциферол. Перорально холекальциферол вводили коровам до корму щоденно у добовій дозі 30 МО на 1 кг маси тіла впродовж місяця, починаючи за 7−10 днів до прогнозованої дати отелення, та з 5−7-го дня після отелення. Внутрішньом’язово вітамін D вводили: перший раз — за 7−10 днів до прогнозованої дати отелення і ще тричі починаючи з 5−7-го дня після отелення через кожні 7 днів у дозі 210 МО на 1 кг маси тіла за одне введення. Для досліджень у телят брали кров на 5–7 і 28–30-й день після народження. У крові досліджували The Animal Biology, 2015, vol. 17, no. 2 179 The Animal Biology, 2015, vol. 17, no. 2 179 gy, 179 Біологія тварин, 2015, т. 17, № 2 вміст 25-гідроксивітаміну D, кальцію загального і його фракцій, фосфору неорганічного, магнію та активність лужної фосфатази. у ф ф Встановлено, що телята, отримані від корів, яким в останні дні тільності та після отелення вводили холекальциферол перорально і внутрішньом’язово, характеризувалися більшим вмістом у крові 25-ОНD3, кальцію загального, протеїнзв’язаного і ультрафільтрованого, неорганічного фосфору, магнію та нижчою активністю лужної фосфатази, ніж телята, отримані від корів контрольної групи. Телята, отримані від корів, яким вітамін D вводили внутрішньом’язово характеризувалися вірогідно вищим вмістом 25-ОНD3, кальцію і неорганічного фосфору на 5–7-й і 28–30-й день після народження, порівняно із контрольними. Додавання коровам холекальциферолу до корму проявляло свій вплив на досліджувані показники у крові телят лише на 28–30-й день після народження. р Отже, введення холекальциферолу коровам в кінці тільності і на початку лактації є важливим для забезпечення їхніх телят цим вітаміном у ранній постнатальний період. Ключові слова: КОРОВИ, ТЕЛЯТА,ВІТАМІНD, МЕТАБОЛІЗМ, КРОВ 25- ГІДРОКСИХОЛЕКАЛЬЦИФЕРОЛ, КАЛЬЦІЙ, ФОСФОР, МАГНІЙ, ЛУЖНА ФОСФАТАЗА D-ВИТАМИННЫЙ СТАТУС ТЕЛЯТ В ПЕРВЫЙ МЕСЯЦ ЖИЗНИ ПРИ РАЗНЫХ СПОСОБАХ ВВЕДЕНИЯ ХОЛЕКАЛЬЦИФЕРОЛА КОРОВАМ Л. Л. Юскив, В. В. Влизло l_yuskiv@inenbiol.com.ua Институт биологии животных НААН, ул. В. Стуса, 38, 79034, Украина Институт биологии животных НААН, ул. В. Стуса, 38, 79034, Украина In young cattle 1–12 months old, mild D-deficient condition is diagnosed in more than 40 %. Calves born in the autumn and winter are sick more often [1–4]. In addition, today the issue of normal values of concentration of 25-OH D3 in serum is debatable. There is no doubt in fact that «normal» can be considered such level of 25- OH D3, which ensures the implementation of cholecalciferol effects in all organs that contain specific receptors for its hormonally active form — 1,25 (OH)2 D3 [15]. Research has established that in the early postnatal period animals, can adapt to low levels of vitamin D in their body variously, which depends on the species [16]. It is known that newborn calves have a low level of vitamin D in the blood plasma and liver [5]. The supplement of their mothers by vitamin D and its levels in colostrum and milk consumed the offspring has a direct impact on D-vitamin status of the body in the early postnatal period [3, 6–11]. Research by B. W. Hollis et al. (1983) found that there is a relationship between level of 25- hydroxyvitamin D in blood of cows and its level in milk [12]. p p [ ] Despite the large number of studies that being conducted by different research groups in order to establish normal levels of vitamin D in the blood of humans and animals, remains important questions regarding the optimal level of vitamin D in the body of calves in early postnatal period because of various factors and ways of correction. This issue is topical not only in Ukraine, but also in other European countries, because of very little information about the actual content of vitamin D in the feed in various regions, the loss of it’s prolonged storage ability and genetic ability of cows to accumulate vitamin D in the liver and adipose tissue by the action sunlight during the grazing period. Moreover, no data about the levels of 25-OH D3 in plasma, which define the lower limit of adequacy or sufficiency of the body and its effects on metabolism in calves during the first month of life. Vitamins D2 and D3 are biologically inert compounds. They pass a series of consistent transformations for the manifestation of theirs biological actions. The first stage of this transformation occurs in the liver under the influence hydroxylized enzymes to form 25-OH D. Институт биологии животных НААН, ул. В. Стуса, 38, 79034, Украина В зимне-стойловый период исследовали D-витаминный статус телят раннего постнатального периода развития, которые были получены от высокопродуктивных коров, которым перорально и парентерально вводили холекальциферол. Перорально холекальциферол вводили коровам в корм ежедневно в дозе 30 МЕ на 1 кг массы тела в течение месяца, начиная за 7– 10 дней до прогнозируемой даты отела, и с 5–7-го дня после отела. Внутримышечно витамин D вводили: первый раз — за 7–10 дней до предполагаемой даты отела и еще трижды начиная с 5–7-го дня после отела через каждые 7 дней в дозе 210 МЕ на 1 кг массы тела за одно введение. Для исследований в телят брали кровь на 5–7 и 28–30-й день послерождения. В крови исследовали содержание 25-гидроксивитамина D, кальция общего и его фракций, фосфора неорганического, магния и активность щелочной фосфатазы Установлено, что телята, полученные от коров, которым в последние дни стельности и после отела вводили холекальциферол перорально и внутримышечно, характеризировались высшим содержанием в крови 25-ОН D3, кальция общего, протеинсвязанного и ультрафильтруючегося, неорганического фосфора, магния и более низкой активностью щелочной фосфатазы, чем телята полученные от коров контрольнойг руппы. Телята, полученные от коров, которым витамин D вводили внутримышечно; характеризировались достоверно высшим содержанием 25-ОН D3, кальция и неорганического фосфора на 5–7-й и 28–30-й день после рождения, по сравнению с контрольными. Добавление коровам холекальциферола в корм проявляло своевлияние на исследуемые показатели в крови телят только на 28–30-й день после рождения. р р Следовательно, введение холекальциферола коровам в конце стельности и в начале лактации является важным для обеспечения телят этим витамином в ранний постнатальный период. Ключевые слова:КОРОВЫ, ТЕЛЯТА, ВИТАМИН D, МЕТАБОЛИЗМ, КРОВЬ, 25- ГИДРОКСИХОЛЕКАЛЬЦИФЕРОЛ, КАЛЬЦИЙ, ФОСФОР, МАГНИЙ, ЩЕЛОЧНАЯ ФОСФАТАЗА The Animal Biology, 2015, vol. 17, no. 2 180 The Animal Biology, 2015, vol. 17, no. 2 180 gy 180 Біологія тварин, 2015, т. 17, № 2 Біологія тварин, 2015, т. 17, № 2 in the range of 20 to 50 ng/ml. Values less than 5 ng/ml is considered as a sign of deficiency of vitamin D; if the concentration exceeds 200 to 300 ng/ml, it is evidence of the development of hypervitaminosis, and causes intoxication by vitamin D [14]. Feeding and housing conditions of pregnant cows have a significant impact on the viability of newborn calves and their physiological maturity, growth, development and implementation of the genetic productivity potential. The vitamin D plays important role in ensuring vital functions of calves in early postnatal periods. The Animal Biology, 2015, vol. 17, no. 2 Materials and methods The content of calcium (total, bounded with protein and ultrafiltrated), inorganic phosphorus, magnesium and alkaline phosphatase (ALP) activity were detected using the biological test kits produced by the Pliva Lachema firm (the Czech Republic) applying the techniques described in the mentioned paper [17]. The AP isoenzymes activities were detected using techniques described in the mentioned paper [18] and expressed as U/l — the number of micromoles of 4-nitrophenol released by the enzyme contained in 1 liter of serum for 1 min under these conditions. The obtained data were processed statistically by the Statistica software. The results of the mean values was considered statistically significant at: p<0.05 — , p<0.01 — * and p<0.001 — **, compared to a control group of calves. Studies were conducted in the three groups of dairy calves in pilot farm «Pasichna» of Institute of forage and agricultural Podillya NAAS of Ukraine, located in the natural geographical areas of Podillya. The experiment was performed during the winter housing period. The calves of all groups were obtained from high-yielding cows of the Ukrainian Black-and-White dairy breed that were kept in and the same conditions and got with balanced feeding. Calves born from these cows were divided into three groups. The 1st group of calves (the control one) were obtained from cows that never got additional cholecalciferol. The calves of 2nd (experimental) group derived from cows that received the daily dose of vitamin D3 (30 IU per each kg of body weight) every day during a month per oral starting from 7−10 day, up to the expected calving date, and later — since 5−7 day after calving. The calves of 3rd (experimental) group derived from cows that injected with vitamin D3 intramuscular: the first injection — 7−10 days before calving and later — three more times since 5−7 day after calving (each seven days, dose — 210 IU per each kg of body weight for one injection). Институт биологии животных НААН, ул. В. Стуса, 38, 79034, Украина Further it’s transformation to active metabolites depends on the level of calcium and phosphorus in the blood. During hypocalcemia and hypophosphatemia, 25 OH D is converted to 1,25-(OH)2 D in kidney, but for norm- and hypercalcemia hydroxylation occurs on 24- or 26-th position of carbon to form 24,25-(OH)2 D and 26,25-(OH)2 D, respectively. Partially the transport form of 25-OH D enters into the fat and muscle tissues, where it can create tissue depot of indefinite existence [13]. In this context, the aim of this study was to investigate the contents of the active metabolite of vitamin D3 — 25-OH D3, the concentration of calcium and its fractions, inorganic phosphorus, magnesium and alkaline phosphatase activity in the blood of calves recieved from high-yielding cows which were рarenteral injection and oral supplementation of vitamin D3 in the last days of gestation and after calving. The criterion for evaluation of the need for vitamin D, which suggested R. L. Horst et al. (1994) is the concentration of 25- hydroxycholecalciferol in blood. The level of 25-hydroxycholecalciferol is considered as a total reflection of the endogenous formation of cholecalciferol in the skin and its receipts from feed or vitamin preparations. In healthy dairy cows concentration of 25-OH D3 in plasma is The Animal Biology, 2015, vol. 17, no. 2 The Animal Biology, 2015, vol. 17, no. 2 The Animal Biology, 2015, vol. 17, no. 2 181 Біологія тварин, 2015, т. 17, № 2 Results and discussion Vitamin D provision rate of an animal organism is detected by 25-OH D3 concentration in blood, which reflects the total number of vitamin D of endogenous and exogenous origin [1, 5, 13, 15]. 25-ОH D3 content in blood serum of the cattle ows depends upon age, breed, housing conditions and clinical state [1, 5–9, 15]. It was established that the content of 25-OHD3 in the blood of calves aged 5–7 days was lower, and the concentration of calcium and inorganic phosphorus — higher than in the blood of their mothers after parturition [19]. We found a lower concentration of 25-OH D3 in the blood of newborn calves compared to the content of their mothers for 5–7 days after calving apparently confirmed by studies of several authors, that the activity of 25- hydroxylase in the liver of newborns was very low [20]. The blood for research was collected from the jugular vein of calves in the following dates: at 5th−7th days old (after the first intramuscular injection) and at 28–30th days old (after five days after the final injection). The concentration of 25-ОН D3 in the blood of the examined animals was detected by means of the enzymelinked immunoassay using the test system developed by the Immunodiagnostik (Germany). The method is based on the competitive binding of 25-OH D3 serum and 25OH D3-biotin with vitamin D3- binding protein (VDBP), that immobilized on 96-well immunological plates. The Animal Biology, 2015, vol. 17, no. 2 182 The Animal Biology, 2015, vol. 17, no. 2 182 The Animal Biology, 2015, vol. 17, no. 2 182 gy 182 Біологія тварин, 2015, т. 17, № 2 р , , , Fig. 1. The content of 25-ОН D3 (nmol/l) in the blood serum of the calves conditional upon various modes of vitamin D3 administration to cows (M ± m, n = 5) * * 0 5 10 15 20 25 30 35 40 45 50 5-7 d of age 28-30 d of age nmol/l 1 group 2 group 3 group Fig. 1. The content of 25-ОН D3 (nmol/l) in the blood serum of the calves conditional upon various modes of vitamin D3 administration to cows (M ± m, n = 5) The Animal Biology, 2015, vol. 17, no. 2 of The content of 25- hydroxycholecalciferol in serum of calves in the control group at 5–7-days age old was 31.42.56 nmol/l and slightly increased for the 28–30-days (Fig. 1, 2). The injection of cholecalciferol to cows by different ways led to an increase of 25-OH D3 concentration in blood up their calves at 5–7th and 28–30th days old. Thus, at 28–30th days after birth,concentration of 25-OH D3 in the calves blood in 2nd and 3rd groups were 25 % and 29 % higher (p<0.05) in comparison to calves of the control group (p<0.05). In 5–7th days age the 25-hydroxycholecalciferol content was significantly higher only in calves derived from cows that vitamin D was administered intramuscularly (p<0.05). its level in the blood of their offspring [6–8; 10, 12]. The content of total calcium in the blood of all groups calves in the 5–7-th day after birth was higher in comparisant to its rates in 30-day age (Fig. 2, 3). The high concentration of total calcium in the blood of calves during the first days after birth is also likely to be the result of a high concentration of 1,25-(OH)2 D in maternal blood and its effect on placental calcium transport, as confirmed by studies on sheep [21]. The injection of cholecalciferol to cows before and after calving was accompanied by an increase in total calcium and its fractions in the blood of their calves. Thus, at the 5–7 days after birth the total calcium content in the serum of calves from the 3rd group was higher by 11 % (p<0.05), protein-bound — 15 % (p<0.01) and ultrafiltrated — 9 % (p<0.05) compared to that of the control group calves. In the 2nd group of calves at this age, significant difference in the contents protein- bound calcium was only observed. Thus, the 25-hydroxycholecalciferol concentration in the blood of calves during first days after birth depends on the content of this metabolite in the blood of their mothers and in the consumed colostrum and milk. Our data are consistent with studies on other animal species and human, the level of vitamin D in the blood of pregnant women is shown in The Animal Biology, 2015, vol. 17, no. 2 The Animal Biology, 2015, vol. 17, no. 2 183 Біологія тварин, 2015, т. 17, № 2 Fig. 2. of The content of mineral elements (mmol/l) in the blood serum of the calves in the 5th−7th day age old (M ± m, n = 5) * * ** * 0 0,5 1 1,5 2 2,5 3 3,5 Ca (total) Ca (bounded with protein) Ca (ultrafiltrated) P (inorganic) Mg mmol/l 1 group 2 group 3 group 3,5 Fig. 2. The content of mineral elements (mmol/l) in the blood serum of the calves in the 5th−7th day age old (M ± m, n = 5) In the 28–30-day after birth the total calcium content in the blood serum of calves from the 2nd group was higher by 11 % (p<0.05) and 3rd group — by 14 % (p<0.01) in comparison to parameters of the control group (Fig. 3). Thus, the content of protein-bound calcium in serum of calves from the 2nd group was higher by 22 % (p<0.001) and 3rd — 26 % (p<0.001). Content ultrafiltrated calcium in this age was significantly higher only in calves from the 3rd experimental group. control as well as in experimental groups of calves aged 28–30 days and amounted in accordance: 1.830.05; 1.910.06 and 2.020.05 mmol/l (Fig. 3). The injection of cholecalciferol to cows before and after calving of vitamin D by different ways was leading to a significant increase in the concentration of inorganic phosphorus in the blood of calves only the 3rd group on the 28– 30th day after birth. Also, there were found no significant differences on the concentration of magnesium in the blood of calves of both research groups in the 5–7-day as well as in the 28–30-day age, in comparisant to the performance of calves in the control group. In the control and experimental groups of calves aged 5–7-days, there was insignificant difference between values of concentration of inorganic phosphorus (1.77– 1.82 mmol/l) in the blood (Fig. 2). The content of phosphorus in the blood was increased in The Animal Biology, 2015, vol. 17, no. 2 184 gy, 184 Біологія тварин, 2015, т. 17, № 2 Fig. 3. The content of mineral elements (mmol/l) in the blood serum of the calves in the 28th−30th day age old (M ± m, n = 5) * * *** * * 0 0,5 1 1,5 2 2,5 3 3,5 Ca (total) Ca (bounded with protein) Ca (ultrafiltrated) P (inorganic) Mg mmol/l 1 group 2 group 3 group Fig. 3. The Animal Biology, 2015, vol. 17, no. 2 The Animal Biology, 2015, vol. 17, no. 2 of The content of mineral elements (mmol/l) in the blood serum of the calves in the 28th−30th day age old (M ± m, n = 5) The activity of alkaline phosphatase in the blood serum of calves in 5–7th after birth was 160–166 IU/L (Fig. 4). By the 28–30 days age the enzyme activity in the blood of calves somewhat increased especially due to increasing bone isoenzyme (Fig. 5). The administration of cholecalciferol to cows before and after calving by different ways were accompanied by a decrease in total alkaline phosphatase activity and bone isoenzyme activity and increased intestinal isoenzyme in the blood of calves experimental groups. Significant difference was only observed in the intestinal isoenzyme activity in the blood of calves at the 28–30th-day of age that were obtained from cows injected intramuscularly vitamin D. Fig. 4. The activity of alkaline phosphatase (IU/l) and its isoenzymes in the 5th−7th days age old (M ± m, n = 5) 0 20 40 60 80 100 120 140 160 180 200 ALP (total) ALP (intestinal) ALP (bone) U/l 1 group 2 group 3 group Fig. 4. The activity of alkaline phosphatase (IU/l) and its isoenzymes in the 5th−7th days age old (M ± m, n = 5) The Animal Biology, 2015, vol. 17, no. 2 The Animal Biology, 2015, vol. 17, no. 2 185 Біологія тварин, 2015, т. 17, № 2 Fig. 5. The activity of alkaline phosphatase (IU/l) and its isoenzymes in the 28th−30th days age old (M ± m, n = 5) * 0 20 40 60 80 100 120 140 160 180 200 ALP (total) ALP (intestinal) ALP (bone) U/l 1 group 2 group 3 group Fig. 5. The activity of alkaline phosphatase (IU/l) and its isoenzymes in the 28th−30th days age old (M of alkaline phosphatase (IU/l) and its isoenzymes in the 28th−30th days age old (M ± m, n = 5) The obtained results indicate that the provision of vitamin D and calcium and phosphorus metabolism in calves aged up to month depended on the content of cholecalciferol in the body of cows in late pregnancy and early lactation periods. Also it indicates about more active absorption of this vitamin from maternal colostrum and milk due to the beneficial effects of active metabolites cholecalciferol on the functional state of organs (intestine, liver, and kidneys) participating at its absorption and metabolism. of from cows that were introduction cholecalciferol orally and intramuscularly were characterized with higher levels of 25- OH D3 in blood, total calcium and its fractions, inorganic phosphorus, magnesium and lower activity of alkaline phosphatase than calves from cows of the control group. The level of these changes depends on way of introduction, the quantity of injected vitamin and age of animals. To prospect of further researches. The prospect for further research is the study the biological action of vitamin D in the body of cattle in different geographical areas of Ukraine in different age periods of development and different physiological periods in health and disease. 1. Levchenko V. І., Vlіzlo V. V., Kondrahіn І. P. et al. ; za red. Levchenka V. І. The clinical diagnosis of internal diseases. Bіla Cerkva, 2004. 608 р. (In Ukrainian). 2. Levchenko V. І., Tihonjuk L. A., Apuhovska L. І. Diagnosis of early forms of D- hypovitaminosis in calves over the content of phosphorus and 2.3 dyfosfohlitseratu in erythrocytes. Bulletin of Agricultural Science, 1981, № 9, pp. 73–76 (in Ukrainian). 1. Levchenko V. І., Vlіzlo V. V., Kondrahіn І. P. et al. ; za red. Levchenka V. І. The clinical diagnosis of internal diseases. Bіla Cerkva, 2004. 608 р. (In Ukrainian). Сonclusions The intramuscular injection of cholecalciferol to cows: the first time — for 7– 10 days before the predicted calving date and three more times every 7 days starting from 5– 7 day after calving at a dose of 210 IU per kg of body weight for one injection, and by daily adding of cholecalciferol to the feed every day at a daily dose of 30 IU per kg of body weight for a month, starting 7–10 days before the predicted date of calving, and 5–7 day after calving in winter-stall period, is accompanied by increase in D-vitamin status of their calves from birth to 30 days of age. Calves derived 1. Levchenko V. І., Vlіzlo V. V., Kondrahіn І. P. et al. ; za red. Levchenka V. І. The clinical diagnosis of internal diseases. Bіla Cerkva, 2004. 608 р. (In Ukrainian). 1. Levchenko V. І., Vlіzlo V. V., Kondrahіn І. P. et al. ; za red. Levchenka V. І. The clinical diagnosis of internal diseases. Bіla Cerkva, 2004. 608 р. (In Ukrainian). 2. Levchenko V. І., Tihonjuk L. A., Apuhovska L. І. Diagnosis of early forms of D- hypovitaminosis in calves over the content of phosphorus and 2.3 dyfosfohlitseratu in erythrocytes. Bulletin of Agricultural Science, 1981, № 9, pp. 73–76 (in Ukrainian). The Animal Biology, 2015, vol. 17, no. 2 gy 186 Біологія тварин, 2015, т. 17, № 2 phosphorus metabolism. M. F. Holick, T. K. Ray, and C. S. Anaste, ed. Elsevier Sci. Publ., B. V., 1983.The Netherlands. 3. Kurtjak B. M., Janovich V. G. Fat-soluble vitamins in veterinary medicine and animal husbandry. Lvіv, Trіada Pljus, 2004. 426 p. (In Ukrainian). 13. DeLuca H. F. The vitamin D system in the regulation of calcium and phosphorus metabolism. Nutr. Rev., 1979, 37, pp. 161–193. 4. Maslova T. V., Egorova G. G. Etiological factors of development of D defytsyt status of calves. Advances modern natural science, 2005, № 10, p. 68 (in Russian). 14. Horst R. L., Goff J. P., Reinhardt T. A. Calcium and vitamin D metabolism in the dairy cow. J. Dairy Sci., 1994, 77, pp. 1936–1951. 5. Horst R. L., Reinhardt T. A. Vitamin D metabolism in ruminants and its relevance to the periparturient cow. J. Dairy Sci, 1983, Vol. 66, pp. 661–678. 15. Norman A. W. From vitamin D to hormone D: Fundamentals of the vitamin D endocrine system essential for good health. Am. J. Clin. Сonclusions Nutr., 2008, 88, pp. 491–499. 6. Goff J. P., Horst R. L., Littledike E. T. Effect of maternal vitamin D status at parturition on the vitamin D status of the neonatal calf. J. Nutr., 1982, 112, pp. 1387–1393. 16. Goff J. P., Horst R. L., Littledike E. T. Effect of sow vitamin D status at parturition on the vitamin D status of the neonatal piglets. J. Nutr., 1984, pp. 114–163. 7. Barlet J. P., Argemi B., Davicco M. & Lefaivre J. Plasma concentrations of 25- hydroxyvitamin D in pregnant and lactating ewes and foetal and newborn lambs. J. Endocrinol., 1978, 79, pp. 149–150. 17. Vlіzlo V. V., Fedoruk R. S., Ratych І. B. et al. ; za red. Vlіzlо V. V. Laboratory methods of research in biology, veterinary medicine: a guide. Lvіv: SPOLOM, 2012. 764 p. (In Ukrainian). 8. Van Saun R J. Vitamin D-responsive rickets in neonatal lambs. Can Vet J., 2004, vol. 45, pp. 841–844. 18. Vagner V. K., Putilin V. M., Harabuga G. G. Questions of medical chemistry, 1981, 27, № 6, pp. 752–754 (in Russian). 19. Yuskiv L. L., Vlizlo V. V. Vitamin D Provision in High-Yield Dairy Cows During Winter Housing Period. Agricultural Science and Practice, 2014, vol. 1 (1), pp. 42–46. 9. Reinhardt T. A., Horst R. L., Goff J. P. Calcium, phosphorous, and magnesium homeostasis in ruminants. Vet. Clin. North Am. Food Anim. Pract., 1988, 4, pp. 331–350. 20. Gascon-Barre M., Plourdo V., Haddad P., Martial J. Fetal and neonatal uptake and microsomal C-25 hydroxylation of vitamin D by the rat liver. Page 654 in Vitamin D. Chemical, biochemical and clinical update. A. W. Norman, K. Schaefer, H.-G. Grigolt, and D. V. Herra, h,ed. de Gruyter, 1985, Berlin. 10. Luk'janova E. M., Antipkin Ju. G., Omel'chenko L. I., Apuhovskaja L. I. Vitamin D & its role in the Provision of health of children and pregnant women. K., Еkspert B, 2005. 230 p. (In Ukrainian). 11. Dittmer K. E., Thompson K. G. Vitamin D Metabolism and Rickets in Domestic Animals : A Review. Vet. Pathol, 2011, vol. 48, pp. 389–407. 11. Dittmer K. E., Thompson K. G. Vitamin D Metabolism and Rickets in Domestic Animals : A Review. Vet. Pathol, 2011, vol. 48, pp. 389–407. 12. Hollis B. W., Lambert P. W., Horst R. L. Factors affecting the antirachitic content of native milk. Page 157 in Perinatal calcium and 21. Durand, D., G. D. Сonclusions Braithwaite, and J.-P. Baler. The effect of la-hydroxycholecalciferol on the placental transfer of calcium and phosphate in sheep. Br. J. Nutr., 1983, 49, pp. 475–483. A Review. Vet. Pathol, 2011, vol. 48, pp. 389 407. 12. Hollis B. W., Lambert P. W., Horst R. L. Factors affecting the antirachitic content of native milk. 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https://openalex.org/W2603141154	https://europepmc.org/articles/pmc5410207?pdf=render	English	null	Potential of used frying oil in paving material: solution to environmental pollution problem	Environmental science and pollution research international	2,017	cc-by	5,660	1 The University of Trinidad and Tobago, O’Meara Industrial Park, 74-98 Churchill Roosevelt Highway, Arima, Trinidad and Tobago Environ Sci Pollut Res (2017) 24:12220–12226 DOI 10.1007/s11356-017-8793-z Environ Sci Pollut Res (2017) 24:12220–12226 DOI 10.1007/s11356-017-8793-z RESEARCH ARTICLE Potential of used frying oil in paving material: solution to environmental pollution problem Dimple Singh-Ackbarali1 & Rean Maharaj1 & Nazim Mohamed1 & Vitra Ramjattan-Harry1 Received: 27 May 2016 /Accepted: 9 March 2017 /Published online: 28 March 2017 # The Author(s) 2017. This article is published with open access at Springerlink.com Abstract The improper disposal of used frying oil (UFO) presents numerous ecological, environmental and municipal problems. Of great concern is the resultant blockage of mu- nicipal drainage systems and water treatment facilities, harm to wildlife when they become coated in it and detriment to aquatic life and ecosystems due to the depletion of the oxygen content in water bodies such as rivers and lakes that have become contaminated. Statistics show that in Trinidad and Tobago, in excess of one million liters of used cooking oil is collected annually from various restaurant chains. This paper investigated the potential of using UFO as a performance en- hancing additive for road paving applications utilizing Trinidad Lake Asphalt (TLA) and Trinidad Petroleum Bitumen (TPB) as a mitigation strategy for improper UFO disposal. Modified blends containing various additions of UFO (2–10% wt) were prepared for the TLA and TPB asphal- tic binders. Results demonstrated in terms of stiffness, increas- ing the dosage of UFO in TLA and TPB base binders resulted in a gradual decrease in stiffness (G* value decreased). In terms of elasticity, increasing the dosage of the UFO additive in TLA resulted in a general decrease in the elasticity of the blends indicated by an increase in phase angle or phase lag (δ). Increasing dosages of the UFO additive in TPB resulted in a significant decrease in δ where the most elastic blend was at the 6% UFO level. TLA and UFO-TLA modified blends ex- hibited significantly lower values of δ and higher values of G* confirming the superiority of the TLA material. Incorporation of the UFO in the blends led to a decrease in the rutting resistance and increase in the fatigue cracking resistance (de- crease in G/sinδ and Gsinδ, respectively). This study highlighted the potential for the reuse of UFO as an asphalt modifier capable of producing customized UFO modified as- phaltic blends for special applications and confirms its feasi- bility as an environmentally attractive means of reusing the waste/hazardous UFO material locally. Keywords Used frying oil . Trinidad Lake Asphalt . Trinidad Petroleum Bitumen . Rutting resistance . Fatigue cracking Responsible editor: Philippe Garrigues * Dimple Singh-Ackbarali dimple.singh@utt.edu.tt Responsible editor: Philippe Garrigues The environmental problem In order for a reduction in neg- ative impacts of improper disposal of UFO in Trinidad and Tobago the following must be done: & Reduction in dissolved oxygen content and the death of aquatic plants and animals as a result of the existence of the layer of oil on water bodies which prevents sunlight from getting to aquatic plants stifling photosynthesis & Rancid odor production & Clogging of drainage systems and water treatment plants & Isolation of soil from air and water, killing the earth worms, and bacteria necessary for regeneration of plants & Proliferation of rats and vermin that feed on the solidified waste cooking oil which creates a pest control problem or health hazard. In Trinidad and Tobago, the amended Water Pollution Rules (WASA 2006) targets commercial business activities using cooking oil such as restaurants, food service companies, and even households where these entities are required by law to register with the Environmental Management Authority (EMA). However according to an interview with a managing director of a company that collects and recycles used cooking oil, the manager stated that the laws on cooking oil disposal is not enforced as it is either frozen and thrown away or poured down the drain. & Update national inventory of use and disposal Information collected in 2010 by the Trinidad and Tobago Central Statistical Office reported that there were 317 food and drink processing establishments and 297 hotel and guest establishments in the country. A study found that a popular internationally based fried chicken fast food outlet used ap- proximately 151.4 L of oil per week and when this is translat- ed to all of its 52 outlets; over 409,000 L of UFO can be collected annually from this one franchise alone (John and Seetahal 2008). It is estimated that commercial establishments would use 30% of the available edible oil, 55,315 L of edible oil a day (Wyse-Mason and Beckles 2012). The remaining 70% of the consumed edible oil are utilized by residential households that are not required to have disposal facilities such as oil separators, grease traps, waste water sumps, or have their used cooking oil collected by recycling or treatment and disposal companies. This allows a significant quantity of UFO to be disposed of down the sink and drain, onto the ground, and into the garbage. Challenges with past solutions Integrating UFO into the food chain through animal feed, can be a potential cause of human health problems as there is some evidence that highly oxidized fats formed during frying where oils are exposed to high temperatures in the presence of atmo- spheric oxygen, may have carcinogenic properties (Chang and Peterson 1978, Azpilicueta and Remirez 1991, Costa Neto et al. 2000, Panadare and Rathod 2015). The use of waste or used fats and oils in animal feedstock as an additive can also be problematic as when it becomes rancid, it imparts an objectionable odor and decreases palatability of the feed. Additionally, when excess fat or oil exceeds 6% of the feeds dry matter, inhibition of fiber digestion in rumens can occur (Engstrom et al. 1994, Panadare and Rathod 2015). The environmental problem Frying oil is vegetable and animal oil that is used to fry food at high temperatures by the food industry, restaurants/food ser- vice establishments, and homes. The used frying oil (UFO) generated by these activities has become a major environmen- tal and ecological issue, especially since it is usually indis- criminately discarded after use, into municipal landfills or poured down drains without any treatment (Patil et al. 2012, Zhang et al. 2012, Dias et al. 2014). Petroleum oils, vegetable oils, and animal fats share com- mon physical properties and produce similar environmental effects as outlined by literature (EPA 2015, Rodewald 2015). They include: Responsible editor: Philippe Garrigues & Suffocation of animals and plants that have been coated with oil & Eutrophication due to micro-organisms, phytoplankton, and algae which use the UFO as a food source 12221 Environ Sci Pollut Res (2017) 24:12220–12226 oil due to inadequate collection services and limited utilization of recycling. Third world countries are lagging behind in this regard as they have low awareness regarding recycling of waste materials, not yet developed effective legislation, and have not yet selected lead agencies responsible for rules, reg- ulations, and enforcement legislation (Batayne et al. 2008, Kahn et al. 2009). Progress is being made in this region, how- ever, as the Basel Convention Regional Centre for Training and Technology Transfer for the Caribbean Region (BCRC- Caribbean) has a new central focus which shifts away from the strict prohibition of the movement of hazardous wastes from one party to another, towards the recognition of waste as a resource which can stimulate economic development and cre- ate new employment opportunities, more so among civil so- ciety groups and small business entrepreneurs. This new focus encourages waste prevention and minimization at source and waste recovery, reuse, and recycling as downstream value added components of the waste stream (Basel Convention Region Business Plan 2012). The environmental problem Currently, the UFO generated commercially from Trinidad restaurants is contracted to one company who indicated that they collect up to one million liters of UFO annually. p y p & Review policy and enable legislation to facilitate waste oil collection, re-refining, disposal and destruction. & Conduct strategic assessment of appropriate technologies that can be applied & Develop pilot project with private sector investors & Review fuel subsidies in Trinidad and Tobago so that local market will be inviting to the use of alternative fuel. Many researchers have studied the potential use of recycled UFO by integration into the food chain through animal feeds, production of soaps, or conversion to biodiesel; however, lim- ited information exist on its use of UFO as an additive in asphalt pavement binders (Bronislaw 2014, Deba et al. 2015, Panadare and Rathod 2015). Possible solutions Used oil is the Bsingle largest environmentally hazardous re- cyclable material^ (MARRC 2001) and a spill of used oil as small as 1 L can potentially contaminate a million liters of fresh water. The recycling of waste oil is becoming a viable alternative in mitigating the associated environmental and ecological problems (El-Fadel and Khoury 2001). However, developing countries struggle to properly manage their used Environ Sci Pollut Res (2017) 24:12220–12226 12222 Digestive disturbances, diarrhea, and reduced feed intake may occur if excessive levels of fat are fed to animals. There are several positives when looking at UFO as a fuel source for biodiesel (Sunde et al. 2011, Thamsiriroj and Murphy 2011, Bronislaw 2014) however, before the UFO can be used in saponification and biodiesel production, investments have to be made to pre-treat the waste material via filtration and ester- ification to remove any free fatty acids (Chang and Peterson 1978, Bronislaw 2014). While pre-treatment for the UFO to be converted to biodiesel may not be expensive, the cost of converting a diesel engine to run on UFO can cost up to TT$15000.00 (Trinidad and Tobago Newsday 2010), which may be a deterrent for citizens/individuals. Digestive disturbances, diarrhea, and reduced feed intake may occur if excessive levels of fat are fed to animals. There are several positives when looking at UFO as a fuel source for biodiesel (Sunde et al. 2011, Thamsiriroj and Murphy 2011, Bronislaw 2014) however, before the UFO can be used in saponification and biodiesel production, investments have to be made to pre-treat the waste material via filtration and ester- ification to remove any free fatty acids (Chang and Peterson 1978, Bronislaw 2014). While pre-treatment for the UFO to be converted to biodiesel may not be expensive, the cost of converting a diesel engine to run on UFO can cost up to TT$15000.00 (Trinidad and Tobago Newsday 2010), which may be a deterrent for citizens/individuals. refinery bitumen such as TPB. TLA contains kaolinitic clay not present in TPB and other refinery bitumen. These compositional differences have been shown to influence the flow, colloidal characteristics, and rheological properties of asphaltic systems which ultimately influences their performance attributes. A liter- ature survey shows that previous studies measuring the influence of UFO on the rheological properties of the asphaltic materials TLA and TPB, indigenous to Trinidad and Tobago have proven to be limited. Possible solutions Despite the reported enhancement of asphaltic materials mod- ified with polymeric additives, there are some associated difficul- ties. Polymer modified asphaltic materials have been linked to increased amounts of polycyclic aromatic hydrocarbons (PAHs) being leached into storm water and contaminating water bodies. PAHs consist of over a hundred organic compounds with two or more aromatic rings that occur together as mixtures. They can be concentrated by incomplete burning of carbon-containing mate- rial; sources include tyres and crumbling asphalt. Road pavement material and car park sealants can contribute significant amounts of PAHs to water ways via storm water which can be toxic to aquatic animals (Wright et al. 2009). A 2006 evaluation of PAHS in frying oils found that both before and after frying, the benzo-a- pyrene concentration in edible oils ranged from trace to 0.7 ppb, well below the 2 ppb limit for PAHs in foods recently proposed by the European Community (Purcaro et al. 2006). Research showed that crumb rubber samples analyzed had high levels of PAHs and Zinc (Marsili et al. 2015). Background and new proposed solution Asphalts and bitumen are both used together with mineral aggre- gates to construct roads/pavements. The performance of these road pavements depend on the properties of the asphalt and the bitumen which are the only deformable components in the mix- ture. Both these systems have thermal susceptibilities and can become deformed due to weathering, moisture damage, heavy traffic, or embrittlement due to the chemical oxidation of func- tional groups within the asphalt. These limitations can be over- come as their performance characteristics significantly modified by modification with polymeric materials (Zhu et al. 2014, Maynard et al. 2015). It has been reported that polymer modified asphalt can increase the shelf life of pavements by up to 10 years (Dwyer and Betts 2011, Boyer 2013). The blending of recycled asphalt with UFO has been shown to improve the performance qualities of the resulting blends as the fatty acids present in UFO has been shown to act as cohesive agent, reducing the high viscosity of the aged, recycled binders, facilitating homogenous mixing and reducing surface tension of the aggregate and coated binder, when integrated with new pavement materials (Huh 2012). Other past evaluations of binder performance (Asli et al. 2012, Zargar et al. 2012) showed that a 3–4% by weight addition of the UFO gave similar viscosity results compared to the orig- inal bitumen material. It was also reported that the used of veg- etable oil decreases the stiffness of the aging mixture (Bailey and Philips 2010). This paper seeks to fill the gap of research investigating the influence of UFO on the rheological properties of TLA and TPB asphaltic materials indigenous to Trinidad and Tobago, and hence assess its potential as an environmentally attractive means of reusing the waste/hazardous UFO material locally. Materials sources A gallon of used frying oil (UFO) was obtained from a com- mercial restaurant in South Trinidad. Trinidad Lake Asphalt (TLA) and Trinidad Petroleum Bitumen (TPB) were sourced from the Lake Asphalt Company of Trinidad and Tobago and the Petroleum Company of Trinidad and Tobago Limited, respectively. Despite the existence of studies using other asphaltic binders from other sources, the influence of polymeric additives on the rheological properties of Trinidad asphaltic materials cannot be generalized and must be independently investigated as a clear relationship between the differences in the quality of asphalt (different compositions) from different sources and the resulting performance qualities of the binders exist; asphaltic materials with the same specifications can often produce pavements of varying physical properties, performance, and serviceability (Oyenkunle 2006, Oyenkunle 2007, Mohamed et al. 2016). TLA is an asphaltic material of unique composition containing significantly higher asphaltene content compared to other Sample preparation Aluminum cans of approximately 500 cm3 were filled with 250– 260 g of asphalt and put in a thermoelectric heater Thermo Scientific Precision (Model 6555) where the temperature was raised to 200 °C. A digital IKA (Model RW20D) high shear mixer was then immersed in the can and set to 3000 rpm. The UFO was added gradually while the system was kept at a Environ Sci Pollut Res (2017) 24:12220–12226 12223 temperature of 200 ± 1 °C. Each TLA-UFO and TPB-UFO blend was formed from 0, 2, 4, 6, 8, and 10% of UFO by weight. At the end of mixing, each blend was split into different cans, transferred to a desiccator and stored under static conditions and in an oxygen-free environment. After 24 h period of curing, the cans were taken out, remixed using high shear mixer, and the molten mixtures were then cast into a ring stamp with 25 mm diameter and 1 mm thickness for subsequent rheological testing. Before testing, the samples were cooled at room temperature and stored in a Fisher Isotemp freezer at −20 °C. temperature of 200 ± 1 °C. Each TLA-UFO and TPB-UFO blend was formed from 0, 2, 4, 6, 8, and 10% of UFO by weight. At the end of mixing, each blend was split into different cans, transferred to a desiccator and stored under static conditions and in an oxygen-free environment. After 24 h period of curing, the cans were taken out, remixed using high shear mixer, and the molten mixtures were then cast into a ring stamp with 25 mm diameter and 1 mm thickness for subsequent rheological testing. Before testing, the samples were cooled at room temperature and stored in a Fisher Isotemp freezer at −20 °C. attributes of fatigue cracking and rutting resistance (Hosein et al. 2013, Maharaj and Maharaj 2015, Maynard et al. 2015). attributes of fatigue cracking and rutting resistance (Hosein et al. 2013, Maharaj and Maharaj 2015, Maynard et al. 2015). attributes of fatigue cracking and rutting resistance (Hosein et al. 2013, Maharaj and Maharaj 2015, Maynard et al. 2015). Rheological measurements The rheological characterization of the various asphalt blends were studied using an oscillatory dynamic shear rheometer (ATS RheoSystems) operated within the linear domain under strain control. The test geometries were plate to plate (diame- ters 25 and 1 mm gap). Viscosity measurements were conduct- ed in the temperature range 40–90 °C and frequency range was 0.1–15.91 Hz. The analysis was performed under the strain control mode and the complex modulus (G) and phase angle (δ) values at the different oscillating frequencies and temperatures were calculated using the instruments software. Figure 1a, b show the changes in complex shear modulus (G) at various oscillating load frequencies at 60 °C, as the concentration of the added UFO was increased for TLA and TPB binders, respectively. A comparison of Fig. 1a, b shows that unmodified TLA and the UFO-TLA blends exhibited higher G* values than TPB and UFO-TPB blends indicating that they are generally stiffer. This observation was consistent with the findings of previous researchers (Hosein et al. 2013, Maharaj et al. 2014, Maynard et al. 2015). The results show that for both the TLA and TPB base binders, as the concentration of the added UFO was increased gradually, the stiffness generally decreased (G* value decreased). A similar observation was recorded by Raghavan and Kaler (2001), Borhan et al. (2009), and Singh-Ackbarali and Maharaj (2011), and it has been sug- gested that the decrease in G* observed can be attributed to an increased solvency of the maltenes present in the asphaltic materials in the fatty acids present in the UFO; softening the intermolecular cross-linkages which resulted in the modified blends having reduced ability to withstand elongation. When aggregate is added to these UFO modified asphaltic blends, it is expected that the mechanical properties of the pavement Sample preparation Deformation in asphalt material consists of three types: Deformation in asphalt material consists of three types: – Instant elastic recoverable strain – Delayed elastic recoverable strain – Permanent non-recoverable strain (or viscous flow) – Instant elastic recoverable strain – Delayed elastic recoverable strain – Permanent non-recoverable strain (or viscous flow) Most critical among these is the permanent non- recoverable strain or viscous flow parameter which deter- mines the permanent deformation of the traffic asphalt pave- ment due to repeated loading forces. Table 1 below describes the different parameters that were tested with the DSR, and introduces the characteristics of the material that can be interpreted when analyzing the results. Results and discussion The use of measurements using dynamic shear rheometer (DSR) rheological properties of Trinidad asphalt materials at temperatures from high to intermediate values are an important consideration in understanding pavement distress characteristics such as pavement deformation due to rutting and shearing. The understanding and application of this technique is well- documented by Kim (2009) and has been successfully utilized for the rheological characterization of polymer modified asphal- tic materials including the measurement of key performance Table 1 The different parameters and characteristics that were tested and analyzed using the DSR Parameter Characteristics that can be interpreted Complex shear modulus, G* Represents the total resistance of the asphalt/bitumen sample to deformation (or stiffness) caused by repeated pulses of small angle oscillations by the plates of the DSR, high values are desirable for a stiffer material low values are associated with a softer material Phase angle or the phase lag, δ Represents the degree of the elasticity of the material, high values are associated with high viscosity materials, low values are associated with highly elastic materials resistance Rutting resistance parameter G/sinδ Higher values of G/sinδ will result in higher rutting resistance of material, lower values of G/sinδ will result in lower rutting resistance of the material Fatigue cracking parameter Gsinδ Lower values of Gsinδ will result in higher fatigue cracking resistance of material higher values of Gin lower fatigue cracking resistance of the material 12224 Environ Sci Pollut Res (2017) 24:12220–12226 Fig. 1 The variation of G* with increasing concentration of UFO for TLA and TPB binders at various oscillating load frequencies and at 60 °C Fig. 1 The variation of G* with increasing concentration of UFO for TLA and TPB binders at various oscillating load frequencies and at 60 °C Fig. 1 The variation of G* with increasing concentration of UFO for TLA and TPB binders at vari would be improved as the reduced viscosity will result in a reduction in the surface tension between the aggregate and the binder coating, expelling trapped air and increasing interfacial cohesion between the asphaltic binder and aggregate. and its consequential use as an additive to improve the properties of other refinery bitumen including TLA (Widyatmoko and Elliott 2008). The effect of increasing the concentration of the UFO additive in TLA resulted in a general decrease in the elas- ticity of the blends indicated by an increase in δ. Conclusion The rheological analysis of modified TLA and TPB blends containing various additions of UFO (2–10% wt) demonstrat- ed the following: & In terms of stiffness, increasing the concentration of UFO in the TLA and TPB base binders resulted in a correspond- ing decrease in stiffness (G* value decreased). & In terms of elasticity, increasing the concentration of the UFO additive in TLA resulted in a general decrease in the elasticity of the blends indicated by an increase in δ. Increasing the concentration of the UFO additive in TPB resulted in a significant decrease in δ where the most elas- tic blend was at the 6% UFO level. Fig. 3 The variation of the fatigue cracking resistance parameter (Gsinδ) with increasing concentration of UFO in TLA and TPB mix at various oscillating frequencies at 60 °C TPB and UFO-TPB blends. The variation of the rutting resis- tance parameter (G/sinδ) with increasing concentration of UFO in TLA and TPB blends at three oscillating frequencies, respectively are shown in Fig. 4. & TLA and UFO-TLA modified blends exhibited signifi- cantly lower values of δ and higher values of G* confirming the superiority of the TLA material. The results clearly indicate that the addition of UFO in the TLA and TPB binders resulted in a decrease in the rutting resistance (a decrease in G/sinδ values) of the resulting blends. The unmodified TLA and TPB exhibited superior rut- ting resistance properties compared to the UFO modified blends. The superiority of the TLA and its UFO-TLA blends was again evident as the G/sinδ (rutting resistance) values were significantly higher than TPB and the UFO-TPB blends. & Incorporation of the UFO in the blends led to a decrease in the rutting resistance and increase in the fatigue cracking resistance (decrease in G/sinδ and Gsinδ, respectively). This study demonstrated the potential reuse of UFO as an asphalt modifier capable of producing customized UFO mod- ified asphaltic blends for special applications. It also demon- strates the feasibility of the reuse strategy as an environmen- tally attractive means of disposal of the waste/hazardous UFO material locally. Results and discussion The effect of increasing the concentration of the UFO additive in TPB resulted in a significant decrease in δ at the 6% UFO level indicating a superior elastic UFO blend. Figures 2a, b show the variation of the phase angle δ with increasing concentration of UFO for TLA and TPB, respec- tively at various oscillating load frequencies and at 60 °C. The results demonstrated that δ was generally higher for the TPB and UFO-TPB blends indicating that these blends had low- er elasticity. The observation that δ was almost 90° for unmodi- fied TPB indicated that the material behaved almost like a vis- cous liquid. On the other hand, the TLA and UFO-TLA blends had significantly lower values of δ indicating that these blends were significantly more elastic. The relatively higher values of G* and lower values of δ observed for the TLA based binder (relatively stiffer and more elastic) offer supporting rheological evidence confirming TLA’s world renowned superior qualities The variation of the fatigue cracking resistance parameter (Gsinδ) with increasing concentration of UFO in TLA and TPB at various oscillating frequencies at 60 °C is shown in Fig. 3. The trend of decreasing Gsinδ values as the % UFO was increased for both TLA and TPB indicates higher fatigue cracking resistance as the UFO component was increased. The TLA and the UFO-TLA blends exhibited higher Gsinδ values (lower fatigue cracking resistance values) compared to Fig. 2 a, b The variation of δ with increasing concentration of UFO for TLA and TPB binders at various oscillating load frequencies and at 60 °C Fig. 2 a, b The variation of δ with increasing concentration of UFO for TLA and TPB binders at various oscillating Fig. 2 a, b The variation of δ with increasing concentration of UFO for TLA and TPB binders at various oscillating load frequencies and at 60 °C Environ Sci Pollut Res (2017) 24:12220–12226 12225 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07 0 5 10 G sin δ % added UFO TLA TPB Fig. 3 The variation of the fatigue cracking resistance parameter (Gsinδ) with increasing concentration of UFO in TLA and TPB mix at various oscillating frequencies at 60 °C Compliance with ethical standards Conflict of interests The authors declare that they have no conflict of interest. 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 appro- priate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07 0 5 10 G/sin PA % added UFO TLA TPB Fig. 4 The variation of the rutting resistance parameter (G/sinδ) with increasing concentration of UFO in TLA and TPB blends at three oscillating frequencies 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07 0 5 10 G/sin PA % added UFO TLA TPB Conclusion Differences between the rheological properties observed for the TLA and TPB blends offer supporting evidence for previous studies where it has been shown that the influence of additives on the rheological properties of asphaltic mate- rials from different sources cannot be generalized and must be independently investigated as there exists a clear proven rela- tionship between the differences in the quality of asphalt (dif- ferent compositions) from different sources and the resulting performance qualities of the binders exist; asphaltic materials with the same specifications can often produce pavements of varying physical properties, performance, and serviceability (Mohamed et al. 2016, 28). 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https://openalex.org/W2810924560	https://bmcpediatr.biomedcentral.com/track/pdf/10.1186/s12887-018-1186-8	English	null	Emergency department 72-hour revisits among children with chronic diseases: a Saudi Arabian study	BMC pediatrics	2,018	cc-by	5,059	Ahmed et al. BMC Pediatrics (2018) 18:205 https://doi.org/10.1186/s12887-018-1186-8 Ahmed et al. BMC Pediatrics (2018) 18:205 https://doi.org/10.1186/s12887-018-1186-8 Open Access © 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. 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: Emergency Department (ED) revisits have often been used as an indicator of medical care quality. This study aimed to quantify the frequency of ED revisits within 72 h of discharge and identify its factors among children with chronic diseases. Methods: We designed a retrospective cohort study of children with at least one chronic disease who were also under 18 years of age and had attended and were discharged from the ED at King Abdullah Specialist Children’s Hospital (KASCH-RD), Riyadh, Saudi Arabia between April 19, 2015 and July 29, 2017. The outcome measure was the frequency of ED revisits during a period of 72 h after discharge. Results: The study included 11,057 ED discharges of children with at least one chronic disease. Their revisit rate was 1211 (11%), with 83 (6.9%) having had a second ED revisit within 72 h of ED discharge. According to ICD-10 codes, the most common causes of ED revisits were respiratory, digestive, genitourinary, symptoms, and external causes. Factors of frequent ED revisits within 72 h were young age, institutional health insurance coverage, year of new health information system (2015), external causes, and genitourinary. Conclusion: The rate of 72-h ED revisits after discharge of children with chronic diseases treated at KASCH-RD was relatively high, and was associated with young age, institutional health insurance coverage, year of a new health information system implementation, and external causes of ED visit. These study findings amplify the need for intervention to reduce the rate of early ED revisits among children with chronic diseases. Keywords: Emergency department, ED revisit, Chronic diseases, Children, Saudi Arabia Keywords: Emergency department, ED revisit, Chronic diseases, Children, Saudi Arabia Emergency department 72-hour revisits among children with chronic diseases: a Saudi Arabian study Anwar E. Ahmed1,2,5, Bashayr I. ALMuqbil2, Manair N. Alrajhi2, Hend R. Almazroa2, Doaa A. AlBuraikan2, Monirah A. Albaijan1, Maliha Nasim1, Majid A. Alsalamah2, Donna K. McClish3 and Hamdan AL-Jahdali2,4,5 Background ED visits were observed in patients of various age groups, with a large amount having been noticed in chil- dren [8]. Earlier studies have assessed the rate of ED revisits among children in general: it ranges between 2.7 and 19% [9–17]. However, a high frequency of ED revisits was observed in children with chronic diseases [14, 15]. The large variation rates of ED revisits among children highlights the need for more evaluation, particularly in unstudied populations. Overcrowding in pediatric emergency departments (EDs) places a heavy burden on healthcare systems in terms of financial costs [1, 2] and potential infection-related ED visits [3]. Recently, significant interest and research has focused on the number of return-to-emergency depart- ment (ED) visits, as it represents a quality benchmark for patient safety and care [4–6]. This number also contrib- utes to overcrowding in EDs, as some visits are unneces- sary [4, 7]. In several international reports, the greater rate of ED revisits in children has been attributed to young age [9, 12, 15, 18] and health insurance coverage [18–20]. Recent Saudi Arabian studies, however, have documented that chronic diseases are the top cause of death among * Correspondence: ahmeda5@vcu.edu; jahdalih@gmail.com 1King Abdullah International Medical Research Center (KAIMRC), Riyadh, Saudi Arabia 2King Saud bin Abdulaziz University for Health Sciences, National Guard Health Affairs, P.O. Box 22490, Riyadh 11426, Saudi Arabia Full list of author information is available at the end of the article 2King Saud bin Abdulaziz University for Health Sciences, National Guard Health Affairs, P.O. Box 22490, Riyadh 11426, Saudi Arabia Full list of author information is available at the end of the article 2King Saud bin Abdulaziz University for Health Sciences, National Guard Health Affairs, P.O. Box 22490, Riyadh 11426, Saudi Arabia Full list of author information is available at the end of the article Methods This is a retrospective study of all ED discharges of chil- dren with chronic diseases under the age of 18 years who attended the ED at King Abdullah Specialist Chil- dren’s Hospital, Riyadh (KASCH-RD) between April 19, 2015 and July 29, 2017. The study obtained approval from the Institutional Review Board (IRB) at the Minis- try of National Guard - Health Affairs (MNG-HA), ap- proval #RC17/081/R. The ED records of eligible children were retrieved from the institution’s BESTCare database. In 2015, the children’s hospital implemented a new integrated health information system, BESTCare. It is a unified database that allows integration of all main hospital units: in- patient, outpatient, emergency, intensive care, and oper- ating room. All medical documentation, medical orders, medication histories, and radiology and lab results are stored in chronological order [23]. Housing a wide range of administration and clinical activity (including a clin- ical data warehouse), it also benefits from a clinical deci- sion support system that highlights standardized clinical guidelines of procedures to be followed for certain con- ditions, and issues alerts to physicians, on ordering in- vestigations or prescribing medication that may be contraindicated or unsafe [23]. We defined the study population as children with at least one chronic disease who were discharged from the ED at KASCH-RD during the study period. We included the main types of chronic diseases that require long-term control [24]. These chronic diseases were defined by the Saudi Ministry of Health as national health priorities for prevention and treatment, such as cardiovascular disease, diabetes melli- tus, hypertension, cancer, pulmonary disease, and asthma [25]. Ahmed et al. BMC Pediatrics (2018) 18:205 Page 2 of 7 Page 2 of 7 children in Saudi Arabia [21, 22], and have become a major priority in Saudi Vision 2030. no), year of ED visit (2015, 2016, or 2017). Patients’ ages were classified into 4 groups: < 3 years, 3 ≤age < 6 years, 6 ≤age < 14 years, and ≤14 age < 18 years old. Despite the fact that revisits to the ED are well rec- ognized as a key for quality improvement [4–6], no data exists in Saudi Arabia regarding the rate of ED revisits among children with chronic diseases. This study attempts to investigate the frequency of ED re- visits at a major Saudi hospital and the common causes, and to identify factors among children with chronic diseases associated with the high rate of 72-h ED revisits after discharge. We assessed the hypoth- esis that younger children, health insurance coverage, year of a new health information system implementa- tion, and causes of ED visits may be associated with the high rate of ED revisits within 72 h among the study population. In our center the health coverage includes 3 categor- ies: 1) Ministry of National Guard employees and their dependents who are fully covered. 2) those with private health insurance coverage and 3) those without insur- ance but covered exceptionally based on their case com- plexity. We reclassified health insurance status into two groups; institutionally insured or privately insured. In our center the emergency department is open to all emergency cases. Case that sever or need urgent admis- sion will be admitted to our hospital regardless of their insurance coverage, or nationalities. The study units of analysis are ED discharges of chil- dren with at least one chronic disease. The study out- come was the number of ED revisits within 72 h of discharge (0,1,2 etc). The causes of initial ED visit/re- visits were classified according to the International Stat- istical Classification of Diseases and Related Health Problems (10th version, Australian modification) code, chapters I “Certain infectious and parasitic diseases” to XXII “Codes for special purposes” [26]. The ICD-10 is publically available at http://apps.who.int/classifications/ icd10/browse/2016/en#/XVIII. Data analysis y The data analysis was performed using IBM SPSS v. 25 (IBM Corp., Armonk, NY, USA). Subject characteristics were illustrated as frequency and percentage (Table 1). The most common causes reported at the first ED revisit and the second ED revisit within 72 h were presented in bar charts (Figs. 1 & 2). The bar charts were generated using Microsoft Excel 2010. A univariate Poisson regres- sion model was used to calculate the unadjusted relative rate (uRR) and assess differences in the rate of ED re- visits within 72 h of ED discharge across children’s char- acteristics. Multiple Poisson regression models were used to calculate the adjusted relative rate (aRR) and identify independent factors that were associated with the high rate of ED revisits within 72 h of ED discharge. Table 2 shows the findings of Poisson regression models: p-value (P), RR, and confidence intervals (CI) for RR. A P ≤0.05 was considered significant. Results The KASCH-RD ED medical records of 11,057 ED dis- charges for children with chronic diseases were analyzed. Of the ED discharges, 1211/11,057 (11%) had a first ED revisit, and 83/1211 (6.9%) had a second ED revisit within 72 h. The median age was 4.9 (25 percentile = 2.3 and 75 percentile = 8.7 years), with 60.1% being males. Among the ED discharges, 618 (5.6%) children were ad- mitted to the hospital. Table 1 summarizes the sample The study data included age, gender, institutional health insurance status (yes/no), new patient or patient has not received healthcare services in our facility (yes/ Ahmed et al. BMC Pediatrics (2018) 18:205 Page 3 of 7 Table 1 Characteristics of ED discharges at KASCH-RD between September 13, 2015 and July 29, 2017 Characteristics Levels Number Percentage Age < 3 years 2735 32.1 3 ≤Age < 6 years 2192 25.8 6 ≤Age < 14 years 3151 37.0 ≥14 years 432 5.1 Gender Female 3394 39.9 Male 5116 60.1 Institutional health insurance coverage Yes 8223 96.6 No 287 3.4 Year of ED visit 2015 2538 23.0 2016 5663 51.2 2017 2856 25.8 New patient ED visit Yes 533 17.4 No 2526 82.6 Causes of initial ED visit Circulatory 362 3.3 Congenital malformations 371 3.4 Digestive 2046 18.5 Ear 478 4.3 External causes 851 7.7 Genitourinary 1954 17.7 Respiratory 2606 23.6 Other 2389 21.6 Initial ED discharges (N = 11,057) Had first revisit within 72 h No 9846 89.0 Yes 1211 11.0 Had a second revisit within 72 h No 1128 93.1 Yes 83 6.9 Fig. 1 The most common causes at the first ED revisit within 72 h of discharge Fig. 1 The most common causes at the first ED revisit within 72 h of discharge Ahmed et al. BMC Pediatrics (2018) 18:205 Page 4 of 7 Fig. 2 The most common causes at the second ED revisit within 72 h of discharge mortality (3.6%) (Fig. 1). The most common causes on the second ED revisit were respiratory (34.9%), digestive (22.9%), genitourinary (16.9%), external causes of morbid- ity and mortality (4.8%), along with symptoms, signs, and abnormal clinical and laboratory findings (2.4%) (Fig. 2). characteristics. Among children studied, the 6 most com- mon chronic diseases were asthma (8.9%), allergy (3.6%), heart disease (1.6%), eczema (1%), anemia (0.6%), and dia- betes (0.2%). . Significant at α = 0.05 Results Among children with chronic diseases, institutional health insur- ance coverage was associated with a higher frequency of revisits within 72 h (uRR = 4.239, P = 0.001). The risk of revisits within 72 h was higher for children with chronic diseases who visited the ED in 2015 (uRR = 1.363, P = 0.001) and 2016 (uRR = 1.288, P = 0.001) compared to children who visited the ED in 2017. Causes for ED visits, such as digestive diseases (uRR = 1.490, P = 0.001), genitourinary diseases (uRR = 1.765, P = 0.001), respira- tory diseases (uRR = 1.435, P = 0.001), and ear diseases (uRR = 1.380, P = 0.026) were also significant predictors for a higher frequency of revisits within 72 h. study we calculated the rate of ED revisit in a definitive group, specifically children with chronic diseases. How- ever, this comparison indicates that the presence of chronic diseases is associated with a high rate of ED utilization. A similar ED revisit rate was found in earlier investiga- tions: two studies among young children with gastro- enteritis [13] and common illnesses [15] showed 16%. In our study, we evaluated revisits within 72-h of ED dis- charge, and it should be noted that these two studies have evaluated longer time spans of ED revisits. For in- stance, Freedman et al. [13] evaluated revisits within 7 days. Reducing early ED revisits at KASCH-RD must be a hospital priority to reduce unnecessary costs [16] and improve quality of services [17]. Understanding the rea- sons for ED revisits among children with chronic dis- eases, based on the initial ED visit discharge, may allow implementing intervention or guidelines to reduce the ED revisit rates. To reduce avoidable ED revisits, this could involve applying a predetermined framework on early follow-up and parent education on home management. g q y Independent risk factors for ED revisits within 72 h in- cluded age, year, institutional health insurance coverage, external causes, and genitourinary diseases (Table 2). Children with chronic diseases of a younger age were as- sociated with a higher risk of ED revisits within 72 h: age < 3 years (aRR = 6.056, P = 0.001), 3 ≤age < 6 years (aRR = 4.831, P = 0.001), and 6 ≤age < 14 years (aRR = 2.663, P = 0.036) compared to children of age ≥14 years. Results The most common causes at the initial ED visit were re- spiratory (23.6%), digestive (18.5%), genitourinary (17.7%), and external causes of morbidity and mortality (7.7%). The most common causes on the first ED revisit were re- spiratory (52.8%), digestive (12.6%), genitourinary (11.2%), symptoms, signs, and abnormal clinical and laboratory findings (5.3%), and external causes of morbidity and The results of unadjusted and adjusted Poisson ana- lyses of the number of revisits within 72 h after ED dis- charge are illustrated in Table 2. The unadjusted relative risk (uRR) significantly increased in children with chronic diseases of age < 3 years (uRR = 3.293, P = 0.001), 3 ≤age < 6 years (uRR = 3.114, P = 0.001), and 6 ≤ Table 2 Factors associated with higher rate of ED revisits within 72 h of discharge Univariate Multivariate 95% Wald CI for RR 95% Wald CI for aRR Factor Reference P RR Lower Upper P aRR Lower Upper Age < 3 years ≥14 years 0.001 3.293 2.103 5.158 0.001* 6.056 2.461 14.904 3 ≤Age < 6 years ≥14 years 0.001* 3.114 1.982 4.893 0.001* 4.831 1.930 12.091 6 ≤Age < 14 years ≥14 years 0.001* 2.392 1.524 3.754 0.036* 2.663 1.068 6.641 Female Male 0.830 1.013 0.898 1.143 0.094 1.253 0.962 1.633 Institutional health insurance coverage None 0.001* 4.239 2.200 8.168 0.025* 3.132 1.152 8.518 Year 2015 Year 2017 0.001* 1.363 1.166 1.593 0.001* 2.040 1.390 2.993 Year 2016 Year 2017 0.001* 1.288 1.124 1.475 0.050* 1.427 1.000 2.037 New patient ED visit No 0.341 0.833 0.572 1.213 0.950 0.988 0.681 1.434 Circulatory Other 0.687 1.075 0.756 1.529 0.595 0.858 0.487 1.511 Congenital malformations Other 0.296 1.195 0.856 1.667 0.102 1.401 0.935 2.099 Digestive Other 0.001* 1.490 1.249 1.777 0.141 1.456 0.883 2.399 Ear Other 0.026* 1.380 1.039 1.832 0.518 1.217 0.671 2.204 External causes Other 0.449 0.902 0.690 1.178 0.039* 1.944 1.034 3.657 Genitourinary Other 0.001* 1.765 1.487 2.095 0.002* 1.814 1.251 2.631 Respiratory Other 0.001* 1.435 1.212 1.699 0.001* 0.111 0.035 0.351 *. Significant at α = 0.05 Table 2 Factors associated with higher rate of ED revisits within 72 h of discharge Univariate Multivariate Ahmed et al. BMC Pediatrics (2018) 18:205 Page 5 of 7 age < 14 years (uRR = 2.392, P = 0.001) compared to chil- dren with chronic diseases of age ≥14 years. Results The rate of ED revisits within 72 h was higher for chil- dren with institutional health insurance than for children without health insurance (aRR = 3.132, P = 0.025). The year of implementing a new health information system (2015) significantly predicted a high rate of ED revisits within 72 h (aRR = 2.040, P = 0.001), and the following year 2016 (aRR = 1.427, P = 0.050), compared to 2017. Among children with chronic diseases, external causes of morbidity and mortality (aRR = 1.944, P = 0.039) and genitourinary (aRR = 1.814, P = 0.002) were associated with a higher rate of revisits within 72 h after ED dis- charge. Gender was not associated with 72-h ED revisits among children with chronic diseases. In the KASCH-RD center, respiratory conditions were found to be the most frequent cause of initial ED visits. This is in agreement with Goh et al., where it reported that respiratory conditions were linked with higher ED utilization [10]. Focusing on ED revisits related to re- spiratory diseases may reduce ED revisit rates by identi- fying non-urgent ED visits. In this large cohort study, we noted that the rate of ED revisits decreased with age in children with chronic diseases. The greater ED revisit rate in younger children noted in the study is in agreement with earlier studies showing that children of a younger age are associated with frequent ED revisits [9, 12, 15, 18]. Interventions are needed to reduce ED revisits in younger children, such as referrals or early follow-up appointments in the outpatient clinic setting to specialty clinics, and parent involvement in the ED discharge process. Acknowledgements The authors would like to thank the Ministry of National Guard - Health Affairs, Riyadh, Saudi Arabia, for approving this study. Competing interests Competing interests The authors declare that they have no competing interests. Competing interests The authors declare that they have no competing interests. The authors identified several limitations to the study. Al- though the number of ED visits studied was high, the findings were based on a single-center and retrospective study rather than a multi-center and prospective assess- ment of within 72 h of ED revisits. We did not record ED revisits occurring at another health facility. Furthermore, the study has not collected data on important details such as mode of arrival to the ED, number of chronic diseases, physician-related causes, and patient-related causes. Despite these limitations, to our knowledge, this study represents an initial investigation on early ED revisits and their causes among Saudi Arabian children with chronic diseases. Discussion In this retrospective study, we included all ED dis- charges of patients with chronic diseases under the age of 18 years who were admitted to the ED at KASCH-RD between April 19, 2015 and July 29, 2017. This represents the first Saudi Arabian evalu- ation of children with chronic diseases re-attending the ED within 72 h after ED discharge. Our main aim was to determine the frequency of ED revisits and the main causes, as well as to identify characteristics associated with the high rate of 72-h ED revisits among children with chronic diseases. In this study, institutional health insurance coverage was found to be an important determinant of ED revisits in children with chronic diseases. In several reports, public health insurance coverage as compared with un- insured (self-pay) or privately insured has been cited as an important predictor of ED revisits. Walsh-Kelly et al. [18] noted that public insurance was associated with greater ED revisits. Scales et al. [19] and Jacobstein et al. [20] reported that the odds of ED revisits were 52 and 86% higher in the Medicaid group as compared to the private insurance group, respectively. The impact of in- stitutional health insurance coverage on ED revisits in children with chronic diseases must be evaluated to identify preventable reasons for ED revisits. A rather high ED revisit rate (11%) was observed among children with chronic diseases in Saudi Arabia as compared to the general global children’s population: USA 2.7% [9], Singapore 4.3% [10], Canada 4.4% [11], and Taiwan 6.47% [12]. It may not be possible to com- pare this study’s findings with these studies, as in our Ahmed et al. BMC Pediatrics (2018) 18:205 Page 6 of 7 Ahmed et al. BMC Pediatrics (2018) 18:205 A higher rate of ED revisits in children with chronic diseases appeared during the year 2015 as compared to the more recent year of 2017. This indicates that the rate of ED revisits in the Saudi facility decreased with time. This may be due to the 2015 implementation in the KASCH-RD facility of a new computerized hospital sys- tem, BESTCare, which is a unified electronic health in- formation system that includes a clinical decision support system. The reduction of the ED revisit rate over time could be due to implementation of a wide array of training programs to promote and evaluate the use BESTCare among physicians. Availability of data and materials The electronic health records dataset analyzed in the current study are not publicly available due to concerns regarding security and privacy of the health records. The original health records dataset pertaining to this study can be obtained from the Ministry of National Guard - Health Affairs. Conclusions A high rate of early ED revisits was found at KASCH-RD among children with chronic diseases, occurring fre- quently in one in ten children. In children with chronic diseases, higher ED revisit rates are associated with young age, institutional health insurance coverage, and causes for ED visits. Intervention could be implemented to examine whether parent education on home management, early follow-up appointment in an outpatient clinic setting, and clear discharge guidance could reduce the rate of early ED revisits among children with chronic diseases. Author details 1King Abdullah International Medical Research Center (KAIMRC), Riyadh, Saudi Arabia. 2King Saud bin Abdulaziz University for Health Sciences, National Guard Health Affairs, P.O. Box 22490, Riyadh 11426, Saudi Arabia. 3Department of Biostatistics, School of Medicine, Virginia Commonwealth University, Box 980032, Richmond, VA 23298, USA. 4McGill University, 5 Montreal, Canada. 5Pulmonary Division Medical Director of sleep disorders, Center King Abdulaziz Medical City, Riyadh, Saudi Arabia. Received: 1 April 2018 Accepted: 21 June 2018 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Authors’ contributions AEA and DKM developed the study, analyzed the data, and wrote the manuscript. DAA, HRA, MNA, and BIA retrieved the data from the BESTCare records and used the International Classification of Diseases (ICD-10) to determine the cause of ED visits. MAA1 retrieved and reviewed data quality and cross-checked analysis. MAA2, HJ, and MN commented on the methods and clinical findings. All authors read and approved the final manuscript. Studies have shown that electronic health information systems, particularly those that incorporate clinical deci- sion tools, have the advantage of improving the health- care provider’s adherence to evidence-based clinical guidelines and care protocols [27, 28]. Improvements in adherence can lead to improved clinical outcomes [27– 31] and potentially better quality of patient care, which may help to explain the reduction in ER revisits. How- ever, it should be noted that these studies have taken place at inpatient and outpatient settings and not the ER, and thus have not directly assessed the effect of clin- ical decision tools. Abbreviations aRR: Adjusted relative rate; CI: Confidence interval; ED: Emergency Department; IRB: Institutional Review Board; KASCH-RD: King Abdullah Specialist Children’s Hospital, Riyadh; MNG-HA: Ministry of National Guard - Health Affairs; P: p-value; uRR: Unadjusted relative rate Consent for publication Not applicable. 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https://openalex.org/W2599226013	https://research.tilburguniversity.edu/files/24448152/Child_Robot_Interactions_for_Second_Language_Tutoring_to_Preschool_Children.pdf	English	null	Child-Robot Interactions for Second Language Tutoring to Preschool Children	Frontiers in human neuroscience	2,017	cc-by	6,020	Child-Robot Interactions for Second Language Tutoring to Preschool Children Vogt, Paul; de Haas, Mirjam; de Jong, Chiara; Baxter, Peta; Krahmer, Emiel Published in: Frontiers in Human Neuroscience DOI: 10.3389/fnhum.2017.00073 Publication date: 2017 Document Version Publisher's PDF, also known as Version of record Link to publication in Tilburg University Research Portal Citation for published version (APA): Vogt, P., de Haas, M., de Jong, C., Baxter, P., & Krahmer, E. (2017). Child-Robot Interactions for Second Language Tutoring to Preschool Children. Frontiers in Human Neuroscience, 11, Article 73. https://doi.org/10.3389/fnhum.2017.00073 Child-Robot Interactions for Second Language Tutoring to Preschool Children Vogt, Paul; de Haas, Mirjam; de Jong, Chiara; Baxter, Peta; Krahmer, Emiel Child-Robot Interactions for Second Language Tutoring to Preschool Children Vogt, Paul; de Haas, Mirjam; de Jong, Chiara; Baxter, Peta; Krahmer, Emiel Document Version Publisher's PDF, also known as Version of record Link to publication in Tilburg University Research Portal Citation for published version (APA): Vogt, P., de Haas, M., de Jong, C., Baxter, P., & Krahmer, E. (2017). Child-Robot Interactions for Second Language Tutoring to Preschool Children. Frontiers in Human Neuroscience, 11, Article 73. https://doi.org/10.3389/fnhum.2017.00073 Citation for published version (APA): Vogt, P., de Haas, M., de Jong, C., Baxter, P., & Krahmer, E. (2017). Child-Robot Interactions for Second Language Tutoring to Preschool Children. Frontiers in Human Neuroscience, 11, Article 73. https://doi.org/10.3389/fnhum.2017.00073 Tilburg University General rights C i ht d General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private st • You may not further distribute the material or use it for any profit making activity or commercial gain y y p g y • You may freely distribute the URL identifying the publication in the public portal Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim Download date: 24. Oct. 2024 PERSPECTIVE published: 02 March 2017 doi: 10.3389/fnhum.2017.00073 PERSPECTIVE Child-Robot Interactions for Second Language Tutoring to Preschool Children Paul Vogt , Mirjam de Haas , Chiara de Jong , Peta Baxter and Emiel Krahmer Tilburg Center for Cognition and Communication, Tilburg University, Tilburg, Netherlands In this digital age social robots will increasingly be used for educational purposes, such as second language tutoring. In this perspective article, we propose a number of design features to develop a child-friendly social robot that can effectively support children in second language learning, and we discuss some technical challenges for developing these. The features we propose include choices to develop the robot such that it can act as a peer to motivate the child during second language learning and build trust at the same time, while still being more knowledgeable than the child and scaffolding that knowledge in adult-like manner. We also believe that the ﬁrst impressions children have about robots are crucial for them to build trust and common ground, which would support child-robot interactions in the long term. We therefore propose a strategy to introduce the robot in a safe way to toddlers. Other features relate to the ability to adapt to individual children’s language proﬁciency, respond contingently, both temporally and semantically, establish joint attention, use meaningful gestures, provide effective feedback and monitor children’s learning progress. Technical challenges we observe include automatic speech recognition (ASR) for children, reliable object recognition to facilitate semantic contingency and establishing joint attention, and developing human-like gestures with a robot that does not have the same morphology humans have. We brieﬂy discuss an experiment in which we investigate how children respond to different forms of feedback the robot can give. Edited by: Mila Vulchanova, Norwegian University of Science and Technology, Norway Reviewed by: Ramesh Kumar Mishra, University of Hyderabad, India Vera Kempe, Abertay University, UK Keywords: social robots, second language tutoring, education, child-robot interaction, robot assisted language learning SOCIAL ROBOTS FOR SECOND LANGUAGE TUTORING Correspondence: Paul Vogt p.a.vogt@uvt.nl *Correspondence: Paul Vogt p.a.vogt@uvt.nl Given the globalization of our society, it is becoming increasingly important for people to speak multiple languages. For instance, the ability to speak foreign languages fosters people’s mobility and increases their chances for employment. Moreover, immigrants to a country need to learn the official host language. Since young children are most flexible at learning languages, starting second language (L2) learning in preschool would provide them a good opportunity to acquire the second language more fluently at a later age (Hoff, 2013). Received: 26 October 2016 Accepted: 06 February 2017 Published: 02 March 2017 Keywords: social robots, second language tutoring, education, child-robot interaction, robot assisted language learning Peer-Like Tutoring embodied interactions that young children naturally engage in and learn from (Glenberg, 2010; Leyzberg et al., 2012). Social robots represent an emerging technology that provides situatedness and embodiment, and thus have potential benefits for educational purposes. In essence, social robots are autonomous physical agents, often with human-like feature, that can interact socially with humans in a semi-natural way for prolonged periods of time (Dautenhahn, 2007). The use of social robots, in comparison to more traditional digital technologies, allows for the development of tutoring systems more akin to human tutors, especially with respect to the situated and embodied social interactions between child and robot. Thus, this offers the opportunity to design robots such that they interact in a way that optimizes the child’s language learning. g One of the first questions that comes up when designing a robot tutor is whether the robot should take the role of a teacher or a peer. Research on children’s language acquisition has demonstrated that children learn more effectively from an adult who can use well-defined pedagogical methods for teaching children using clear directions, explanations and positive feedback methods (Matthews et al., 2007). However, designing and framing the robot as an adult tutor has the disadvantage that children will form expectations about the robot’s behavior and proficiency that cannot be met with current technology (Kennedy et al., 2015). Due to technological limitations of the robot and underlying software, communication breakdowns are more likely to occur than with a human. For a peer robot introduced as a fellow language learner, breakdowns in communication are more acceptable. Moreover, interacting with robots acting as peers is conceived as more fun (Kanda et al., 2004), allows for learning-by-teaching (Tanaka and Matsuzoe, 2012) and has a proven to be efficient in teaching children how to write (Hood et al., 2015). Furthermore, there is some evidence that children’s learning can benefit from interacting with peers (Mashburn et al., 2009). Given these considerations, we believe it is desirable to frame or introduce the robot as a peer and friend, yet design its interactions insofar possible based on pedagogically well-established strategies to scaffold language learning. Recently, an increasing interest has emerged to develop social robots to support children with learning a second language (Kanda et al., 2004; Belpaeme et al., 2015; Kennedy et al., 2016). First Impressions While there is a fair body of research on robot tutors, a comprehensive description of the design features for a second language robot tutor based on what is known about children’s language acquisition is lacking. What are the design features of child-robot interactions that would support second language learning? And, to what extent can these interactions be implemented in today’s social robot technologies? In this perspective article, we try to answer these questions based on theoretical accounts from the literature on children’s language acquisition in combination with our own experiences in designing a tutor robot. To implement effective tutoring, the robot needs to interact with children in multiple sessions, so they have to be motivated to engage in long-term interactions with the robot. Establishing common ground between child and robot can contribute to this (Kanda et al., 2004), but first impressions to establish trust and rapport are also crucial (Hancock et al., 2011). pp Despite the wealth of studies regarding the introduction of entertainment robots as toys to children (e.g., Lund, 2003), surprisingly little research has been conducted on designing protocols on how to introduce a robot tutor to a group of preschool children. Fridin (2014) presents one exception, and found that introducing a robot tutor to children in group sessions improved subsequent interactions compared to introducing the robot to children in individual sessions. Another study by Westlund et al. (2016) found that the way a robot is framed, either as a machine or a social entity, affected the way children later engaged with the robot. They concluded that introducing the robot as a machine could create a more distant relation between child and robot, thus reducing acceptance. We therefore decided to frame the robot in our project as a social playmate for the children and introduced the robot in a group session. However, the NAO robot is slightly taller and more rigid than the fluffy huggable Tega robot, which Westlund et al. (2016) used, and we observed that some 3-year-old children were somewhat intimidated by the NAO robot on their first encounter. Such a first impression of the robot could reduce the trust that the child had for the robot, which could negatively affect their willingness to interact with the robot in the short-term, but also in the long-term. To develop a successful first encounter and to build Citation: Vogt P, de Haas M, de Jong C, Baxter P and Krahmer E (2017) Child-Robot Interactions for Second Language Tutoring to Preschool Children. Front. Hum. Neurosci. 11:73. One trend in the digital age of the 21st century is that technologies are being developed for educational purposes, including technologies to support L2 tutoring. There exist many forms of digital technologies for PCs, laptops or tablet computers that support second language learning, although there is little evidence about their efficacy (Golonka et al., 2014; Hsin et al., 2014). While children can benefit from playing with such technologies, these systems lack the situated and March 2017 \| Volume 11 \| Article 73 Frontiers in Human Neuroscience \| www.frontiersin.org 1 Child-Robot Interactions for Second Language Tutoring Vogt et al. Peer-Like Tutoring While a social robot cannot provide tutoring to the level humans can, recent studies suggest that using social robots can result in an increased learning gain compared to digital learning environments for tablets or computers (Han et al., 2008; Leyzberg et al., 2012). It is, however, unclear why this is the case. Perhaps the physical presence of the robot draws the attention of children for longer periods of time, but the embodiment and situatedness of the learning environment perhaps also helps the children to ground the language more strongly than interactions with virtual objects do. Frontiers in Human Neuroscience \| www.frontiersin.org DESIGNING CHILD-ROBOT INTERACTIONS In our project, we aim to design a digital learning environment in which preschool children interact one-on-one with a social robot that supports either their learning of English as a foreign language, or the school language for those children who have a different native language (Belpaeme et al., 2015). In particular, the project aims to develop a series of tutoring sessions revolving around three increasingly complex domains (numbers, spatial relations and mental vocabulary). In each session, the child will engage with the robot (a Softbank Robotics NAO robot) in a game-like scenario focusing on learning a small number of target words. The contextual setting is generally displayed on a tablet computer that occasionally also provides some verbal support, however, the robot acts as the interactive tutor. Below we discuss the design features and considerations that we believe are crucial to design a successful tutoring system. Frontiers in Human Neuroscience \| www.frontiersin.org March 2017 \| Volume 11 \| Article 73 2 Child-Robot Interactions for Second Language Tutoring Vogt et al. trust between the child and robot, we designed the following strategy for introducing the robot to 3-year-old children at their preschool. (Garvey and Berninger, 1981) and having framed the robot as a peer children made the delays more plausible or expected. Nevertheless, while a lag in temporal contingency may not harm the interaction with children, it may harm learning. One way to remedy this may be to have the robot start responding by providing a backchannel signal, such as ‘‘uhm’’ to indicate the robot is (still) taking his turn, but requires more time to process (Clark, 1996). p Pilot studies revealed that some children got anxious when the robot was introduced and then suddenly started to move. To familiarize children prior to their first encounter with the robot, it is therefore advisable to prepare them well. For our study, we sent coloring pages of the robot to the preschools during recruitment and asked the pedagogical assistants to talk a little bit about the robots to the children. About 1 week before the experimental trials, the experimenters introduced the robot in class during their daily ‘‘circle time’’, as this provided a safe and familiar environment with the whole group in which the pedagogical assistants usually introduce new topics or new activities. Monitoring Children’s Behavior g To understand children’s communication bids, as well as to test their pronunciation of the L2, it is important that the robot be equipped with well-functioning automatic speech recognition (ASR). However, the performance of state-of-the-art ASR for children is still suboptimal, especially for preschool-aged children (Fringi et al., 2015; Kennedy et al., 2017). Reasons for this include that children’s pronunciation is often flawed and that their speech has a different pitch than adults. Moreover, relatively little research has been carried out in this domain and not much data exist to train ASR on. While it can be expected that the performance of ASR for children will improve in the not too distant future (Liao et al., 2015), until then alternative strategies need to be developed that do not (exclusively) rely on ASR. In our project, we explore various strategies to achieve this, both based on monitoring non-verbal behaviors of the children and focusing on comprehending rather than producing L2. The first strategy relies on providing children tasks they have to perform in the learning environment, such as placing ‘‘a toy cow behind a tree’’ when teaching spatial language. This, however, requires the visual object recognition on the robot to work well, which is only the case when the scene contains a limited set of distinctively recognizable objects, such as distinctly colored objects (Nguyen et al., 2015). A potential solution explored in our project is to use objects with build-in RFID sensors that can be tracked automatically. The second solution we explore is to use a touch screen tablet that displays scenes the child can manipulate, which not only has the advantage of avoiding the problem of object recognition, but also allows us to control DESIGNING CHILD-ROBOT INTERACTIONS One experimenter first introduced the robot by telling a story about Robin, the name of our robot, using a makeshift picture book. In this story we explained the similarities and dissimilarities between the robot and children to construct the type of common ground considered to have a positive effect on the learning outcome (Kanda et al., 2004). For example, we told that Robin enjoys dancing and wants to meet new friends, and even though he does not have a mouth and because of that cannot smile, he can smile using his eye LEDs. Semantic Contingency Robots should not only respond to children in a timely fashion, but also in a semantically contingent fashion (i.e., consistent with the child’s focus of attention), as this too has a positive effect on children’s language acquisition (Bornstein et al., 2008; McGillion et al., 2013). For instance, research has shown that by responding in a semantically contingent manner, either verbally or by following children’s gaze, (joint) attention is sustained for a longer duration (Yu and Smith, 2016), allowing children to learn more about a situation. To achieve semantically contingent responses, the robot should be able to understand the child’s communication bids, construct joint attention with the child, or at least identify what the child is attending to. Monitoring children’s behavior and establishing joint attention are therefore considered crucial for designing a successful robot tutor. After this story, another experimenter entered the room with the robot while it was actively looking at faces to provide an animate feeling. The robot introduced itself with a small story about itself and by performing a dance in which the children were encouraged to participate. The end of the circle time consisted of getting a blanket for the robot so it could ‘‘sleep’’. This introduction was repeated later on the days we conducted the experiment in one-on-one sessions. While by then most children were comfortable interacting with the robot, some were still timid and anxious. To encourage these children to feel comfortable, one of the experiment leaders would sit next to the child during the warm-up phase of the experiment and motivate the child to respond to the robot when necessary until the child was sufficiently comfortable to interact with the robot by herself/himself. We found that the younger 3-year olds required more support from the experimenters than the older 3-year olds (Baxter et al., 2017). Although we are still analyzing the experiments, preliminary findings suggest that our introduction helped children to build trust and common ground with the robot effectively. Frontiers in Human Neuroscience \| www.frontiersin.org Joint Attention and Gestures Joint attention, where interlocutors attend on the same referent, is a form of social interaction that has been shown to support children’s language learning (Tomasello and Farrar, 1986). One way to establish joint attention with a child is to guide their attention to a referent using gestures, such as pointing or iconic gestures. The ability to produce gestures in the real world is potentially one of the main advantages of using physical robots as opposed to virtual agents, who may have a harder time to establish joint attention. However, many robots’ physical morphologies do not correspond one-to-one to the human body. Hence, many human gestures cannot be translated directly to robot gestures. For instance, the NAO robot that we use in our research has a hand with three fingers that cannot be controlled independently, so index finger pointing cannot be Temporal Contingency Research has shown that it is crucial for children’s language development that their communication bids are responded to in a temporally contingent manner (Bornstein et al., 2008; McGillion et al., 2013). This, however, faces a technological challenge. While adults tend to take over turns very rapidly, robots require relatively long processing time to produce a response. Nevertheless, in our first experiment (de Haas et al., 2016), we observed that children were at first surprised by the delayed responses, but quickly adapted to the robot and waited patiently for a response. Perhaps this is because children also require longer than adults to take turns March 2017 \| Volume 11 \| Article 73 Frontiers in Human Neuroscience \| www.frontiersin.org 3 Child-Robot Interactions for Second Language Tutoring Vogt et al. achieved (see Figure 1). Will children still recognize NAO’s arm extension as a pointing gesture? And if so, will they be able to identify the object the robot refers to? We are currently running an experiment to investigate how NAO’s pointing gestures are perceived, and preliminary findings show that participants have difficulty identifying the referred object on a small tablet screen. Similar issues arise when developing other gestures. One of the other non-verbal behaviors we are using is the coloring of NAO’s eye LEDSs to indicate the robot’s happiness as a form of positive feedback, since the robot cannot smile with its mouth. the robot’s responses and vary the scenes in real time. A downside, however, is that it takes away the 3-dimensional physical aspect of embodied cognition that would help the children to better entrench what they learn (Glenberg, 2010). Currently, experiments are underway to investigate the effect of using real vs. virtual objects. These solutions not only aid in understanding the child’s communication bids, it also helps in identifying their attention and can thus contribute to establishing joint attention. Feedback Feedback, too, is an interactional feature known to help language learning (Matthews et al., 2007; Ates¸ -S¸en and Küntay, 2015). The question is how should the robot provide feedback, such that it is both pleasant and effective for learning? While adults provide positive feedback explicitly, they usually provide negative feedback implicitly by reformulating children’s errors in the correct form. In child-child interactions, however, Long (2006) found that there was a clear advantage in learning from explicit negative feedback (e.g., by saying ‘‘no, that’s wrong, you need to say ‘he ran’’’) when compared to reformulating feedback (the learner says ‘‘he runned’’ and the teacher reacts with ‘‘he ran’’). FIGURE 1 \| NAO pointing to a block with three ﬁngers. (Note that written, informed consent was obtained from the parents of the child for the publication of this image). FIGURE 1 \| NAO pointing to a block with three ﬁngers. (Note that written, informed consent was obtained from the parents of the child for the publication of this March 2017 \| Volume 11 \| Article 73 Frontiers in Human Neuroscience \| www.frontiersin.org Child-Robot Interactions for Second Language Tutoring Vogt et al. To investigate how children experience feedback from a peer robot, we carried out an experiment among 85 3-year-old Dutch- speaking children at preschools in Netherlands (de Haas et al., 2016, 2017). In this experiment, the children interacted with a NAO robot during which they received a short lesson on how to count from 1 to 4 in English. After a short training phase, in which the children were presented with the four counting words twice in relation to body parts and wooden blocks, they were given instructions by the robot to pick up a given number of blocks. While the instructions were given in their native language, the numbers were uttered in English. In response to the child’s ability to achieve the task, the robot provided feedback. The experiment followed a between-subjects design with three conditions: adult-like feedback (explicit positive and implicit negative), peer-like feedback (no positive and explicit negative) and no feedback. We did not find significant differences in learning gain between the conditions, probably because the target words were insufficiently often repeated. CONCLUSION This perspective article presented some design features that we consider crucial for developing a social robot as an effective second language tutor. We believe the robot is most effective when it is framed as a peer, i.e., as a fellow language learner and playmate, but that is designed to use adult-like interaction strategies to optimize learning efficacy. In order to establish common ground and trust to facilitate long-term interactions, we consider it essential that the robot be introduced with appropriate care on the first encounter. As an example, we AUTHOR CONTRIBUTIONS PV, MH and EK designed the conceptual aspects of the article; PV, MH, CJ and PB carried out the literature review; PV, EK and MH designed the feedback study; MH, CJ and PB designed the introduction study; MH, CJ and PB carried out the studies; PV and MH wrote the article; CJ, PB and EK revised the article critically. ETHICS STATEMENT y p y Finally, from a pedagogical point of view it is desirable that the interactions between child and robot be sufficiently challenging and varied so that the child has a target to learn from, but at the same time interactions should not be too difficult, because that may frustrate the child causing it to lose interest in the robot (Charisi et al., 2016). In other words, the robot should remain in Vygotsky’s Zone of Proximity that supports an effective learning environment (Vygotsky, 1978). In order to achieve this, the robot should be able to keep track of the children’s advancements in language learning and perhaps their emotional states during the tutoring sessions, and adapt to these. While the former can be monitored as discussed previously, it may be possible to detect emotional states known to influence learning (e.g., concentration, confusion, frustration and boredom) using methods from affective computing (D’Mello and Graesser, 2012). Using this type of information, it is possible to adapt the tutoring sessions by either reducing or increasing the number of repetitions, and/or change the subject (Schodde et al., 2017). The Research Ethics Committee of Tilburg School of Humanities approved this study, and the parents of all participating children gave written informed consent in accordance with the Declaration of Helsinki. FUNDING This work has been supported by the EU H2020 L2TOR project (grant 688014). CJ and PB thank the research trainee program of the Tilburg School of Humanities for their support. Feedback However, we explored the way in which the children engaged with the robot after they received feedback and we found that children looked less often at the experimenter in the feedback conditions than in the no feedback condition. Further analyses are carried out to evaluate how the children responded to the various forms of feedback to find out what type of feedback would be most effective for achieving both acceptable and effective tutoring interactions. outlined our strategy for introducing a robot to preschool children. Interactions between child and robot should be contingent and multimodal, and provide appropriate forms of feedback. We argued that the robot should remain within Vygotsky (1978) Zone of Proximal Development and thus should adapt to the individual level of the child. We also discussed some technical challenges that need to be solved in order to implement contingent interactions; the most important of which we believe is ASR, which presently does not work well for children’s speech. While various technical challenges still remain, we expect that social robots will provide effective digital technologies to support second language development in the years to come. The present list of design features covers many aspects that need to be considered when developing a tutor robot, but it is not yet comprehensive. One aspect that has not been covered, for instance, concerns the design of robots for children from different cultures, which could require different design choices (Shahid et al., 2014). For example, in some cultures education is more teaching-centered (Hofstede, 1986) and thus designing the tutor as a peer robot may be less effective or acceptable (Tazhigaliyeva et al., 2016). 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https://openalex.org/W2083350169	https://europepmc.org/articles/pmc3663989?pdf=render	English	null	Chewing ability in an urban and rural population over 40 years in Shandong Province, China	Clinical oral investigations	2,012	cc-by	8,227	Clin Oral Invest (2013) 17:1425–1435 DOI 10.1007/s00784-012-0822-1 Clin Oral Invest (2013) 17:1425–1435 DOI 10.1007/s00784-012-0822-1 ORIGINAL ARTICLE Abstract Objectives This study aimed to assess chewing ability relat- ed to dental status. Results Seventy-eight to 91 % of subjects reported no or minor chewing problems. The conditions ‘≥10 teeth in each jaw’, and ‘complete anterior regions’ were not associated, whereas ‘sufficient’ premolar regions’ and ‘sufficient molar regions’ were associated with chewing problems (Ors, 0.33– 0.58). If classified hierarchically, the condition ‘≥10 teeth in each jaw’ was relevant for chewing problems (likelihood ratios 3.3–3.7). ‘Sufficient premolar region’ and ‘sufficient molar region’ were relevant to reduce the likelihood ratios for having chewing problems (both approximately with a factor 2), both for soft and for hard foods. Subjects with artificial teeth added had similar chance for chewing prob- lems compared to counterparts with natural teeth only. However, if comparing replaced teeth with natural teeth, subjects with tooth replacement showed higher chance for chewing problems. Material and methods One thousand four hundred sixty- two Chinese subjects over 40 years, dentate in both jaws, were categorized in a hierarchical functional classification system with and without tooth replacements. Chewing abil- ity was analyzed using multivariable logistic regression including five dental conditions (≥10 teeth in each jaw’; ‘complete anterior regions’; “sufficient premolar regions’ (≥3 posterior occluding pairs (POPs)); ‘sufficient molar regions’ (bilaterally ≥1 POP); and tooth replacement), ad- justed for six background variables. Likelihood ratios for chewing problems were assessed at each level of the Q. Zhang (*) Department of Prosthetic Dentistry, Affiliated Hospital of Medical School, Qingdao University, Jiangsu Road 16#, Qingdao, People’s Republic of China e-mail: habebeq@hotmail.com Conclusions Chewing ability was strongly associated with dental conditions. Clinical relevance The presence of at least 10 teeth in each jaw had highest impact on chewing ability. D. J. Witter: N. H. J. Creugers Department of Oral Function and Prosthetic Dentistry, College of Dental Science, Radboud University Nijmegen Medical Centre, Philips van Leydenlaan 25, 6525 EX Nijmegen, The Netherlands Keywords Chewing ability . Occlusal status . Hierarchical dental functional classification system . Chinese adults D. J. Witter e-mail: d.witter@dent.umcn.nl D. J. Witter e-mail: d.witter@dent.umcn.nl Chewing ability in an urban and rural population over 40 years in Shandong Province, China Qian Zhang & Dick J. Witter & Ewald M. Bronkhorst & Nico H. J. Creugers Received: 27 June 2012 /Accepted: 2 August 2012 /Published online: 2 September 2012 # The Author(s) 2012. This article is published with open access at Springerlink.com hierarchical classification system based on these dental conditions. Introduction N. H. J. Creugers e-mail: n.creugers@dent.umcn.nl One of the most immediate and important functional con- sequences of tooth loss is a reduction in chewing ability. Oral rehabilitation might restore partially the objective chewing capacity and might lead to an increased apprecia- tion of chewing ability. The relationship among chewing ability and the state of dentition has been subject of numer- ous studies, of which the majority of studies report a strong E. M. Bronkhorst Department of Preventive and Restorative Dentistry, College of Dental Science, Radboud University Nijmegen Medical Centre, Philips van Leydenlaan 25, 6525 EX Nijmegen, The Netherlands e-mail: e.bronkhorst@dent.umcn.nl Clin Oral Invest (2013) 17:1425–1435 1426 relationship [1–4]. Other studies, however, are less clear about a direct relationship and reported ambiguous findings regard- ing the number and location of teeth needed for a satisfactory chewing function [5, 6]. People’s satisfaction with chewing ability is not determined entirely by their mechanical chewing function. Instead, it is a complex measure that embraces broad physical, social, and psychological components [7]. It has been stated that the chewing process is an individually deter- mined and adaptive process [8, 9]. For instance, in elderly populations chewing ability has been associated with several non-dental functional impairments, such as decreased biting force and reduced saliva flow, as well as with factors such as general health and psychological and social well-being [10–13]. Also, recent studies have indicated that impaired chewing ability affects oral health-related quality of life (OHRQoL) [6, 14]. However, it has also been reported that the correlation between perceived chewing ability and OHR- QoL is not substantially influenced by number of teeth and age, but by gender, level of education, treatment demand, and prosthodontic status [2, 6]. lower jaw, (2) completeness of anterior regions, (3) number of premolar occluding pairs, and (4) number of molar occluding pairs. This classification system has not yet been used to assess chewing ability in relation to dental status in China. The aim of this study was to assess the relationship among dental conditions according to the proposed dental functional classification system (without and with FDP or RPD) and chewing ability of Chinese subjects of 40 years and older. It was hypothesized that subjects with FDP will have more benefit from added artificial teeth with respect to chewing ability than subjects with RDP. Materials and methods The study was conducted in the Qingdao area, located at the east coast of Shandong Province, the latter situated in East- ern China. Shandong is one of the largest provinces in China in terms of population (94 million in 2008) and economy. Qingdao City has approximately 3 million inhabitants. Qingdao City has direct jurisdiction over the surrounding rural territory in Shandong Province, including five county- level cities (approximately 200.000–400.000 residents each) and the counties surrounding these cities. Each rural county comprises 40–80 small rural villages. The total area of Qingdao (urban and rural) is approximately 10,000 km2 with a coastline of approximately 730 km and has approx- imately 8 million inhabitants. A systematic review based on DMFT data reported that the number of missing teeth in Chinese adults increases with age from approximately 2 at the age of 40 years to approx- imately 12 at the age of 65 years [15]. Information about the relationship of number of teeth and chewing ability for Chinese people is scarce. Zeng et al. [16] have described the effect of clinical dental status on chewing ability for a Chinese population in Guangxi. In that study decreased chewing ability was significantly related with having fewer (occluding) teeth, both with natural teeth only as well as with natural plus replaced teeth. However, that study reported on an elderly population (55 years and older) and did not distinguish between fixed (FDP) and removable dental prostheses (RDP). Moreover, although number of teeth and number of occluding anterior and posterior teeth were used as a measure for dental status, the actual config- urations of the dentitions involved remains unclear. Sampling method For this study, a cross-sectional survey, representing 1,588 subjects aged ≥40 years living in urban and rural areas in Qingdao, Shandong Province, was conducted. To calculate the sample size needed, it was decided that the sample should allow for multivariable logistic regression with at least 12 independent variables among dentate subjects. This implies that at least 120 observations of the least prevalent part of a dichotomous variable among dentate subjects are necessary. Using 8 % prevalence as a worst-case scenario, a total sample size of 1,500 is needed to attain the 120 observations needed. To allow for an estimated 5 % prevalence of edentulous subjects, the targeted population was increased to 1,575. Several authors have made attempts to classify dentitions according to the number of teeth, tooth location, or number of occluding pairs. For example, the Eichner index has been validated for an elderly Japanese population with respect to chewing ability [17]. However, the Eichner index does not specify the number and location of teeth present and is therefore not reflecting oral functionality in detail, for example aesthetics. According to a systematic review sufficient oral functional depends on the presence of minimally 20 teeth with nine to 10 occluding pairs, no tooth loss in the anterior region, and the retention of premolars, whereas there may be little increase in satisfaction seen in subjects with presence of molars [18]. Recently, a hierarchical classification system has been de- scribed that reflects oral functionality according to the conclu- sions of this review [19, 20]. In this classification system, oral functionality is expressed by (1) number of teeth in upper and Subjects were selected randomly from administrative lists of residents of communities or villages provided by local authorities and lists of employees of factories. Inclusion aimed at proportional distribution according to age catego- ries, gender, and place of residence (urban or rural). Data were collected in 2009 and 2010. The urban sample was constructed after consulting local authorities on the basis of accessibility, and comprised 11 communities and four factories in Qingdao City. Administra- tors of the communities informed and invited their residents 1427 Clin Oral Invest (2013) 17:1425–1435 Table 1 Number (%) of included subjects (n01,462) dentate in upper and lower jaw according to gender and place of residence, distribution of SES, age (minimum, maximum, and mean), and OHIP-14CN total score (minimum, maximum, and mean) for participation in this study. Questionnaire Subjects were asked to complete a structured questionnaire that was used previously in a study in Vietnam [19] and translated into Mandarin. The Chinese version was checked for language adequacy by a panel of dentists and pilot tested on 20 Chinese subjects to assess clarity. Some minor mod- ifications were made based on the results of the pilot. The questionnaire included the Chinese short version of the Oral Health Impact Profile (OHIP-14CN) [22], demographic in- formation (age, gender, and place of residence), SES (mod- ified Kuppuswami’s SES classification [23]), and questions that asked whether the subject was able to chew eight different foods common for Chinese people. The eight foods were listed randomly in the questionnaire and included four foods that Chinese people consider as soft (cooked rice, steamed bread, Shaobing (Chinese style baked roll), meat) and four that are considered as hard (raw vegetables, raw carrots, apples, and nuts). Perceived difficulty of chewing was scored as: 1 0 very easy to chew; 2 0 minor problems with chewing, got used to it; 3 0 minor problems, cannot get used to it; 4 0 difficult to chew, not avoiding this food; 5 0 very difficult to chew, not avoiding; 6 0 very difficult to chew, avoiding this food; 7 0 not avoiding this food, never eating it. OHIP-14CN was included in the questionnaire to assess OHRQoL. Responses on each OHIP question were given on a 5-point Likert scale (0 0 never, 1 0 hardly ever, 2 0 occasionally, 3 0 fairly often, 4 0 very often) for a reference period of 3 months. The research was carried out in compliance with the Hel- sinki Declaration and was approved by the ethics committee of the medical school at Qingdao University, Qingdao, China. Sampling method The examination venue usually was a neighborhood community office or a social center for the elderly. A total of 570 community inhabitants and 193 employees from factories were included on the basis of vol- untary participation. As truly representative sampling was not feasible the pathfinder sampling method was adopted incor- porating sufficient examination sites to cover relevant groups of the population intended [21]. It appeared that subjects of certain age categories were underrepresented in the initial urban sample (mostly males). Therefore, a complementary convenient sub-sample, drawn from community residents at- tending a health centre while they were waiting for a period- ical check-up, was eventually included. Fifty-three subjects were included in this way. Urban Rural Total Female 405 (58) 297 (42) 702 (48) Male 385 (51) 375 (49) 760 (52) Total 790 (54) 672 (46) 1,462 (100) SES high SES middle SES low SESa 583 449 428 Minimum Maximum Mean (SD) Age 39 87 54.95 OHIP-14CN 0 54 7.76 a SES data of two subjects missing For the rural sample, one county (Zhugou) considered representative for northeast Shandong Province was chosen on the basis of accessibility for investigating dental health status and cooperation from local authorities. This county (a predominantly agrarian area with a low population density and a total population of approximately 36,000) is located approximately 120 km northwest from Qingdao City and comprises 56 villages ranging from 153 to 1,583 inhabi- tants. On the basis of information from the local authorities, it appeared that there were large differences in income among the villages. As gross domestic product (GDP) was expected to be related with socio-economic status (SES), 10 villages with different GDP were selected randomly: three villages out of 19 with highest 2008 GDP; four out of 18 with middle GDP, and three out of 19 with lowest GDP. Next, subjects from these villages were randomly selected using administrative name-lists. In cases where subjects were invited but did not show up (n0347, 45 %), other subjects were randomly drawn from the same sampling lists. Clinical examination Clinical examination separately. In the second approach, chewing ability was related to the hierarchical functional classification system, in which the dental regions are considered in the context of the conditions of the dentition as a whole. After obtaining verbal consent from the participants, a clinical examination was conducted by a calibrated examiner follow- ing the procedures and diagnostic criteria recommended by the World Health Organization [24]. Inter-observer agree- ments between the principal investigator and experienced researchers in the field on DMFT variables were excellent (kappa’s≥0.89). Of all variables recorded, only the presence of teeth (including third molars), tooth type, number and location of natural POPs, and tooth replacements were con- sidered in the present study. Roots were considered non- functional teeth with respect to chewing ability, and therefore considered as missing teeth. A natural POP was considered as a posterior occluding pair of natural teeth. A distinction was made between teeth replaced by FDP and those replaced by RDP. A replaced tooth was defined as a missing tooth replaced by FDP or RDP. Mean numbers of POPs added by FDP or RDP were also considered. In the first approach—in which the relationship between chewing ability and the separate dental regions is analyzed— multivariable logistic regression models were used. In these models ‘chewing problems’ was the dependent variable; the conditions at the levels II to V (≥10 teeth in each jaw, anterior region complete, premolar region sufficient, and molar re- gion sufficient) and tooth replacement were the independent variables. Possible associations between the condition of the separate dental regions and chewing problems were adjusted for a number of background variables. The following back- ground variables were included in the models: OHIP-14CN total score (dichotomized using the sample median as the cut-off point); questionnaire administration (completely self- administered vs. (partly) assisted by a dental assistant); the demographic variables age (age categories 40–49, 50–59, 60–69, and 70 years and older), gender, and place of resi- dence (urban or rural); and socio-economic status (three levels). Dental functional status classification system Dental functional status classification system With respect to chewing ability of the eight separate foods, the answers ‘very easy to chew’, ‘minor problems with chewing, got used to it’, and ‘minor problems, cannot get used to it’ were considered ‘no or minor problems with chewing’; the answers ‘difficult to chew, not avoiding this food’, ‘very difficult to chew, not avoiding’, and ‘very difficult to chew, avoid this food’ were considered ‘chewing problems’. In the analysis of chewing ability of the respec- tive foods, outcomes were dichotomized as follows: ‘no or minor chewing problems’ (scores 1,2, and 3) vs. ‘chewing problems’ (scores 4, 5, and 6). Score 7 (‘not avoiding this food, never eating it’) was considered missing. In the classification system [19], dentitions were classified on the basis of a dichotomized five level branching hierar- chy in which the criteria applied on the levels are based on conditions that reflect functionality (Table 2). With regard to each level in the branching hierarchy, the number of natural teeth, the tooth types present, and the number of natural POPs were calculated. Subjects were classified in two ways. First, subjects were classified on the basis of their configu- ration of natural teeth only (Classnat). Next they were reclas- sified on the basis of configurations including natural teeth plus teeth replaced by FDP (ClassF) and/or RDP (ClassR) [20]. Combined soft and hard foods were analyzed at a differ- ent level in which a more stringent criterion was applied. Cut-off for dichotomization here was defined: ‘no chewing problems’ (score 1 (‘very easy to chew’) for each of the four combined foods) vs. ‘chewing problems’ (score >1 for at least one of the combined foods). Data analyses Participants Of the 1,588 subjects participating in the epidemiological study, 126 subjects (8 %) were edentulous in one or both jaws and were excluded from the present analyses. The remaining 1,462 subjects dentate in upper and lower jaw were included (Table 1). A previous report of this survey revealed that of all dentate subjects, 59 % (n0861) had a natural dentition without any tooth replacement, 30 % had an FDP (n0441), and 11 % (n0160) had an RDP. Forty- three subjects (3 % of the total sample) had both FDP and RDP. The majority of subjects with FDP (57 %) had one or two teeth replaced; the majority of RDPs replaced three or more teeth (78 %) [20]. More details with respect to number of teeth and tooth replacements have been described in that report. Subjects not able to complete the questionnaire them- selves (e.g., because of illiteracy or visual impairment) were helped by an assistant who read aloud the questions and recorded the answers. After completion, the questionnaire was checked for unrecorded items, and if applicable, sub- jects were requested to complete the form. Clin Oral Invest (2013) 17:1425–1435 1428 Data analyses Two approaches were used to analyze chewing ability in relation to dental conditions. First, the relationships were analyzed for the conditions of the different dental regions Table 2 Levels and criteria for dichotomization of the step-by-step branching hierarchy used and percentages of subjects (n01,462) classified in the subsequent categories based on natural teeth only (Classnat) Level Meeting criterion Dichotomy Yes No I Dentition level ≥1 Tooth present in each jaw 100 % ≥1 Tooth vs. no teeth II Jaw level ≥10 Teeth in both upper and lower jaw 82 % <10 Teeth in upper or lower jaw 18 % ≥10 Teeth vs. <10 teeth III Anterior level All 12 anterior teeth present 72 % <12 Anterior teeth 28 % Complete vs. incomplete IV Premolar level 3 or 4 Occluding pairs of premolars 75 % ≤2 Occluding pairs of premolars 25 % ‘Sufficient’ vs. ‘impaired’ V Molar level ≥1 Occluding pairs of molars at both left and right side of the dentition 74 % No occluding pairs of molars at left or right side of the dentition 26 % ‘Sufficient’ vs. ‘impaired’ Table 2 Levels and criteria for dichotomization of the step-by-step branching hierarchy used and percentages of subj he subsequent categories based on natural teeth only (Classnat) dichotomization of the step-by-step branching hierarchy used and percentages of subjects (n01,462) classified in on natural teeth only (Classnat) 1429 Clin Oral Invest (2013) 17:1425–1435 problems ranged from 65.6 (for meat) to 85.8 % (for cooked rice); AUCs ranged from 0.673 to 0.715, showing a reason- ably high level of predictability of the model. The full model, including all 10 variables, predicted 73.8 (again for meat) to 93.5 % (again for cooked rice) of the subjects having chewing problems; AUCs ranged from 0.803 to 0.844. The performance of the multivariable logistic models was expressed as the percentages of subjects having chewing problems predicted by (1) the dental conditions only and (2) all variables. To express the performance of the logistic models, the area under the curve (AUC) statistic is used. An AUC of 0.5 indicates a total absence of model fit; an AUC of 1 belongs to a situation where model fit is perfect. Although the models are etiologic by nature and not meant as a predictive tool, the percentage predicted correctly are presented as an additional indication of the model fit. Data analyses The multivariable logistic regression analysis for com- bined soft and combined hard foods revealed that subjects with the condition ‘sufficient premolar region’ and subjects with the condition ‘sufficient molar region’ had significantly less chance to have chewing problems compared to subjects not meeting the dental conditions (Table 4). The condition ‘anterior regions complete’ showed significantly less chew- ing problems for hard foods. On the basis of the dental conditions only, the percentage of correctly predicted sub- jects having chewing problems was 65.0 % for soft foods and 64.1 % for hard foods (both AUCs 0.670). The full model predicted 73.8 % for soft foods and 74.4 % for hard foods correctly (AUCs 0.805 and 0.808, respectively). In the second approach—in which the relationship between chewing ability and dental functional status is analyzed— likelihood ratios were calculated after dichotomization (meet- ing vs. not meeting the respective dental conditions). These likelihood ratios express the extent to which a given condition, for instance having at least 10 teeth in each jaw, discriminates between people with and without chewing problems with soft (Ls) and hard foods (Lh), respectively. A likelihood ratio of 1 indicates a classification criterion that is not discriminatory. The branching hierarchy shown in Fig. 2 describes 82 % of all subjects dentate in each jaw (n01462) up to level IV (premolar region) and 72 % up to level V (molar region). Categories not meeting the dental conditions in the ‘≥10 teeth in each jaw’ branch and categories meeting the dental conditions in the ‘<10 teeth in each jaw’ branch were not further dichotomized to the next level. At level II of the classification system (‘≥10 teeth in each jaw’) the likelihood ratio for chewing problems with combined soft foods (Ls) was 3.33. The likelihood ratio with hard foods (Lh) at this level was 3.74. In the branch of ‘≥10 teeth in each jaw’, subjects with incomplete anterior region had a higher chance for having chewing problems (level III: Ls01.50; Lh01.59). In this branch likelihood ratios for the premolar regions (level IV) were: Ls02.06 and Lh01.86; for the molar region (level V) they were Ls02.30 and Lh02.20, respectively. In the branch of ‘<10 teeth in each jaw’, likelihood ratios for having chewing problems ranged from 0.99 to 1.06 for the anterior and premolar regions. For the molar region, Ls and Lh were 1.15 and 1.23, respectively. Results Chewing ability and dental conditions based on natural teeth only Data analyses Both approaches were applied to the dental conditions based on (1) natural teeth only (Classnat) and subsequently (2) on levels based on natural teeth plus teeth replaced by FDP (ClassF) and/or RDP (ClassR). R software version 2.13.1 was used for the statistical analyses [25]. Chewing ability and dental conditions based on natural teeth only The majority of subjects (78–91 %) reported no or minor problems with chewing of the foods (Fig. 1). More prob- lems, in terms of frequency as well as severity, were reported for hard foods than for soft foods except for meat. The percentage of subjects reporting ‘easy to chew’ was lowest for meat (53 %) and highest for cooked rice (91 %), whereas ‘very difficult to chew, but not avoiding this food’ was lowest for cooked rice (0.5 %) and highest for meat (7 %). The percentage of subjects avoiding foods because of difficulties with chewing was low (<5 %) except for carrots (12 %) and nuts (10 %). Multivariable logistic analysis in which the variable ‘tooth replacement’ was added in the model based on natu- ral teeth (Classnat) revealed that tooth replacement was clearly not significantly related with the chance for having chewing problems. In all four models (two models for soft foods with FDP and/or RDP and two for hard foods), the p value for this variable was larger than 0.5 (not in Tables). Having ‘sufficient’ molar region compared to not meeting this criterion reduced the chance for having chewing problems significantly for all foods with a factor 0.47 (Table 3: OR for Shaobing, 0.53) to 0.67 (OR for carrots, 0.33). Having ‘suffi- cient’ premolar region reduced the chance for having chewing problems with a factor 0.42 to 0.53 for all foods except for cooked rice and steamed bread. Incomplete anterior regions was only associated with problems about chewing apples (OR00.64). On the basis of dental conditions only, the per- centages of correctly predicted subjects having chewing Chewing ability and dental conditions based on natural teeth plus teeth replaced by FDP or RDP After reclassification to categories based on natural teeth plus tooth replacements (ClassF and/or ClassR) subjects with FDP had more chewing problems with hard foods than their Clin Oral Invest (2013) 17:1425–1435 1430 Fig. 1 Percentage distribution of subjects dentate in each jaw (n01,462) reporting ease or various difficulties with chewing the eight foods investigated Fig. 1 Percentage distribution of subjects dentate in each jaw (n01,462) reporting ease or various difficulties with chewing the eight foods investigated Fig. 1 Percentage distribution of subjects dentate in each jaw (n01,462) reporting ease or various difficulties with chewing the eight foods investigated as well as the ‘sufficient molar region’ were significant for all foods. Bold figures indicate significant relationships. Functional levels based on natural teeth only (Classnat) Chewing ability and dental conditions based on natural teeth only Functional levels based on natural teeth only (Classnat) Table 4 Odds ratios [95 % CI] for having ‘chewing problems’ for the combined soft (cooked rice, steamed bread, Shaobing, and meat) and combined hard (raw vegetables, raw carrots, apples, and nuts) foods according to the dental condition in the multivariable logistic regression model, adjusted for the background variables OHIP-14CN total score, age, gender, place of residence, SES, and questionnaire administration format Conditiona (level) Soft foods Hard foods OR 95 % CI OR 95 % CI ≥10 Teeth in each jaw (II) 1.10 [0.65–1.84] 0.78 [0.46–1.33] Anterior regions complete (III) 0.76 [0.55–1.05] 0.68 [0.50–0.95] Premolar region ‘sufficient’ (IV) 0.54 [0.37–0.78] 0.63 [0.43–0.92] Molar region ‘sufficient’ (V) 0.44 [0.31–0.62] 0.41 [0.29–0.59] AUC dental conditions 0.670 0.670 Percentage correctly predicted by dental conditions only 65.0 64.1 AUC dental conditions plus background variables 0.805 0.808 Percentage correctly predicted by dental conditions plus background variables 73.8 74.4 Bold figures indicate significant relationships. Functional levels based on natural teeth only (Classnat) AUC area under curve a Reference = condition not present Fi 2 Di t ib ti f bj t d t t i h j ( 1 462) d t th i h j III t i i l t IV l i Clin Oral Invest (2013) 17:1425–1435 1431 1431 Clin Oral Invest (2013) 17:1425–1435 Table 4 Odds ratios [95 % CI] for having ‘chewing problems’ for the combined soft (cooked rice, steamed bread, Shaobing, and meat) and combined hard (raw vegetables, raw carrots, apples, and nuts) foods according to the dental condition in the multivariable logistic regression model, adjusted for the background variables OHIP-14CN total score, age, gender, place of residence, SES, and questionnaire administration format Conditiona (level) Soft foods Hard foods OR 95 % CI OR 95 % CI ≥10 Teeth in each jaw (II) 1.10 [0.65–1.84] 0.78 [0.46–1.33] Anterior regions complete (III) 0.76 [0.55–1.05] 0.68 [0.50–0.95] Premolar region ‘sufficient’ (IV) 0.54 [0.37–0.78] 0.63 [0.43–0.92] Molar region ‘sufficient’ (V) 0.44 [0.31–0.62] 0.41 [0.29–0.59] AUC dental conditions 0.670 0.670 Percentage correctly predicted by dental conditions only 65.0 64.1 AUC dental conditions plus background variables 0.805 0.808 Percentage correctly predicted by dental conditions plus background variables 73.8 74.4 Bold figures indicate significant relationships. Functional levels based on natural teeth only (Classnat) AUC area under curve a Reference = condition not present Fig. Chewing ability and dental conditions based on natural teeth only On the basis of the dental conditions only, the percent- age of correctly predicted subjects with chewing problems was between 63.7 and 65.1 %. The full model predicted 73.6–74.5 % of subject with chewing problems correctly. counterparts with natural teeth only: OR01.45 (p00.009; Table 5). Subjects with RDP had more chewing problems than their counterparts with natural teeth only: OR01.96 (p<0.001) for soft foods and OR02.20 (p<0.001) for hard foods. In this model, the condition ‘sufficient premolar region’ variables OHIP-14CN total score, age, gender, place of residence, SES, and questionnaire administration format variables OHIP-14CN total score, age, gender, place of residence, SES, and questionnaire administration format Table 3 Odds ratios [95 % CI] for having ‘chewing problems’ with chewing for the eight foods according to the dental condition in the multivariable logistic regression model, adjusted for the background multivariable logistic regression model, adjusted for the background Conditiona (level) OR [95 % CI] Rice Steamed bread Shaobing Meat Vegetables Carrots Apples Nuts ≥10 Teeth in each jaw (II) 1.17 0.60 0.70 1.14 0.89 0.94 0.89 0.83 [0.50–2.74] [0.35–1.04] [0.42–1.18] [0.68–1.92] [0.53–1.48] [0.55–1.60] [0.53–1.50] [0.49–1.40] Anterior regions complete (III) 0.65 0.87 0.96 0.73 0.79 0.78 0.64 0.67 [0.38–1.13] [0.59–1.27] [0.68–1.36] [0.53–1.00] [0.57–1.09] [0.56–1.08] [0.46–0.89] [0.48–0.93] Premolar region ‘sufficient’ (IV) 0.96 0.66 0.57 0.48 0.58 0.56 0.50 0.47 [0.48–1.92] [0.43–1.02] [0.39–0.84] [0.33–0.70] [0.40–0.85] [0.38–0.83] [0.34–0.73] [0.32–0.69] Molar region ‘sufficient’ (V) 0.46 0.44 0.53 0.48 0.46 0.33 0.46 0.45 [0.26–0.80] [0.29–0.65] [0.37–0.77] [0.34–0.68] [0.33–0.65] [0.24–0.47] [0.32–0.66] [0.32–0.65] AUC dental conditions 0.682 0.715 0.693 0.673 0.676 0.704 0.705 0.698 Percentage correctly predicted by dental conditions only 85.8 79.5 74.0 65.6 69.8 70.6 73.2 67.9 AUC dental conditions plus background variables 0.821 0.844 0.829 0.808 0.803 0.816 0.834 0.830 Percentage correctly predicted by dental conditions plus background variables 93.5 84.1 79.0 73.8 75.9 75.8 78.8 76.3 Bold figures indicate significant relationships. Chewing ability and dental conditions based on natural teeth only Dark columns indicate status of not meeting the criterion 1432 Clin Oral Invest (2013) 17:1425–1435 Table 5 Odds-ratios, p values, and 95 % confidence intervals (CI) of the multivariable logistic regression analysis for having chewing prob- lems with combined soft and combined hard foods with dental status after reclassification to ClassF and ClassR, adjusted for the background variables OHIP-14CN total score, age, gender, place of residence, SES, and questionnaire administration format lems with combined soft and combined hard foods with dental status and questionnaire administration format Conditiona (level) Soft foods Hard foods In classF In classR In classF In classR OR P 95 % CI OR P 95 % CI OR P 95 % CI OR P 95 % CI ≥10 teeth in each jaw (II) 1.22 0.516 0.67–2.23 1.36 0.294 0.77–2.40 1.12 0.713 0.60–2.09 0.84 0.565 0.47–1.51 Anterior regions complete (III) 0.83 0.279 0.58–1.17 0.69 0.028 0.50–0.96 0.65 0.015 0.45–0.92 0.64 0.008 0.46–0.89 Premolar region ‘sufficient’ (IV) 0.60 0.017 0.39–0.91 0.50 <0.001 0.34–0.74 0.58 0.013 0.38–0.89 0.62 0.015 0.42–0.91 Molar region ‘sufficient’ (V) 0.38 <0.001 0.26–0.56 0.41 <0.001 0.29–0.59 0.32 <0.001 0.22–0.48 0.42 <0.001 0.29–0.60 Teeth replaced 1.29 0.071 0.98–1.70 1.96 <0.001 1.31–2.94 1.45 0.009 1.10–1.92 2.20 <0.001 1.45–3.33 AUC dental conditions 0.670 0.672 0.683 0.671 Percentage subjects correctly predicted by dental conditions only 63.7 65.1 64.4 64.3 AUC dental conditions plus background variables 0.804 0.806 0.813 0.806 Percentage subjects correctly predicted by dental conditions plus background variable 73.9 73.6 74.5 74.0 Bold figures indicate significant relationships AUC area under curves a Reference = condition not present instance, the predictor ‘anterior region incomplete’ revealed likelihood ratios for having chewing problems of 1.50 and 1.55, respectively, for soft foods if the subjects had ‘≥10 teeth in each jaw’; when subjects did not meet the condition ‘≥10 teeth in each jaw’ likelihood ratios were 1.06 and 1.36, respectively. In general, likelihood ratios for having chewing problems for subjects classified on the basis of natural teeth plus replaced teeth (ClassF/ClassR) were higher than likelihoods for subjects classified on the basis of natural teeth only (Classnat), except for ‘premolar region impaired‘ and ‘molar region impaired’ for both soft and hard foods (Table 6). Likelihood ratios for having chewing problems were highest at the level of ‘≥10 teeth in each jaw’ (Ls03.33 in Classnat to Lh05.12 in ClassF/ClassR). Chewing ability and dental conditions based on natural teeth only For the subsequent predictors, likelihood ratios for having chewing problems were gener- ally higher if the conditions at proceeding levels were met than if the conditions at preceding levels were not met. For Chewing ability and dental conditions based on natural teeth only 2 Distribution of subjects dentate in each jaw (n01,462) accord- ing to the functional classification system [19] and likelihood ratios for having problems with chewing: I dentate in each jaw, II ≥10 natural teeth in each jaw, III anterior region complete, IV premolar region ‘sufficient’, V molar region ‘sufficient’. Dark columns indicate status of not meeting the criterion Fig. 2 Distribution of subjects dentate in each jaw (n01,462) accord- ing to the functional classification system [19] and likelihood ratios for having problems with chewing: I dentate in each jaw, II ≥10 natural teeth in each jaw, III anterior region complete, IV premolar region ‘sufficient’, V molar region ‘sufficient’. Discussion This study aimed to investigate chewing ability in Chinese adults without and with prosthodontic replacements. As this the cut-off for the next level, based on natural teeth only (Classnat) and on natural teeth plus replaced teeth (ClassF/ClassR) Table 6 Likelihood ratios for having chewing problems with com- bined soft and hard foods according to the condition of meeting/not meeting a functional level in the hierarchical classification system at g y Condition Predictor Soft foods Hard foods ≥10 teeth in each jaw Anterior region complete Premolar region ‘sufficient’ Classnat ClassF/classR Classnat ClassF/classR <10 teeth in each jaw 3.33 (62) 4.14 (23) 3.73 (53) 5.12 (18) Yes Anterior region incomplete 1.50 (100) 1.55 (78) 1.59 (93) 1.68 (71) No Anterior region incomplete 1.06 (15) 1.36 (10) 1.03 (12) 1.75 (8) Yes Yes Premolar region ‘impaired’ 2.06 (47) 1.96 (34) 1.86 (48) 1.83 (34) No No Premolar region ‘impaired’ 0.99 (5) 1.10 (2) 1.05 (6) 1.24 (2) Yes Yes Yes Molar region ‘impaired’ 2.30 (47) 2.74 (34) 2.20 (46) 2.43 (35) No No No Molar region ‘impaired’ 1.15 (9) 1.33 (1) 1.23 (9) 0.87 (0) n smallest number in the four cells in the respective comparisons Clin Oral Invest (2013) 17:1425–1435 1433 study is part of a larger epidemiological survey, sample construction aimed at proportional distribution of subjects according to place of residence, gender, and age categories. With the aid of the local governmental administrative sys- tem this goal was reasonably well met in the rural area; therefore, the rural sample is considered to reflect the rural population in Shandong Province. In the urban area inclu- sion of the intended subjects through administrative lists appeared to be more complicated. To deal with this, a pathfinder sampling method was used to find subjects from randomly chosen communities and factories. Eventually, unfilled cells were filled with community residents attend- ing a health center for periodical check-up. Although the composition of this convenient sub-sample (which is 6 % of the total urban sample) was slightly different from the total urban sample with respect to SES and gender (i.e., males above the age of 70 were not represented in the sub-sample), we consider that the urban sample reflects the population of Qingdao City. only (Classnat) significantly reduced the chance to have chewing problems with all soft and hard foods as compared to an impaired molar region. Discussion The condition of the premolar region appeared to be not relevant for the two softest foods (cooked rice and steamed bread) but affected chewing abil- ity for the other foods. The importance of sufficient POPs in premolar and molar regions for chewing ability has been demonstrated in other studies, both for natural and artificial POPs [3, 16, 33]. Moreover, it has been suggested that impaired molar areas cause more chewing problems than impaired premolar areas [33], which is in accordance with the present result showing the importance of molars when considered as an isolated dental condition. The condition ‘complete anterior region’ did not signifi- cantly affect chewing ability, with the exception of ‘chew- ing’ apples; obviously because anterior teeth are needed for biting off apples. Notably, the condition ‘≥10 teeth in each jaw’ was not associated with chewing problems when con- sidered as an isolated condition. However, the odds ratios in the multivariable regression analyses—in which the condi- tions at the separate levels of the dental functional system sec are the independent variables—only describe the chance for having chewing problems related to that specific (isolated) dental condition. Apparently having at least 10 teeth in each jaw does not affect chewing ability if other dental conditions such as complete anterior region or suffi- cient premolar region are not taken into account. In other words, expressing the dental status only by having more or less than 10 teeth in each jaw did not discriminate with respect to chewing problems. The hierarchical dental functional classification system applied in the present study has been previously evaluated [19, 20]. The homogeneities of the dichotomized groups appeared to be satisfactory; we therefore consider the clas- sification system as appropriate to describe the dental func- tional status of a population. The Chinese diet, unlike western diets, contains few hard fibrous foods and most foods frequently eaten by Chinese people are steamed, fried, or boiled [16]. For this reason we included uncooked foods in the questionnaire, which were assigned to the category ‘hard foods’. Depending on its preparation, meat could be considered soft or hard as reflected in the outcomes of this study. In contrast, when subjects’ dentitions are classified hier- archically within the frame of the classification system and taking into account the other dental conditions, the condi- tion ‘≥10 teeth in each jaw’ was highly correlated with having chewing problems (Fig. References 1. Peek CW, Gilbert GH, Duncan RP (2002) Predictors of chewing difficulty onset among dentate adults: 24-month incidence. J Public Health Dent 62:214–221 2. Gilbert GH, Meng X, Duncan RP, Shelton BJ (2004) Incidence of tooth loss and prosthodontic dental care: effect on chewing diffi- culty onset, a component of oral health-related quality of life. J Am Geriatr Soc 52:880–885 3. Ueno M, Yanagisawa T, Shinada K, Ohara S, Kawaguchi Y (2008) Masticatory ability and functional tooth units in Japanese adults. J Oral Rehabil 35:337–344 4. Morita I, Nakagaki H, Karo K, Murakami T, Tsuboi S, Hayashizaki J, Sheiham A (2007) Relationship between number of natural teeth in older Japanese people and health related functioning. J Oral Rehabil 34:428–432 5. Takata Y, Ansai T, Awano S, Fukuhara M, Sonoki K, Wakisaka M, Fujisawa K, Akifusa S, Takehara T (2006) Chewing ability and quality of life in an 80-year-old population. J Oral Rehabil 33:330–334 of life in an 80-year-old population. J Oral Rehabil 33:330–33 6. Inukai M, John MT, Igarashi Y, Baba K (2010) Association be- tween perceived chewing ability and oral health-related quality of life in partially dentate patients. Health Qual Life Outcomes 8:118 However, also the quality of the tooth replacements, including the indications for FDP and RDP might play a role in this matter. It has been stated before that, especially in rural areas in China, dental care providers (which are not necessarily dentists) prefer to extract affected teeth above treatments that aim at the retention of such teeth. They seem to practice rather unconventional prosthodontic principles, in which they tend to provide FDPs for low prices rather than RDPs, even when only very few teeth are available as abutment teeth [38]. Indeed, FDPs were more often found among rural subjects than among urban subjects, whereas the reverse tendency was seen for RDP [39]. 7. Meng X, Gilbert GH (2007) Predictors of change in satisfaction with chewing ability: a 24-month study of dentate adults. J Oral Rehabil 34:745–758 8. Van der Bilt A, Engelen L, Pereira LJ, Van der Glas HW, Abbink JH (2006) Oral physiology and mastication. Physiol Behav 89:22–27 9. Ueda T, Sakurai K, Sugiyama T (2006) Individual difference in the number of chewing strokes and its determinant factors. J Oral Rehabil 33:85–93 10. Conflict of interest The authors declare that they have no conflict of interest. Conflict of interest The authors declare that they have no conflict of interest. Open Access This article is distributed under the terms of the Crea- tive Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited. However, when comparing chewing ability in denti- tions with natural plus artificial teeth, artificial teeth seem to perform less (more chewing problems) than natural teeth. The hypothesis that subjects with FDP have more benefit from added teeth than subjects with RDP has to be accepted for soft foods only. This finding is in line with findings from a randomized controlled clinical trial, in which patients with shortened dental arches extended with FDP were more satisfied than patients with RDP [36]. Also it was suggested that RDPs add little in avoiding chewing problems [3]. The wide confidence intervals found for the odds ratios (Table 5) are most probably due to the wide variations in numbers of teeth replaced by RPD. In this respect, it is worthwhile to mention that a recent review on chew- ing function reported that an association between the change in objective chewing capacity and self-assessed chewing ability as a result of tooth replacements has not been demonstrated so far [37]. Discussion 2; likelihood ratios ranged from 3.33 to 3.74). If the conditions ‘≥10 teeth in each jaw’ and ‘anterior region complete’ were met, ‘sufficient premo- lar region’ and ‘sufficient molar region’ were relevant to reduce the likelihood ratios for chewing problems (both in the order of a factor 2), both for soft and for hard foods. This reduction of likelihood ratios with approximately a factor 2 might be related with the ‘reduction’ of POPs: from seven to eight POPs when the conditions were met, to four or five when the conditions were not met (see Fig. 2). Although the level of evidence for the shortened dental arch concept has been appraised differently [34, 35], a review indicated that reduced dentitions without molar posterior support could provide sufficient oral function but that complaints on per- ceived oral function may arise, especially for hard foods [34]. The findings of the present study showed that the presence of bilaterally at least one molar POP was relevant for chewing ability, but surprisingly not for hard foods only but also for soft foods. With respect to perceived chewing Although the majority of subjects reported no or minor chewing problems, this study demonstrated a significant as- sociation between dental functional status and chewing diffi- culties with the foods. The present study did not address possible associations between chewing ability and temporo- mandibular disorders (TMD). However, reported difficulties with chewing might include TMD symptoms. It has been suggested that chewing ability is correlated with dysfunction of TMD patients [26]. The present paper is focusing on the relationship between chewing ability and dental (occlusal) conditions. Several studies and reviews reported lack of evi- dence with respect to associations between occlusal factors— such as missing posterior teeth—and TMD problems [27–30]. In contrast, some studies associated higher risk for TMD problems with dentitions with asymmetric occlusal support [31, 32]. Based on these considerations it was expected that TMD patients would be more or less equally distributed among the subjects as categorized by the ‘occlusal’ classifi- cation system and thus not biasing the results. Multivariable logistic regression analyses showed that the condition ‘sufficient molar region’ based on natural teeth Clin Oral Invest (2013) 17:1425–1435 1434 Conclusions ability we expected discriminatory differences between soft and hard foods. However, overall differences in outcomes between soft and hard foods (ORs in Tables 3, 4, 5, and 6 and likelihood ratios in Fig. 2) were relatively small. & A minority of a Chinese adult population in Shandong Province reported serious chewing problems. In the hierarchy branch for the condition ‘<10 teeth in each jaw’, the subsequent dental conditions hardly affected chewing ability. This could be expected as less than 10 teeth in each jaw means a large variation in ‘deficiencies’, as is demonstrated by the large variation (SDs) of POPs in this branch. & The condition ‘≥10 teeth in each jaw’ appeared to have high impact on chewing ability; the conditions ‘suffi- cient premolars region’ and ‘sufficient molar region’ contributed equally to chewing ability. & The condition ‘≥10 teeth in each jaw’ appeared to have high impact on chewing ability; the conditions ‘suffi- cient premolars region’ and ‘sufficient molar region’ contributed equally to chewing ability. & In this population artificial teeth added by FDP and RDP performed less than natural teeth. If missing teeth were replaced by FDP or RDP, subjects had similar chance for chewing problems as their counterparts with similar dental conditions without these replacements. In other words, the natural teeth present predominantly determined chewing ability for the eight foods, and not the artificial teeth added. This indicates that the group of subjects with tooth replace- ment cannot be considered as ‘complainers’ with respect to chewing. References Ono T, Hori K, Ikebe K, Nokubi T, Nago S, Kumakura I (2003) Factors influencing eating ability of old in-patients in a rehabilita- tion hospital in Japan. Gerodontology 20:24–31 11. Peyron MA, Blanc O, Lund JP, Woda A (2004) Influence of age on adaptability of human mastication. J Neurophysiol 92:773–779 12. Shinkawa T, Hayashida N, Mori K, Washio K, Hashiguchi K, Taira Y, Morishita M, Takamura N (2009) Poor chewing ability is associated with lower mucosal moisture in elderly individuals. Tohoku J Exp Med 219:263–267 The present study demonstrated that the hierarchical functional classification system was appropriate to predict subjects with chewing problems to a considerable extent. Clin Oral Invest (2013) 17:1425–1435 1435 13. Kim BI, Jeong SH, Chung KH, Cho YK, Kwon HK, Choi CH (2009) Subjective food intake ability in relation to maximal bite force among Korean adults. J Oral Rehabil 36:168–175 27. Seligman DA, Pullinger AG (2000) Analysis of occlusal variables, dental attrition, and age for distinguishing healthy controls from female patients with intracapsular temporomandibular disorders. J Prosthet Dent 83:76–82 14. Brennan DS, Spencer AJ, Roberts-Thomson KF (2008) Tooth loss, chewing ability and quality of life. Qual Life Res 17:227–235 28. Rinchuse DJ, Rinchuse DJ, Kandasamy S (2005) Evidence-based versus experience-based views on occlusion and TMD. Am J Orthod Dentofacial Orthop 127:249–254 15. Zhang Q, Kreulen CM, Witter DJ, Creugers NHJ (2007) Oral health status and prosthodontic conditions of Chinese adults: a systematic review. Int J Prosthodont 20:567–572 29. Mohlin B, Axelsson S, Paulin G, Pietila T, Bondemark L, Brattström V, Hansen K, Holm AK (2007) TMD in relation to malocclusion and orthodontic treatment. Angle Orthod 77:542–548 16. Zeng X, Sheiham A, Tsakos G (2008) Relationship between clinical dental status and eating difficulty in an old Chinese population. J Oral Rehabil 35:37–44 30. Van ’t Spijker A, Kreulen CM, Creugers NHJ (2007) Attrition, occlusion, (dys)function and intervention: a systematic review. Clin Oral Implant Res 18(Suppl 3):117–126 17. Ikebe K, Matsuda K, Murai S, Maeda Y, Nokubi T (2010) Validation of the Eichner Index in relation to occlusal force and masticatory performance. Int J Prosthodont 23:521–524 31. Wang MQ, Xue F, He JJ, Chen JH, Chen CS, Raustia A (2009) Missing posterior teeth and risk of temporomandibular disorders. J Dent Res 88:942–945 p 18. Gotfredsen K, Walls AWG (2007) What dentition assures oral function? Clin Oral Impl Res 18(suppl3):34–45 32. References Sarita PTN, Kreulen CM, Witter DJ, Creugers NHJ (2003) Signs and symptoms of temporo-mandibular dysfunction in adults with shortened dental arches in Tanzania. Int J Prosthodont 16:265–270 19. Nguyen TC, Witter DJ, Bronkhorst EM, Pham LH, Creugers NHJ (2011) Dental functional status in a Southern Vietnamese adult population—an analysis by a combined quantitative and qualita- tive classification system. Int J Prosthodont 24:30–37 33. Nakatsuka Y, Yamashita S, Nimura H, Mizoue S, Tsuchiya S, Hashii K (2010) Location of main occluding areas and masticatory ability in patients with reduced occlusal support. Aust Dent J 55:45–50 20. Zhang Q, Witter DJ, Bronkhorst EM, Jia M, Creugers NHJ (2012) Dental functional status with and without tooth replacement in a Chinese adult population. Clin Oral Invest 16:1251–1259 34. Kanno T, Carlsson GE (2006) A review of the shortened dental arch concept focusing on the work by the Käyser/Nijmegen group. J Oral Rehabil 33:850–862 21. World Health Organization (1997) Pathfinder sampling. Oral Health Surveys, Basic Methods, 4th edn. WHO, Geneva 35. Faggion CM Jr (2011) The shortened dental revisited: from evi- dence to recommendations by the use of the GRADE approach. J Oral Rehabil 38:940–949 22. Wong MC, Lo EC, McMillan AS (2002) Validation of a Chinese version of the Oral Health Impact Profile (OHIP). Community Dent Oral Epidemiol 30:423–430 36. Jepson N, Allen PF, Moynihan P, Kelly P, Thomasson M (2003) Patient satisfaction following restoration of shortened dental arches in a randomized controlled trial. Int J Prosthodont 16:409–414 23. Kuppuswami’s SES classification. [http://www.scribd.Com/doc/ 18658493/kuppuswamys-SES-Classification?autodown0doc,cited09/ 09/09] 24. World Health Organization (1987) Oral Health Surveys. Basic Methods, 3rd edn. WHO, Geneva 37. Van der Bilt A (2011) Assessment of mastication with implications for oral rehabilitation: a review. J Oral Rehabil 38:754–780 38. 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https://openalex.org/W2952125228	https://europepmc.org/articles/pmc6033539?pdf=render	English	null	Diversification of heart progenitor cells by EGF signaling and differential modulation of ETS protein activity	bioRxiv (Cold Spring Harbor Laboratory)	2,017	cc-by	25,185	Department of Biology, Division of Developmental Biology, Friedrich-Alexander University of Erlangen-Nu¨ rnberg, Erlangen, Germany Abstract For coordinated circulation, vertebrate and invertebrate hearts require stereotyped arrangements of diverse cell populations. This study explores the process of cardiac cell diversification in the Drosophila heart, focusing on the two major cardioblast subpopulations: generic working myocardial cells and inflow valve-forming ostial cardioblasts. By screening a large collection of randomly induced mutants, we identified several genes involved in cardiac patterning. Further analysis revealed an unexpected, specific requirement of EGF signaling for the specification of generic cardioblasts and a subset of pericardial cells. We demonstrate that the Tbx20 ortholog Midline acts as a direct target of the EGFR effector Pointed to repress ostial fates. Furthermore, we identified Edl/Mae, an antagonist of the ETS factor Pointed, as a novel cardiac regulator crucial for ostial cardioblast specification. Combining these findings, we propose a regulatory model in which the balance between activation of Pointed and its inhibition by Edl controls cardioblast subtype-specific gene expression. yp p g p DOI: https://doi.org/10.7554/eLife.32847.001 RESEARCH ARTICLE Introduction For correspondence: ingolf.reim@fau.de Competing interests: The authors declare that no competing interests exist. Funding: See page 30 Received: 16 October 2017 Accepted: 04 June 2018 Published: 05 June 2018 Reviewing editor: Utpal Banerjee, University of California, Los Angeles, United States Copyright Schwarz et al. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited. The heart consists of a variety of cells with distinct molecular and physiological properties in both vertebrates and invertebrates. A complex regulatory network of transcription factors and signaling pathways orchestrates the specification of these different cell populations and their proper arrange- ment within a regionalized working myocardium or other functional structures such as valves, inflow and outflow tracts (reviewed in Greulich et al., 2011; Miquerol and Kelly, 2013; Rana et al., 2013; for the invertebrate Drosophila heart see for example Bodmer and Frasch, 2010; Lehmacher et al., 2012; Lovato and Cripps, 2016; Reim and Frasch, 2010). For example, the ver- tebrate T-box gene Tbx20 promotes working myocardial fate by restricting Tbx2 expression and enabling the expression of chamber myocardium-specific genes (Cai et al., 2005; Singh et al., 2005; Stennard et al., 2005). By contrast, Tbx2 and Tbx3 repress working myocardium-specific gene expression and chamber differentiation in the non-chamber myocardium and thus contribute to the formation of endocardial cushions and structures of the conduction system (Christoffels et al., 2004; Hoogaars et al., 2007; Singh et al., 2012). Normal endocardial cushion formation also requires COUP-TFII, an orphan nuclear receptor transcription factor that regulates cell fate decisions in several tissues (Lin et al., 2012; Wu et al., 2016). In the embryonic mouse myo- cardium, COUP-TFII is restricted to atrial cardiomyocytes, a pattern consistent with a fate determina- tion function that confers atrial over ventricular fate (Lin et al., 2012; Wu et al., 2013). This function appears to involve the up-regulation of Tbx5 (Wu et al., 2013), another T-box gene with non-uni- form cardiac expression and a fundamental role in heart development and human cardiac disease (Basson et al., 1997; Bruneau et al., 1999; Bruneau et al., 2001; Ghosh et al., 2017; Steimle and Reviewing editor: Utpal Banerjee, University of California, Los Angeles, United States Reviewing editor: Utpal Banerjee, University of California, Los Angeles, United States Copyright Schwarz et al. Benjamin Schwarz, Dominik Hollfelder, Katharina Scharf, Leonie Hartmann, Ingolf Reim Benjamin Schwarz, Dominik Hollfelder, Katharina Scharf, Leonie Hartmann, Ingolf Reim* Benjamin Schwarz, Dominik Hollfelder, Katharina Scharf, Leonie Hartmann, Ingolf Reim* Department of Biology, Division of Developmental Biology, Friedrich-Alexander University of Erlangen-Nu¨ rnberg, Erlangen, Germany Department of Biology, Division of Developmental Biology, Friedrich-Alexander University of Erlangen-Nu¨ rnberg, Erlangen, Germany Introduction The balance between Pointed and Edl controls which type of heart cell each cell will become. Many cells in other tissues in fruit flies also produce the Pointed and Edl proteins and respond to EGF signals. This means that this system may help to decide the fate of cells in other organs. The EGF signaling system is also present in other animals, including humans. Future work could reveal whether the same molecular decision making happens in our own hearts. Moskowitz, 2017). Furthermore, FGF-mediated receptor tyrosine kinase (RTK) signaling upstream of the cardiogenic transcription factor Nkx2-5 was recently shown to be required for the mainte- nance of ventricular chamber identity of cardiomyocytes in zebrafish (Pradhan et al., 2017). As emphasized below, spatial restriction of cardiac transcription factors as well as precisely controlled RTK signaling activities are not only important in vertebrate but also invertebrate hearts (Gajewski et al., 2000; Lo and Frasch, 2001; Zaffran et al., 2006; this work). j The Drosophila heart (dorsal vessel) comprises several types of cardiomyocytes (in the embryo called cardioblasts, CBs) and non-contractile pericardial cells (PCs) (Bodmer and Frasch, 2010; Lovato and Cripps, 2016). The progenitors of these cells are specified in segmentally repeated heart fields located at the intersection of BMP/Dpp and Wg/Wnt signaling activities (Frasch, 1995; Reim and Frasch, 2005; Wu et al., 1995). Subsequent specification of the definitive cardiogenic mesoderm depends on a conserved group of transcription factors, most importantly those encoded by the Nkx2-5 ortholog tinman (tin), the Gata4 ortholog pannier (pnr) and the Dorsocross1-3 T-box genes (three Tbx6-related paralogs that also share features with Tbx2/3/5; in the following collec- tively called Doc) (Alvarez et al., 2003; Azpiazu and Frasch, 1993; Bodmer, 1993; Gajewski et al., 1999; Junion et al., 2012; Reim and Frasch, 2005; Reim et al., 2003; reviewed in Reim and Frasch, 2010; Reim et al., 2017). While the identification of cardiogenic factors has greatly improved our understanding of early specification events, much less is known about the mechanisms that lead to the diversification of car- diac cell subpopulations. In this study, we mainly focus on the development of the two major cardio- blast subpopulations: generic cardioblasts (gCBs), which build the main portion of the contractile tube (‘working myocardium’), and ostial cardioblasts (oCBs), which form bi-cellular valves (ostia) for hemolymph inflow. Introduction This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited. 1 of 36 Schwarz et al. eLife 2018;7:e32847. DOI: https://doi.org/10.7554/eLife.32847 Research article Research article Developmental Biology eLife digest Organs contain many different kinds of cells, each specialised to perform a particular role. The fruit fly heart, for example, has two types of muscle cells: generic heart muscle cells and ostial heart muscle cells. The generic cells contract to force blood around the body, whilst the ostial cells form openings that allow blood to enter the heart. Though both types of cells carry the same genetic information, each uses a different combination of active genes to perform their role. eLife digest Organs contain many different kinds of cells, each specialised to perform a particular role. The fruit fly heart, for example, has two types of muscle cells: generic heart muscle cells and ostial heart muscle cells. The generic cells contract to force blood around the body, whilst the ostial cells form openings that allow blood to enter the heart. Though both types of cells carry the same genetic information, each uses a different combination of active genes to perform their role. During development, the cells must decide whether to become generic or ostial. They obtain signals from other cells in and near the developing heart, and respond by turning genes on or off. The response uses proteins called transcription factors, which bind to regulatory portions of specific genes. The sequence of signals and transcription factors that control the fate of developing heart muscle cells was not known. So Schwarz et al. examined the process using a technique called a mutagenesis screen. This involved triggering random genetic mutations and looking for flies with defects in their heart muscle cells. Matching the defects to the mutations revealed genes responsible for heart development. Schwarz et al. found that for cells to develop into generic heart muscle cells, a signal called epidermal growth factor (EGF) switches on a transcription factor called Pointed in the cells. Pointed then turns on another transcription factor that switches off the genes for ostial cells. Conversely, ostial heart muscle cells develop when a protein called ‘ETS-domain lacking’ (Edl) interferes with Pointed, allowing the ostial genes to remain on. Introduction EGFR signaling appears to be dispensable for early mesoderm migration events (Wilson et al., 2005) but has been reported to contribute to the specification of particular cell types within the mesoderm, including subsets of adult muscle precursors (AMPs; Figeac et al., 2010) and the Eve+ DA1 muscles (derived from the so-called P15 progenitors in the dorsal mesoderm; Buff et al., 1998; Carmena et al., 1998). By con- trast, Eve+pericardial cells derived from the P2 progenitor were shown to form independent of EGFR activity. The exact contribution of EGFR signaling to Drosophila heart development has been less clear until now, but it was shown that EGFR loss-of-function results in a severe reduction of the numbers of cardioblasts, pericardial nephrocytes, and blood progenitors (Grigorian et al., 2011). Molecularly, the predominant EGFR ligand in the embryo, Spitz (Spi), relies on the protease Rhomboid (encoded by rho) and the chaperon Star (S) for its conversion from a membrane-bound into its active form (reviewed in Shilo, 2014). In contrast to spi, rho expression is restricted to a lim- ited number of cells in a complex and dynamic pattern, including cells of the cardiogenic area (Bidet et al., 2003; Liu et al., 2006), which points to rho expression being the most decisive factor for Spi-mediated EGFR activation. Among the most important downstream effectors of RTK/Ras/ MAPK pathways are the ETS transcription factors PntP2 (encoded by pointed/pnt) and Yan/Aop (encoded by anterior open/aop). While PntP2 becomes an active transcriptional activator upon phos- phorylation by MAPK, the transcriptional repressor Yan is negatively regulated by MAPK (Gabay et al., 1996; O’Neill et al., 1994). Unlike PntP2, a shorter isoform encoded by pnt, PntP1, is constitutively active but was shown to require activated MAPK for its transcriptional activation at least in some cell types (Brunner et al., 1994; Gabay et al., 1996; Kla¨mbt, 1993; O’Neill et al., 1994). Notably, chordate Pnt orthologs (ETS1/2) were shown to contribute to cardiac progenitor for- mation in the tunicate Ciona and during transdifferentiation of human dermal fibroblasts into cardiac progenitors (Davidson et al., 2006; Islas et al., 2012). During early Drosophila cardiogenesis, Pnt favors expression of eve over that of another homeobox gene, ladybird (lbe, expressed in meso- dermal cells immediately anterior of the Eve+ cluster and later in TPCs and two of the four gCBs per hemisegment; Jagla et al., 1997) (Liu et al., 2006). Introduction Due to Hox gene inputs, ostial progenitor specification is limited to the abdomi- nal region (Lo et al., 2002; Lovato et al., 2002; Ponzielli et al., 2002; Ryan et al., 2005; reviewed in Monier et al., 2007). Current research suggests that each abdominal hemisegment generates at Schwarz et al. eLife 2018;7:e32847. DOI: https://doi.org/10.7554/eLife.32847 2 of 36 Research article Research article Developmental Biology Developmental Biology least seven distinct progenitors that give rise to six CBs (4 gCBs + 2 oCBs) and several types of PCs (Tin+/Even-skipped[Eve]+ EPCs, Tin+ TPCs, and Odd-skipped[Odd]+ OPCs; Bodmer and Frasch, 2010 and references therein). Whereas gCBs (a.k.a. Tin-CBs) maintain expression of tin, oCBs (a.k.a. Svp-CBs) specifically express the COUP-TFII ortholog seven-up (svp) and Doc (Gajewski et al., 2000; Lo and Frasch, 2001; Ward and Skeath, 2000; Zaffran et al., 2006). Previous work has shown that Doc is repressed in gCBs in a tin-dependent manner (Zaffran et al., 2006). Robust tin expression in turn depends on the Tbx20 ortholog midline (mid/nmr2). The mid gene is first acti- vated in gCB progenitors, but later, like its paralog H15/nmr1, becomes expressed in all cardioblasts (Miskolczi-McCallum et al., 2005; Qian et al., 2005; Reim et al., 2005). In oCBs, svp represses tin expression thereby permitting continued Doc expression in these cells (Gajewski et al., 2000; Lo and Frasch, 2001; Zaffran et al., 2006). In the abdomen, gCBs and most PCs are preceded by a precursor that undergoes symmetric division, whereas oCBs and half of the OPCs are derived from common, asymmetrically dividing CB/PC progenitors (Alvarez et al., 2003; Han and Bodmer, 2003; Ward and Skeath, 2000). The process of progenitor specification in the somatic and cardiogenic mesoderm involves the antagonistic actions of RTK/Ras/MAPK and Delta/Notch signaling (Carmena et al., 2002; Grigorian et al., 2011; Hartenstein et al., 1992). Two types of RTKs, the fibroblast growth factor (FGF) receptor Heartless (Htl) and the epidermal growth factor (EGF) receptor EGFR, act positively on progenitor selection via MAPK signaling, although they are used by different progenitors to dif- ferent extents (Buff et al., 1998; Carmena et al., 2002; Michelson et al., 1998). Htl and its FGF8- like ligands Pyramus (Pyr) and Thisbe (Ths) have a dual function as regulators of mesodermal cell migration and cell specification, with progenitors of the Eve+ lineage as the most prominent exam- ple for the latter (reviewed in Bae et al., 2012; Muha and Mu¨ller, 2013). Novel EMS-induced mutants reveal a specific requirement of EGF signaling for the specification of generic cardioblasts g g p g In order to identify genes involved in heart and muscle development in an unbiased manner, we have performed an EMS mutagenesis screen for chromosome two in Drosophila melanogaster embryos (Hollfelder et al., 2014). Several of the isolated mutants display a partial loss or irregular alignment of cardioblasts (CBs). Such defects may potentially result from mutations in genes that regulate the specification or differentiation of all CBs or only a particular CB subtype. In the latter case, disturbances in the characteristic ‘2 + 4’ CB pattern of two ostial cardioblast (oCBs; Doc+/Tin-) and four generic CBs (gCBs; Doc-/Tin+) per hemisegment are to be expected. To analyze the cardiac pattern of mutants in more detail, we performed immunofluorescent double stainings for Doc and H15 (or alternatively Mef2) to label oCBs and all CBs, respectively. We then genetically and in part also molecularly mapped the mutations responsible for CB pattern anomalies (for details see the Materials and methods section and Supplementary file 1-Table S1). The class of mutants character- ized by a loss of CBs contained several novel alleles of genes involved in RTK/Ras/MAPK signaling, which is consistent with the assumed role of this pathway in cardiac progenitor selection or mainte- nance (Carmena et al., 2002; Grigorian et al., 2011). However, no specific role for the specification of a particular cardioblast subtype or diversification of gCB versus oCB progenitors had been previ- ously attributed to RTK/Ras/MAPK signaling. Our phenotypic analysis now shows that diminished EGF/EGFR but not FGF/Htl signaling leads to a preferential reduction of gCB numbers. Embryos with partially reduced FGF/Htl signaling, that is mutants lacking both copies of the FGF-encoding gene pyr and one copy of its paralog ths, as well as hypomorphic htl mutants, show an about equal reduction of gCB and oCB numbers (Figure 1B, for quantification see Figure 1M; additional exam- ples in Figure 1—figure supplement 1B,C). This CB reduction can be explained by uneven spread- ing of the early mesoderm to Dpp-receiving areas. By contrast, several mutations mapped to EGF signaling components feature a preferential loss of gCBs. In strong Egfr mutants very few CBs can be found (Figure 1C, Figure 1—figure supplement 1E). Remarkably, the overwhelming majority of the residual CBs express Doc. The few remaining Doc-negative CBs are usually located toward the anterior and thus are possibly remnants of the oCB-free anterior aorta. Introduction In addition, Pnt promotes pericardial cell devel- opment and antagonizes CB fate, especially that of oCBs (Alvarez et al., 2003). Despite the progress in the understanding of cardiac progenitor specification, the mechanisms that diversify progenitors of the oCB and gCB lineages have remained elusive. We have performed an unbiased large-scale mutagenesis screen to identify genes that regulate cardiac development in Drosophila embryos and found several mutants that show CB subtype-specific defects. On this basis, we discovered a novel and rather unexpected function of the EGF pathway in specifying the gCBs of the working myocardium, thus revealing an intimate link between cardioblast specification and Schwarz et al. eLife 2018;7:e32847. DOI: https://doi.org/10.7554/eLife.32847 3 of 36 diversification. Furthermore, we identified ETS domain lacking (Edl a.k.a. Modulator of the activity of ETS, Mae) as a crucial regulator of the specification of inflow valve-forming oCBs. Edl possesses a SAM domain, which mediates binding to the SAM domain-containing ETS factors PntP2 and Yan, thereby inhibiting their activity as a transcriptional activator or repressor, respectively (Baker et al., 2001; Qiao et al., 2006; Qiao et al., 2004; Tootle et al., 2003; Vivekanand et al., 2004; Yamada et al., 2003). Our data imply that Edl enables svp expression and thus oCB fate by limiting the activity of PntP2, thereby blocking subsequent activation of important downstream targets such as pntP1 and mid. Collectively, our data provide the basis for an elaborated model of cardiac cell fate diversification that links MAPK signaling, Pnt activity and the cell-type-specific expression pat- terns of key cardiac transcription factors. Developmental Biology Research article Research article Developmental Biology Developmental Biology diversification. Furthermore, we identified ETS domain lacking (Edl a.k.a. Modulator of the activity of ETS, Mae) as a crucial regulator of the specification of inflow valve-forming oCBs. Edl possesses a SAM domain, which mediates binding to the SAM domain-containing ETS factors PntP2 and Yan, thereby inhibiting their activity as a transcriptional activator or repressor, respectively (Baker et al., 2001; Qiao et al., 2006; Qiao et al., 2004; Tootle et al., 2003; Vivekanand et al., 2004; Yamada et al., 2003). Our data imply that Edl enables svp expression and thus oCB fate by limiting the activity of PntP2, thereby blocking subsequent activation of important downstream targets such as pntP1 and mid. Introduction Collectively, our data provide the basis for an elaborated model of cardiac cell fate diversification that links MAPK signaling, Pnt activity and the cell-type-specific expression pat- terns of key cardiac transcription factors. Novel EMS-induced mutants reveal a specific requirement of EGF signaling for the specification of generic cardioblasts In spitz, rhomboid and Star loss-of-function mutants, the number of Doc-/Tin+ CBs is strongly reduced while that of ostial Doc+/ Tin- CBs is nearly normal or in some cases even increased by a few cells (Figure 1D–G,M, Figure 1— figure supplement 1F, Figure 1—figure supplement 2A–C). In the wild type, the two pairs of sib- ling gCBs within each hemisegment can be further categorized as Lbe+ (anterior pair) or Lbe- (poste- rior pair) subtypes. Since the above-mentioned spitz group mutants often feature a single pair of gCBs in each abdominal hemisegment, we tested whether these cells are preferentially Lbe+ or Lbe-, which would indicate that one of the two gCB progenitor types may be more sensitive to impaired EGF signaling. However, our finding that both types are about equally represented in rho mutants (Figure 1—figure supplement 3) argues against this assumption. Moreover, segment-by-segment analysis in homozygous rhoL68 mutants reveals that residual gCBs most frequently occur either as Lbe+ or Lbe- pairs, whereas none of the analyzed residual gCB duplets consisted of a combination of both gCB types. This suggests that EGF function is required for the formation of gCB progenitors prior to their final division. Notably, progenitors of the oCB lineage apparently do not require activ- ity of the ostial marker gene svp to develop and survive independently of EGF, since total CB Schwarz et al. eLife 2018;7:e32847. DOI: https://doi.org/10.7554/eLife.32847 4 of 36 Research article igure 1. Genetic manipulation of EGF but not FGF signaling leads to cardioblast subtype-specific heart defects. Immunostaining for the cardioblast marker H15 (red) and the ostial cardioblast marker Dorsocross (anti-Doc2+3, green). (HG: hindgut with artificial staining in the lumen). All figures depict orsal views of stage 16 embryos with anterior to the left unless noted otherwise. (A) Wild type (WT) CB pattern with regular alternation of gCBs (red) nd oCBs (yellow) in the posterior aorta and the heart proper. The anterior aorta consists entirely of Doc- CBs. (B) Mutant with reduced FGF activity pyrS3547 over a deficiency, Df(2R)BSC25, that removes pyr and ths) showing a reduction of both CB types. (C) Homozygous EgfrS2561 mutant with a evere loss of CBs. Almost all remaining CBs are Doc+. Research article Developmental Biology Developmental Biology Research article are Doc+). (H) In S B0453 svpAE127 double mutants, total CB numbers are similar to that of S single mutants, even though all CBs are Doc-negative. (I) If the apoptosis inhibitor p35 is artificially expressed in the mesoderm of S mutants a mild increase in the number of CBs can be observed. Compared to the wild type, more Doc+ CBs are present. (J) Pan-mesodermal overexpression of dominant-negative Egfr results in a phenotype similar to spitz group mutants. Expression of rho in the entire mesoderm via how24B-GAL4 (K) or at later time in dorsal mesoderm cells via tinD +tinCD4-GAL4 (L) generates supernumerary gCBs. By contrast, oCB specification is either reduced (K) or unaffected (L) in these backgrounds. (M) Quantification of Doc+ oCBs (green), Doc- gCBs (red) and total cardioblasts (grey). The column bar chart depicts average numbers with standard deviation error bars. Asterisks indicate significant differences compared to the y w control (WT) assessed by Student’s t-test (two-tailed, type 3; =p < 0.05, =p < 0.001; n.s. = not significant). Brackets indicate comparisons between other genotypes. Pie charts display the corresponding average fraction of oCBs and gCBs. DOI: https://doi.org/10.7554/eLife.32847.003 The following source data and figure supplements are available for figure 1: Source data 1. Quantification of Doc+oCBs, Doc- gCBs and total cardioblasts. Source data 1. Quantification of Doc+oCBs, Doc- gCBs and total cardioblasts. DOI: https://doi org/10 7554/eLife 32847 008 Figure supplement 1. Cardiac patterning phenotypes in additional alleles of FGF and EGF pathway mutants. DOI: https://doi.org/10.7554/eLife.32847.004 g nt 2. Extended analysis of cardiac patterning confirming the loss of Tin+cardiac cells in EGF pathway mutants. rg/10.7554/eLife.32847.005 Figure supplement 2. Extended analysis of cardiac patterning confirming the loss of Tin+cardiac cells in EGF p DOI: https://doi.org/10.7554/eLife.32847.005 DOI: https://doi.org/10.7554/eLife.32847.005 Figure supplement 3. Lbe+ and Lbe- subtypes of generic cardioblasts are equally affected in EGF signaling mutants. DOI: https://doi org/10 7554/eLife 32847 006 p g ure supplement 3. Lbe+ and Lbe- subtypes of generic cardioblasts are equally affected in EGF signaling mutant OI: https://doi.org/10.7554/eLife.32847.006 Figure supplement 4. Analysis of apoptosis in Star mutants. numbers are similar in Star single and Star svp double mutants (compare Figure 1H–1G; quantifica- tion in Figure 1M). numbers are similar in Star single and Star svp double mutants (compare Figure 1H–1G; quantifica- tion in Figure 1M). Research article Previous studies in EGF pathway mutants suggested that incorrectly specified mesodermal pro- genitors undergo apoptosis (Buff et al., 1998; Grigorian et al., 2011). Using TUNEL and anti-acti- vated caspase stainings, we could not reliably detect signs of apoptosis in the Tin- or Doc-labeled cardiogenic mesoderm of Star mutants, while numerous signals were observed in other tissues (Fig- ure 1—figure supplement 4 and data not shown). Nevertheless, we obtained indirect evidence for the occurrence of at least some apoptosis by using the baculoviral apoptosis inhibitor p35 (Zhou et al., 1997). If p35 is artificially expressed in the mesoderm of S mutants the number of CBs slightly increases in comparison to S mutants without p35 (Figure 1I,M). Although this is consistent with a pro-survival function of EGF signaling, it does not fully account for the gCBs missing in S mutants. Of note, we detect a small, but statistically significant increase in the average number of Doc+ CBs in comparison to the wild type in spi mutants, in p35-expressing S mutants as well as in embryos overexpressing dominant-negative EGFR (Figure 1M), which suggests that at least some presumptive gCB progenitors adopt oCB-like fates at reduced EGFR activity. However, the observed effects are small and additional explanations such as persistence in an uncommitted dorsal meso- derm cell pool must be considered to fully explain the fate of all lost gCB progenitors (see discus- sion). Collectively, the cardiac patterning phenotypes imply that EGF signaling plays a major role in the correct specification of gCB progenitors, although we cannot exclude an additional function in cardiac cell survival that might be difficult to detect by the applied methods. Novel EMS-induced mutants reveal a specific requirement of EGF signaling for the specification of generic cardioblasts Predominant reduction of gCBs is also observed in the EGF pathway-impairing spitz group mutants spiS3384 (D), rho7M43/rhoL68 (E), SS4550 (F) and SB0453 (G, showing an extreme case in which all retained CBs except for those of the anterior aorta igure 1 continued on next page Research article Developmental Biology Developmental Biology Figure 1. Genetic manipulation of EGF but not FGF signaling leads to cardioblast subtype-specific heart defects. Immunostaining for the cardioblast marker H15 (red) and the ostial cardioblast marker Dorsocross (anti-Doc2+3, green). (HG: hindgut with artificial staining in the lumen). All figures depict dorsal views of stage 16 embryos with anterior to the left unless noted otherwise. (A) Wild type (WT) CB pattern with regular alternation of gCBs (red) and oCBs (yellow) in the posterior aorta and the heart proper. The anterior aorta consists entirely of Doc- CBs. (B) Mutant with reduced FGF activity (pyrS3547 over a deficiency, Df(2R)BSC25, that removes pyr and ths) showing a reduction of both CB types. (C) Homozygous EgfrS2561 mutant with a severe loss of CBs. Almost all remaining CBs are Doc+. Predominant reduction of gCBs is also observed in the EGF pathway-impairing spitz group mutants spiS3384 (D), rho7M43/rhoL68 (E), SS4550 (F) and SB0453 (G, showing an extreme case in which all retained CBs except for those of the anterior aorta Figure 1 continued on next page Schwarz et al. eLife 2018;7:e32847. DOI: https://doi.org/10.7554/eLife.32847 5 of 36 Research article R Generic CBs and a subset of Odd+pericardial cells require spatially and temporally coordinated EGF signals 2G,I; later activity in cardiac cells appears to be less affected; Figure 2L cf. 2K). Similar observations were made for embryos with pan-mesodermal overexpression of the dominant-negative EGFR (data not shown). Altogether, this demonstrates that EGF signaling serves as the major positive input for MAPK activation during early gCB progenitor formation, whereas input from FGFs may gain importance in developing CBs at later stages for CB fate maintenance as was proposed previously (Grigorian et al., 2011). Since half of the odd-expressing pericardial cells (OPCs) are siblings of oCBs, we also analyzed PCs in EGF-related mutants by Odd/Eve as well as Odd/Zfh1 double-stainings (Figure 3A–C,E; Fig- ure 3—figure supplement 1A–D and data not shown). Consistent with the results of previous stud- ies on Eve+ progenitor derivatives (Buff et al., 1998; Carmena et al., 2002; Su et al., 1999), we detected EPCs in almost normal numbers in spi group mutants and in embryos with pan-meso- dermal dominant-negative EGFR, whereas spi-dependent Eve+ DA1 muscles were largely absent (Figure 3B,C,E). OPCs are strongly reduced in these loss-of-function backgrounds. Our quantifica- tion revealed that about half of the OPCs were lost in rho7M43/L68 and other EGF pathway mutants (Figure 3B,C,E). A converse phenotype with many extra OPCs as well as Tin+ PCs (TPCs, excluding the unaffected EPCs) is generated by rho overexpression with tinD +tinCD4-GAL4 (Figure 3D,E; Fig- ure 3—figure supplement 1F). Notably, the number of oCB-sibling OPCs (as identified by svp-lacZ reporter analysis) is not significantly reduced in Star mutants if compared to the wild type (Figure 3F,G), thus implying that the EGF signaling-dependent OPCs are those derived from sym- metrically dividing OPC progenitors. In sum, these data demonstrate that EGF pathway activity is required in the mesoderm specifi- cally for the specification of the symmetrically dividing gCB and OPCs progenitors (and probably also for those of the TPCs, which we did not quantify in detail) but is largely dispensable or even det- rimental for the specification of the svp-expressing oCB/OPC progenitors. Generic CBs and a subset of Odd+pericardial cells require spatially and temporally coordinated EGF signals Because EGF signaling is involved in multiple processes during embryogenesis we next asked whether its impact on gCB specification is directly linked to signaling activity within mesoderm cells. Indeed, mesoderm-specific attenuation of the pathway by expression of a dominant-negative EGFR variant resulted in essentially the same phenotype as with the spitz group mutants (Figure 1J,M). Activation of the EGF pathway in mesoderm cells appears to be largely controlled by the spatially restricted expression of rho (Bidet et al., 2003; Grigorian et al., 2011; Halfon et al., 2000). Over- expression of rho with the pan-mesodermal how24B-GAL4 driver has been previously reported to affect the number of tin-expressing pericardial cells (Bidet et al., 2003), but CBs and their subtypes were not unambiguously labeled in these experiments. We extended these experiments using also other drivers. Consistent with a mesoderm-autonomous function, overexpression of rho in the dorsal Schwarz et al. eLife 2018;7:e32847. DOI: https://doi.org/10.7554/eLife.32847 6 of 36 Research article Research article Developmental Biology Developmental Biology ectoderm (via pnrMD237-GAL4) has no significant effect on CB number or pattern (Figure 1M and data not shown). By contrast, all mesodermal rho overexpression setups increase the gCBs:oCBs ratio in comparison to the wild type (Figure 1K–M and data not shown). The impact on the absolute CB numbers depends on the timing and strength of transgene expression. The later rho is activated in mesodermal cells (with following drivers according to their temporal order and progressive spatial restriction: twist-GAL4, how24B-GAL4 and tinD +tinCD4-GAL4) the larger the total number of CBs (Figure 1K–M and data not shown). This implies that rho activity needs to be tightly regulated, spa- tially as well as temporally. In the wild-type mesoderm, rho expression is first seen in the Eve+ pro- genitor P2 (Buff et al., 1998; Carmena et al., 1995; Halfon et al., 2000) followed by expression in the adjacent CB progenitor-containing clusters C14 and C16 (Bidet et al., 2003; Grigorian et al., 2011; see also Figure 2A–D). Of note, stage 11 rho expression is still robustly observed in all C14/ C16 clusters in S mutants (Figure 2E cf. 2A), showing that earlier patterning events are not disrupted in this situation. Later during stage 12, when rho RNA is normally found in developing CBs along the dorsal mesoderm margin, a reduction of rho expressing cells is apparent in S mutants (Figure 2F cf. 2C), which is consistent with defects in CB progenitor formation. Generic CBs and a subset of Odd+pericardial cells require spatially and temporally coordinated EGF signals Importantly, detection of active diphospho-MAPK is severely reduced in cardiac cells of S mutants already in the cardiogenic clusters at stage 11 as well as during 12 in which dpMAPK is normally detected in both ostial and generic CB progenitors (Figure 2H,J cf. 2G,I; later activity in cardiac cells appears to be less affected; Figure 2L cf. 2K). Similar observations were made for embryos with pan-mesodermal overexpression of the dominant-negative EGFR (data not shown). Altogether, this demonstrates that EGF signaling serves as the major positive input for MAPK activation during early gCB progenitor formation, whereas input from FGFs may gain importance in developing CBs at later stages for CB fate maintenance as was proposed previously (Grigorian et al., 2011). ectoderm (via pnrMD237-GAL4) has no significant effect on CB number or pattern (Figure 1M and data not shown). By contrast, all mesodermal rho overexpression setups increase the gCBs:oCBs ratio in comparison to the wild type (Figure 1K–M and data not shown). The impact on the absolute CB numbers depends on the timing and strength of transgene expression. The later rho is activated in mesodermal cells (with following drivers according to their temporal order and progressive spatial restriction: twist-GAL4, how24B-GAL4 and tinD +tinCD4-GAL4) the larger the total number of CBs (Figure 1K–M and data not shown). This implies that rho activity needs to be tightly regulated, spa- tially as well as temporally. In the wild-type mesoderm, rho expression is first seen in the Eve+ pro- genitor P2 (Buff et al., 1998; Carmena et al., 1995; Halfon et al., 2000) followed by expression in the adjacent CB progenitor-containing clusters C14 and C16 (Bidet et al., 2003; Grigorian et al., 2011; see also Figure 2A–D). Of note, stage 11 rho expression is still robustly observed in all C14/ C16 clusters in S mutants (Figure 2E cf. 2A), showing that earlier patterning events are not disrupted in this situation. Later during stage 12, when rho RNA is normally found in developing CBs along the dorsal mesoderm margin, a reduction of rho expressing cells is apparent in S mutants (Figure 2F cf. 2C), which is consistent with defects in CB progenitor formation. Importantly, detection of active diphospho-MAPK is severely reduced in cardiac cells of S mutants already in the cardiogenic clusters at stage 11 as well as during 12 in which dpMAPK is normally detected in both ostial and generic CB progenitors (Figure 2H,J cf. The SAM domain protein Edl promotes specification of ostial cardioblasts by blocking Pointed activity Our EMS screen also yielded mutants in which the number of ostial cardioblasts was specifically reduced. One such complementation group consisting of three alleles was mapped to the numb gene (alleles listed in Supplementary file 1-Table S1), which is consistent with its well-known func- tion as a Notch suppressor during asymmetric cell division in the oCB lineage (Gajewski et al., 2000; Ward and Skeath, 2000). Preferential reduction of oCBs was also observed in the mutant line S0520. We found that its cardiac phenotype was caused by loss of the gene ETS domain lacking (edl) as part of a multi-gene deletion and named this mutant Df(2R)edl-S0520 (Figure 4A, Supplementary file 2-Table S2). We identified edl as the gene responsible for the oCB losses by obtaining phenocopies with other edl mutants (Figure 4A–D and data not shown). The lacZ enhancer trap insertion allele edlk06602 was used in most edl loss-of-function experiments since its cardiac phenotype is indistinguishable from that of Df(2R)edl-S0520 and Df(2R)edl-L19 (Figure 4C,D and data not shown), and we detected in this strain a small deletion that specifically destroys the edl Schwarz et al. eLife 2018;7:e32847. DOI: https://doi.org/10.7554/eLife.32847 7 of 36 Research article Developmental Biology Figure 2. Expression of rho and MAPK activity in cardiac cells. (A–F) Detection of rho mRNA (green), Mef2 (blue) and Doc (red). (A) At stage 1 detectable in clusters C14/C16 of the cardiac mesoderm (arrowheads) and is fading from the central Doc-negative region containing EPC and muscle progenitors (empty arrowhead). Dashes separate units derived from adjacent mesoderm segments. (B) At late stage 11, rho is expresse evels in at least one cardiac progenitor per cluster close to the dorsal mesoderm segment borders. (C, D) As cardioblasts align near the dorsa Figure 2 continued on next page Figure 2. Expression of rho and MAPK activity in cardiac cells. (A–F) Detection of rho mRNA (green), Mef2 (blue) and Doc (red). (A) At stage 11, rho is detectable in clusters C14/C16 of the cardiac mesoderm (arrowheads) and is fading from the central Doc-negative region containing EPC and somatic muscle progenitors (empty arrowhead). Dashes separate units derived from adjacent mesoderm segments. (B) At late stage 11, rho is expressed at high levels in at least one cardiac progenitor per cluster close to the dorsal mesoderm segment borders. (C, D) As cardioblasts align near the dorsal Figure 2 continued on next page 8 of 36 Schwarz et al. eLife 2018;7:e32847. Figure 2 continued However, Edl is not a direct activator of Doc expression because Doc is found in CBs of edl double mutants with CB-specific ablation of tin (Figure 4I), a phenotype reminiscent of that of CB-specific tin single mutants (Figure 4H; Zaffran et al., 2006). This suggests that edl normally contributes to the activation of Doc in oCBs via suppression of tin. This role of edl in CB patterning is further supported by the observation of some CBs with low levels of both Tin and Doc in edl mutants (Figure 4K; compare to the strictly complementary distribution of Doc and Tin in the wild type, Figure 4J). Next, we analyzed Edl function by ectopic expression. Consistent with a mesoderm-autonomous function, overexpressing edl in the dorsal ectoderm via pnrMD237-GAL4 has no significant effect on cardiogenesis (data not shown). By contrast, overexpression of edl in the entire mesoderm via twist- GAL4 results in an increase of CB numbers (Figure 5A) and a decrease of OPCs (described in the next subsection). The increase in Doc+ CBs is disproportionately high. The extra Doc+ CBs in the heart proper also activate ostial cell differentiation markers such as wg (data not shown). In agree- ment with the proposed function of Edl as a negative regulator of PntP2 (Yamada et al., 2003), our overexpression phenotypes of edl are very reminiscent to that of pntP2-specific mutants (pntRR112 reported in Alvarez et al. (2003); and pntMI03880 shown in Figure 5B) and amorphic pnt mutants (pntD88, pnt2; see Figure 5E,I and Alvarez et al., 2003). Accordingly, overexpression of constitu- tively active PntP2VP16 (Figure 5C) or PntP1 (not shown) via tinD +tinCD4-GAL4 causes a phenotype similar to that of edl loss-of-function mutants (Figure 4C,D). By contrast, analogous overexpression of the potential Edl target Yan/Aop leads to a loss of heart cells irrespective of their subtype (Figure 5D). These losses may result from a more general block in cell specification and differentia- tion since Yan has been related to such functions in several other types of MAPK-dependent progen- itors (Bidet et al., 2003; Caviglia and Luschnig, 2013; Halfon et al., 2000; Rebay and Rubin, 1995). If the predominant function of Edl during CB specification is the inhibition of Pnt, edl pnt double mutants should mimic pnt mutants. In principle, this is what we observed (Figure 5E,F; quan- tifications in Figure 5I). Figure 2 continued mesoderm margin during stage 12, rho continues to be expressed in most CBs. (E,F) Detection of rho RNA in SB0453 mutants showing normal rho expression in cardiogenic clusters at stage 11 (E, compare to A) and reduced cardiac expression at stage 12 (F, compare to C). (G–L) Detection of activated MAPK in the cardiogenic region of wild type (G,I,K) and SB0453 mutant (H,J,L) embryos in immunostainings against diphospho-MAPK (dpMAPK, green), Doc (red) and either Mef2 or Mid (blue) as indicated in each panel. (G) dpMAPK is detectable in the Doc+ cardiogenic clusters (arrowheads) of a stage 11 wild-type embryo. (H) This dpMAPK activity is severely reduced in Star mutants. (I) At stage 12, dpMAPK activity is observed in the Mid-expressing gCB progenitors (arrows) and in the Mid-negative oCBs and their sibling PCs (asterisks). (J) By contrast, both Mid and dpMAPK are severely reduced in stage 12 Star mutants. (K) Early stage 13 embryo after germ band retraction but prior to completion of the final mitotic division of the Mid+ gCB progenitors. dpMAPK is still active in all cardiac cells (oCBs and gCBs labeled as in I). (L) In contrast to earlier stages, dpMAPK staining is prominently observed in both oCBs (asterisks) and the few formed Mid+Doc- gCB progenitors (arrow) of Star mutants at the onset of stage 13. DOI: https://doi.org/10.7554/eLife.32847.009 gene (Figure 4A, Supplementary file 2-Table S2). Furthermore, we were able to rescue the cardiac phenotype of edl by introducing a genomic edl transgene (Yamada et al., 2003; Figure 4E). Pheno- typic rescue was also achieved, albeit with lesser efficiency, by artificially expressing edl in the dorsal mesoderm cells or in cardioblasts using the drivers tinD-GAL4 and tinCD4-GAL4, respectively (Figure 4F,G), demonstrating that Edl is required directly within these cell types. In accordance, edl mRNA is found within the cardiogenic region during stages 10 to 12 (Figure 4—figure supplement 1A–C; Figure 4—figure supplement 2A–D), including prominent expression in early svp-expressing oCB progenitors (Figure 4—figure supplement 2E). Thereafter edl expression shifts to the pericar- dial region, where it persists until stage 15 (Figure 4—figure supplement 1D and data not shown). A distinctive feature of edl mutants is that the normal ‘2 + 4’ pattern of 2 Doc+ CBs + 4 Doc- CBs is often transformed into a ‘1 + 5’ pattern (e.g. bracket in Figure 4D), indicating a fate switch from ostial to generic CBs. Research article Developmental Biology Developmental Biology The SAM domain protein Edl promotes specification of ostial cardioblasts by blocking Pointed activity DOI: https://doi.org/10.7554/eLife.32847 Schwarz et al. eLife 2018;7:e32847. DOI: https://doi.org/10.7554/eLife.32847 Research article Research article R Figure 2 continued By contrast, edl aop double mutants show an additive combination of aop and edl single mutant phenotypes (compare Figure 5H with 5G and 4D; see also quantifications in Figure 5I). Amorphic aop mutants display a reduction in CB number irrespective of CB subtype, which we ascribe to a permissive function during CB development that is probably linked to its well- documented role in restricting eve expression in the early dorsal mesoderm (Bidet et al., 2003; Halfon et al., 2000; Liu et al., 2006; Webber et al., 2013). Importantly, and in contrast to edl and pnt activity changes, manipulating aop activities does not lead to significant shifts in the oCBs:gCBs Schwarz et al. eLife 2018;7:e32847. DOI: https://doi.org/10.7554/eLife.32847 9 of 36 Figure 3. EGF signaling promotes the formation of Odd+PCs. (A–D) Odd/Eve staining to analyze pericardial cells (PCs). (A) In the wild type, each hemisegment contains four OPCs, two EPCs and one Eve+ somatic muscle DA1 (). (B) Amorphic rho7M43/L68 mutant with a loss of about half of all OPCs and all DA1 muscles. (C) Pan-mesodermal overexpression of the dominant-negative Egfr results in a phenotype similar to rho mutants. (D) Overexpression of rho in the dorsal mesoderm generates supernumerary OPCs. The number of EPCs is not affected by altered levels of EGF signaling. E) Quantification of OPCs (green) and EPCs (red). Only abdominal PCs (located posterior to the lymph gland, LG) were included into the analysis. Significant differences compared to the y w control (WT) are designated as in Figure 1. Colored dashed lines mark the average numbers of OPCs and EPCs counted in the wild type. (F,G) Doc2+3/b-galactosidase (LacZ) staining in wild type (F) and Star mutant embryos (G) carrying a heterozygous copy of svpAE127-lacZ and showing presence of normal numbers of oCBs (Doc+/LacZ+) and their OPC siblings (Doc-/LacZ+). Bottom panels show a higher magnification and b-galactosidase single channel view of the upper panel. RG: ring gland, FB: fat body. DOI: https://doi.org/10.7554/eLife.32847.010 The following source data and figure supplement are available for figure 3: Research article Developmental Biology Research article Developmental Biology Figure 3. EGF signaling promotes the formation of Odd+PCs. (A–D) Odd/Eve staining to analyze pericardial cells (PCs). (A) In the wild type, each hemisegment contains four OPCs, two EPCs and one Eve+ somatic muscle DA1 (). (B) Amorphic rho7M43/L68 mutant with a loss of about half of all OPCs and all DA1 muscles. Figure 2 continued (C) Pan-mesodermal overexpression of the dominant-negative Egfr results in a phenotype similar to rho mutants. (D) Overexpression of rho in the dorsal mesoderm generates supernumerary OPCs. The number of EPCs is not affected by altered levels of EGF signaling. (E) Quantification of OPCs (green) and EPCs (red). Only abdominal PCs (located posterior to the lymph gland, LG) were included into the analysis. Significant differences compared to the y w control (WT) are designated as in Figure 1. Colored dashed lines mark the average numbers of OPCs and EPCs counted in the wild type. (F,G) Doc2+3/b-galactosidase (LacZ) staining in wild type (F) and Star mutant embryos (G) carrying a heterozygous copy of svpAE127-lacZ and showing presence of normal numbers of oCBs (Doc+/LacZ+) and their OPC siblings (Doc-/LacZ+). Bottom panels show a higher magnification and b-galactosidase single channel view of the upper panel. RG: ring gland, FB: fat body. DOI: https://doi.org/10.7554/eLife.32847.010 The following source data and figure supplement are available for figure 3: Source data 1. Quantification of OPCs and EPCs. DOI: https://doi.org/10.7554/eLife.32847.012 Figure supplement 1. Extended analysis of pericardial markers in EGF loss- and gain-of-function backgrounds. DOI: https://doi.org/10.7554/eLife.32847.011 Figure 3. EGF signaling promotes the formation of Odd+PCs. (A–D) Odd/Eve staining to analyze pericardial cells (PCs). (A) In the wild type, each hemisegment contains four OPCs, two EPCs and one Eve+ somatic muscle DA1 (). (B) Amorphic rho7M43/L68 mutant with a loss of about half of all OPCs and all DA1 muscles. (C) Pan-mesodermal overexpression of the dominant-negative Egfr results in a phenotype similar to rho mutants. (D) Overexpression of rho in the dorsal mesoderm generates supernumerary OPCs. The number of EPCs is not affected by altered levels of EGF signaling. (E) Quantification of OPCs (green) and EPCs (red). Only abdominal PCs (located posterior to the lymph gland, LG) were included into the analysis. Significant differences compared to the y w control (WT) are designated as in Figure 1. Colored dashed lines mark the average numbers of OPCs and EPCs counted in the wild type. (F,G) Doc2+3/b-galactosidase (LacZ) staining in wild type (F) and Star mutant embryos (G) carrying a heterozygous copy of svpAE127-lacZ and showing presence of normal numbers of oCBs (Doc+/LacZ+) and their OPC siblings (Doc-/LacZ+). Bottom panels show a higher magnification and b-galactosidase single channel view of the upper panel. RG: ring gland, FB: fat body. DOI: https://doi.org/10.7554/eLife.32847.010 10 of 36 10 of 36 Schwarz et al. eLife 2018;7:e32847. DOI: https://doi.org/10.7554/eLife.32847 Schwarz et al. eLife 2018;7:e32847. DOI: https://doi.org/10.7554/eLife.32847 Research article Developmental Biology gure 4. Edl is a decisive factor of ostial cardioblast specification. (A) Map of the edl locus with the used alleles and deficiencies. (B–I) Doc2+3/H15 ainings as in Figure 1. (B) Embryo with transheterozygous combination of Df(2R)edl-S0520 (edl deleted) and Df(2R)ED3636 (edl present) showing a gular ‘2 + 4’ CB pattern of oCBs and gCBs. By contrast, amorphic edl mutants Df(2R)edl-S0520/Exel7157 (C) and edlk06602 (D) have only few oCBs. ote the occurrence of ‘1 + 5’ CB patterns (bracket). (E) The regular CB pattern is restored by a genomic edl+ transgene. A nearly normal CB pattern i bserved in edl mutants upon expression of UAS-edl in the dorsal mesoderm via tinD-GAL4 (F) or only in CBs or their progenitors via tinCD4-GAL4 (G) cardioblast-specific tin mutants (carrying a rescue construct for early tin function) all CBs present become Doc+, irrespective of whether edl is nctional (H) or not (I). Observation of some H15- Doc+ CBs in (H) and (I) suggest that robust H15 expression requires normal tin function. (J) Mutually xclusive expression of Doc and Tin proteins in the wild type at late stage 15. (K) In edl mutants, Doc and Tin are co-expressed in some CBs rrowheads). These oCBs display either low level expression of both Tin and Doc (as exemplified in the magnification) or low levels of Tin concurrent th close to normal levels of Doc Asterisks denote positions of artificial signal overlap due to co projection of oCBs and TPCs gure 4. Edl is a decisive factor of ostial cardioblast specification. (A) Map of the edl locus with the used alleles and deficiencies. (B–I) Doc2+3/H15 ainings as in Figure 1. (B) Embryo with transheterozygous combination of Df(2R)edl-S0520 (edl deleted) and Df(2R)ED3636 (edl present) showing a gular ‘2 + 4’ CB pattern of oCBs and gCBs. By contrast, amorphic edl mutants Df(2R)edl-S0520/Exel7157 (C) and edlk06602 (D) have only few oCBs. ote the occurrence of ‘1 + 5’ CB patterns (bracket). (E) The regular CB pattern is restored by a genomic edl+ transgene. Schwarz et al. eLife 2018;7:e32847. DOI: https://doi.org/10.7554/eLife.32847 A nearly normal CB pattern i bserved in edl mutants upon expression of UAS-edl in the dorsal mesoderm via tinD-GAL4 (F) or only in CBs or their progenitors via tinCD4-GAL4 (G) cardioblast-specific tin mutants (carrying a rescue construct for early tin function) all CBs present become Doc+, irrespective of whether edl is ti l (H) t (I) Ob ti f H15- D + CB i (H) d (I) t th t b t H15 i i l ti f ti (J) M t ll Figure 4. Edl is a decisive factor of ostial cardioblast specification. (A) Map of the edl locus with the used alleles and deficiencies. (B–I) Doc2+3/H15 stainings as in Figure 1. (B) Embryo with transheterozygous combination of Df(2R)edl-S0520 (edl deleted) and Df(2R)ED3636 (edl present) showing a regular ‘2 + 4’ CB pattern of oCBs and gCBs. By contrast, amorphic edl mutants Df(2R)edl-S0520/Exel7157 (C) and edlk06602 (D) have only few oCBs. Note the occurrence of ‘1 + 5’ CB patterns (bracket). (E) The regular CB pattern is restored by a genomic edl+ transgene. A nearly normal CB pattern is observed in edl mutants upon expression of UAS-edl in the dorsal mesoderm via tinD-GAL4 (F) or only in CBs or their progenitors via tinCD4-GAL4 (G). In cardioblast-specific tin mutants (carrying a rescue construct for early tin function) all CBs present become Doc+, irrespective of whether edl is functional (H) or not (I). Observation of some H15- Doc+ CBs in (H) and (I) suggest that robust H15 expression requires normal tin function. (J) Mutually exclusive expression of Doc and Tin proteins in the wild type at late stage 15. (K) In edl mutants, Doc and Tin are co-expressed in some CBs (arrowheads). These oCBs display either low level expression of both Tin and Doc (as exemplified in the magnification) or low levels of Tin concurrent with close to normal levels of Doc. Asterisks denote positions of artificial signal overlap due to co-projection of oCBs and TPCs. DOI: https://doi.org/10.7554/eLife.32847.013 The following figure supplements are available for figure 4: Figure 4 continued on next page 11 of 36 Schwarz et al. eLife 2018;7:e32847. DOI: https://doi.org/10.7554/eLife.32847 Schwarz et al. eLife 2018;7:e32847. DOI: https://doi.org/10.7554/eLife.32847 Figure 4 continued Figure supplement 1. Cardiac edl expression. DOI: https://doi.org/10.7554/eLife.32847.014 Figure supplement 2. Dynamic expression of edl in the cardiogenic mesoderm as detected by an intron-specific probe. Edl and Pnt regulate ostial fate by controlling seven-up expression Edl and Pnt regulate ostial fate by controlling seven-up expression The population of oCBs is characterized by expression of svp. In svp mutants all oCBs are converted into Tin+/Doc- CBs due to de-repression of tin (Gajewski et al., 2000; Lo and Frasch, 2001; Zaffran et al., 2006; Figure 6—figure supplement 1A). Therefore, we tested the possibility that Edl promotes oCB fate by regulating svp. In the wild type, expression of svp is recapitulated by the enhancer trap svpAE127- lacZ (Figure 6A; Lo and Frasch, 2001). In edl mutants, svp-LacZ expression is strongly reduced in cardiac cells (Figure 6B,D). The reduction in numbers of both svp-LacZ+ oCBs and OPCs at late stages (Figure 6D cf. 6C) suggests that edl already affects the fates of their common progenitors. Consistent with a function in promoting svp expression and oCBs fates, mesodermal overexpression of edl leads to larger numbers of svp-LacZ+ cardiac cells, particularly of CBs, where svp expression correlates with expanded Doc expression (Figure 6E,F). As shown for Doc expression, svp expression can be suppressed by PntP2 hyperactivity (green asterisks in Figure 6H). These observations and further evaluation of the epistatic relations between svp and edl (Figure 6—figure supplement 1) demonstrate that edl affects CB patterning by blocking Pnt activity upstream of svp. Schwarz et al. eLife 2018;7:e32847. DOI: https://doi.org/10.7554/eLife.32847 DOI: https://doi.org/10.7554/eLife.32847.015 Research article Research article Developmental Biology Developmental Biology ratio (Figure 5I). Thus, we suggest that Edl acts mainly via negative modulation of PntP2 activity dur- ing cardioblast diversification. ratio (Figure 5I). Thus, we suggest that Edl acts mainly via negative modulation of PntP2 activity dur- ing cardioblast diversification. An additional function of Pnt (and thereby Edl) regarding to the total number of CBs is also apparent in Figure 5. The increase in the total CB number detected in pnt mutants is reminiscent of Notch pathway mutants. Figure 5—figure supplement 1 shows examples of such mutants isolated from our EMS screen. There is an important difference between pnt and Notch pathway mutants regarding the oCBs:gCBs ratio. Whereas oCBs account for about 40–50% of the CBs in pnt mutants (as compared to 27% in the wild type), all Notch pathway mutants for which CB patterning data are available feature a significantly smaller fraction of oCBs than pnt mutants (Figure 5—figure supple- ment 1D). The maximum fraction of oCBs observed was 33% of the total CB number, found in mamS0669. In kuz mutants (data not shown; Albrecht et al., 2006), oCBs even increase by smaller factors than gCBs resulting in oCB fractions below 27%. (Some differences in the oCBs:gCBs ratio between various Notch pathway mutants are likely to arise from variable impact on lateral inhibition and specific functions of Notch in asymmetrically dividing lineages). On a side note, edl expression, which was found to be positively regulated by Notch signaling in a Drosophila cell culture system (Krejcı´ and Bray, 2007), is not negatively affected in the cardiogenic mesoderm of two mam alleles and in bibS1538 mutants (Figure 5—figure supplement 2 and data not shown). Cardioblast subtype-specific expression of the PntP1 isoform is regulated by PntP2 and Edl Proposing a gCB-specific function of Pnt, we next analyzed its cardiac expression. Boisclair Lachance et al. previously reported that the expression of a fully functional genomic pnt-GFP transgene mirrors the combined expression of all Pnt isoforms (Boisclair Lachance et al., 2014). The authors detected Pnt-GFP fusion protein in nearly all cells of the cardiac region, but highest levels were observed in two Yan-nega- tive clusters per hemisegment flanking Eve+ cells. We confirmed and refined these observations showing that high levels of Pnt-GFP are present in the nuclei of gCB progenitors as identified by their position, characteristically enlarged size, presence of only low levels of Doc, and absence of svp-LacZ expression (Figure 7A). We attribute these high total Pnt levels largely to a gCB-specific expression of the PntP1 iso- form since PntP1-specific antibodies (Alvarez et al., 2003) specifically label gCB progenitors (Figure 7B), whereas pntP2 transcripts are present in a rather uniform pattern in the mesoderm including the cardio- genic area (Kla¨mbt, 1993; and data not shown). We further speculated that PntP2 could activate pntP1 transcription in gCB progenitors for a sustained signaling response as found in other tissues (Shwartz et al., 2013). This assumption is indeed supported by our genetic data. First, we detect PntP1 Schwarz et al. eLife 2018;7:e32847. DOI: https://doi.org/10.7554/eLife.32847 12 of 36 Research article Developmental Biology igure 5. Edl promotes oCB fate via inhibition of PntP2. (A–H) CB pattern in embryos with modified activity of edl and/or genes encoding the ETS roteins Pnt and Yan revealed by H15/Doc2+3 stainings. (A) Pan-mesodermal edl overexpression via twist-GAL4 leads to extra CBs with a isproportionately high increase in oCB numbers. This phenotype is reminiscent to that of the pnt mutants pntMI03880 (a PntP2-specific mutant; here in ans with a pnt-deleting deficiency, (B) and pntD88 (without any functional Pnt isoform, (E). (C,C’) Conversely, an edl mutant-like phenotype (loss/ Research article Developmental Biology igure 5. Edl promotes oCB fate via inhibition of PntP2. (A–H) CB pattern in embryos with modified activity of edl and/or genes encoding the ETS roteins Pnt and Yan revealed by H15/Doc2+3 stainings. (A) Pan-mesodermal edl overexpression via twist-GAL4 leads to extra CBs with a isproportionately high increase in oCB numbers. This phenotype is reminiscent to that of the pnt mutants pntMI03880 (a PntP2-specific mutant; here in ans with a pnt-deleting deficiency, (B) and pntD88 (without any functional Pnt isoform, (E). Cardioblast subtype-specific expression of the PntP1 isoform is regulated by PntP2 and Edl (C,C’) Conversely, an edl mutant-like phenotype (loss/ onversion of oCBs, exemplified by arrowheads for one hemisegment, and CBs with low Doc levels marked by asterisks) is generated by verexpression of a constitutively active PntP2 variant in the dorsal/cardiogenic mesoderm. C and C’ depict strong and weak phenotypes, respectively. D) Overexpression of the constitutively active repressor Yan/Aop leads to a loss of both gCBs and oCBs. (E,F) The CB phenotypes of pnt and edl pnt ouble mutants are very similar suggesting that edl acts mainly by blocking Pnt activity during CB specification. (G) Hemizygous aop mutant showing a moderate reduction of both CB types. (H) edl aop double mutant combining aop-like and edl-like defects. (I) Quantification of cardioblasts in various t ff ti Edl P t Y /A ti iti ( t t d i Fi 1M) inhibition of PntP2. (A–H) CB pattern in embryos with modified activity of edl and/or genes encoding the ETS 5. Edl promotes oCB fate via inhibition of PntP2. (A–H) CB pattern in embryos with modified activity of edl and/ Figure 5. Edl promotes oCB fate via inhibition of PntP2. (A–H) CB pattern in embryos with modified activity of edl and/or genes encoding the ETS proteins Pnt and Yan revealed by H15/Doc2+3 stainings. (A) Pan-mesodermal edl overexpression via twist-GAL4 leads to extra CBs with a disproportionately high increase in oCB numbers. This phenotype is reminiscent to that of the pnt mutants pntMI03880 (a PntP2-specific mutant; here in trans with a pnt-deleting deficiency, (B) and pntD88 (without any functional Pnt isoform, (E). (C,C’) Conversely, an edl mutant-like phenotype (loss/ conversion of oCBs, exemplified by arrowheads for one hemisegment, and CBs with low Doc levels marked by asterisks) is generated by overexpression of a constitutively active PntP2 variant in the dorsal/cardiogenic mesoderm. C and C’ depict strong and weak phenotypes, respectively. (D) Overexpression of the constitutively active repressor Yan/Aop leads to a loss of both gCBs and oCBs. (E,F) The CB phenotypes of pnt and edl pnt double mutants are very similar suggesting that edl acts mainly by blocking Pnt activity during CB specification. (G) Hemizygous aop mutant showing a moderate reduction of both CB types. (H) edl aop double mutant combining aop-like and edl-like defects. (I) Quantification of cardioblasts in various genotypes affecting Edl, Pnt or Yan/Aop activities (annotated as in Figure 1M). Cardioblast subtype-specific expression of the PntP1 isoform is regulated by PntP2 and Edl DOI: https://doi.org/10.7554/eLife.32847.016 The following source data and figure supplements are available for figure 5: Source data 1. Quantification of cardioblasts in various genotypes affecting Edl, Pnt or Yan/Aop activities. DOI: https://doi.org/10.7554/eLife.32847.020 Figure supplement 1. The numbers of both generic and ostial cardioblasts increase upon mutation of genes involved in Notch signaling. Figure 5 continued on next page Figure supplement 1. The numbers of both generic and ostial cardioblasts increase upon mutation of genes involved in Notch signaling. Figure 5 continued on next page 13 of 36 13 of 36 The Tbx20 ortholog Midline contributes to Pnt-dependent repression of svp in the working myocardial lineage p g y g According to the common view, we expect Pnt to act as a transcriptional activator also during CB diversi- fication, particularly since overexpression of PntP2 fused to the VP16 activator domain has essentially the same effect on cardiac patterning as PntP1 overexpression (Figure 6H and data not shown). Therefore, its negative impact on svp expression is likely to involve Pnt-dependent activation of a transcriptional repressor. Interestingly, the T-box factor Midline (Mid), like PntP1, shows expression in early gCB progen- itors (Figure 2I,K; Figure 7G). We previously reported that mid functions to maintain tin expression in gCBs, thereby restricting Doc expression to oCBs (Reim et al., 2005). Consistent with this function our EMS screen also generated novel mid alleles showing the same CB patterning defects as previously described alleles (Supplementary file 1-Table S1, Figure 7H and data not shown). While a direct regula- tion of tin by Mid was previously proposed to be responsible for these changes (supported by the gain- and loss-of-function phenotypes of mid; Qian et al., 2005; Reim et al., 2005), another non-exclusive sce- nario could involve repression of svp (encoding a repressor of tin) by Mid. Consistent with the latter, we observe a Doc-like expansion of svp expression in mid loss-of-function mutants (Figure 7I) and a reduc- tion of svp expression upon ectopic overexpression of mid via tinD +tinCD4-GAL4 (Figure 7J). Moreover, persistent tin expression in all CBs of mid svp double mutants (Figure 7—figure supplement 1D, com- pare to control in A and single mutants in B and C) demonstrates that mid is not directly required for tin expression in CBs. Furthermore, the wild type-like expression of svp-lacZ (with nearly no LacZ in gCBs) observed in the same genetic background argues for the involvement of a Svp-dependent positive feed- back loop in ectopic cardiac svp activation in gCBs, as has been predicted previously based on svp over- expression studies (Zaffran et al., 2006). The cardiac pattern phenotype of edl mid double mutants is a composite of the single mutant phenotypes. The number of oCBs (average oCBs: 24.4 ± 3.6; n = 6) is strongly increased as compared to edl mutants, but reduced in comparison with mid mutants, with total CB numbers being similar to those of edl mutants. In some cases, a near wild-type pattern is observed (Figure 7K), although many embryos display an asymmetric arrangement of CBs. Schwarz et al. eLife 2018;7:e32847. DOI: https://doi.org/10.7554/eLife.32847 Schwarz et al. eLife 2018;7:e32847. DOI: https://doi.org/10.7554/eLife.32847 Figure 5 continued DOI: https://doi.org/10.7554/eLife.32847.017 Figure supplement 1—source data 1. Quantification of cardioblasts in Notch signaling-related genotypes. DOI: https://doi.org/10.7554/eLife.32847.018 Figure supplement 2. Expression of edl in the cardiogenic mesoderm is still observed in Notch signaling-related mutants. DOI: https://doi.org/10.7554/eLife.32847.019 Research article Developmental Biology R Research article Developmental Biology Developmental Biology Developmental Biology g pplement 2. Expression of edl in the cardiogenic mesoderm is still observed in Notch signaling-related mutants. //doi.org/10.7554/eLife.32847.019 in an expanded pattern in the cardiogenic mesoderm of edl mutants in which PntP2 activity is assumed to increase (Figure 7C). Second, overexpression of edl (i.e. repression of PntP2 function) as well as genetic disruption of pntP2 resulted in a near-complete loss of cardiac PntP1 (Figure 7D,E; note persistent expression of PntP1 in other cells located more laterally). We conclude that the combined activities of Edl and PntP2 lead to the confined pntP1 expression in gCBs. The EGF Spitz appears to be a major, although not necessarily the sole factor for the MAPK-mediated activation of PntP2 in this context, because PntP1 levels are reduced but not eradicated in cardiac cells of amorphic spi mutants (Figure 7F). The Tbx20 ortholog Midline contributes to Pnt-dependent repression of svp in the working myocardial lineage While the prevalence of many Doc-negative CBs in this background implies that mid is not the only factor that limits oCB fate, it also indicates that edl is normally required in the oCB lineage to restrict mid activity, possibly by blocking a Pnt-dependent activation of mid transcription. This hypothesis is indeed supported by the reversion of ectopic Doc and svp expression in pnt mutants upon forced mid expression (Figure 7L, Figure 7—figure supplement 2C). By contrast, overexpression of the previously assumed Mid target tin in this background only represses Doc, but not svp (Figure 7—figure supplement 2D). To further test the idea that Mid is a repressor of oCB fate downstream of pnt, we analyzed whether it is a direct target of Pnt. Notably, an enhancer identified as a Tin target and named midE19 (mid180 for a shorter minimal version) was recently shown to drive mid expression specifically in gCBs (Jin et al., 2013; Ryu et al., 2011; Figure 8A–C; Figure 9A,C). Since this enhancer does not drive reporter expression in oCBs after germ band retraction as detected for mid in the genomic context, additional cis-regulatory regions must be at work to reproduce all aspects of cardiac mid expression. The characteristic activity pattern of the enhancer suggests that this regulatory region may be specifically (or exclusively) devoted Schwarz et al. eLife 2018;7:e32847. DOI: https://doi.org/10.7554/eLife.32847 14 of 36 Research article Developmental Biology Figure 6. Edl is required for svp expression. (A) In stage 12 control embryos (lateral view) carrying one copy of svpAE127-lacZ, b-galactosidase is detected in oCBs (arrows) and their sibling OPCs (arrowheads) within the Mef2-labeled mesoderm. (B) Cardiac svp-LacZ expression is strongly reduced n edl mutants (Df(2R)edl-S0520/Exel7157;svpAE127-lacZ/+). (C–E) Odd/svp-LacZ staining in stage 16 embryos. (C) In the control, each hemisegment contains two oCB-related svp-LacZ+ OPCs and two svp-LacZ- OPCs. The total number of OPCs decreases if edl is absent (Df(2R)edl-S0520/edl-L19; svpAE127-lacZ/+) (D) or overexpressed (E), but different OPC subpopulations account for these losses: svp-LacZ+ OPCs (arrowheads) are reduced in edl mutants, svp-LacZ- OPCs in edl overexpressing embryos. (E,F) Pan-mesodermal overexpression of edl leads to a drastic increase in the number of svp- LacZ+/Doc+ cardioblasts (Odd-). Compare F to the control in Figure 3F. (G,H) Mef2/Doc2+3/b-galactosidase staining in svp-lacZ/+ controls (G) and embryos overexpressing constitutively active pntP2VP16 in the dorsal mesoderm (H). The Tbx20 ortholog Midline contributes to Pnt-dependent repression of svp in the working myocardial lineage Highest levels are observed in gCB progenitors (large svp-LacZ-negative nuclei with low levels of Doc, arrowheads) and low levels in oCBs and their siblings (small svp-LacZ+ nuclei with igher Doc levels, arrows). (B) At the onset of germ band retraction, PntP1 becomes expressed in gCB progenitors (arrowheads) of wild type embryos Cardiac cells are labeled via anti-Doc3+2 staining. PntP1 is not detected in oCBs and their siblings (arrows). (C) In edl- mutants cardiac PntP1 expressio s generally increased and detected ectopically in some small nuclei that correspond to prospective oCBs and their siblings (arrows). (D) Pan- mesodermal overexpression of edl leads to a strong decrease of cardiac PntP1 expression while other mesodermal tissues are less affected. (E) The ame effect is seen in pntP2 mutants. (F) In spi mutants PntP1 levels are reduced as well, although not as severely as upon loss of pntP2 function. (G) ike PntP1, Mid protein is found in gCB progenitors (arrowheads), but not in prospective oCBs (arrows) at the beginning of germ band retraction. (H,I) The cardiac phenotype of mid mutants is characterized by variable expansion of Doc, which largely correlates with ectopic svp expression in CBs (I, Figure 7 continued on next page Figure 7. PntP1 and Mid are specifically expressed in early gCB progenitors to antagonize oCB fate. (A) Detection of Doc3+2, b-galactosidase and GFP-tagged Pnt (all isoforms) in a pnt-GFP/+; svpAE127-lacZ/+ embryo at the beginning of stage 12 (lateral view). Highest levels are observed in gCB progenitors (large svp-LacZ-negative nuclei with low levels of Doc, arrowheads) and low levels in oCBs and their siblings (small svp-LacZ+ nuclei with higher Doc levels, arrows). (B) At the onset of germ band retraction, PntP1 becomes expressed in gCB progenitors (arrowheads) of wild type embryos. Cardiac cells are labeled via anti-Doc3+2 staining. PntP1 is not detected in oCBs and their siblings (arrows). (C) In edl- mutants cardiac PntP1 expression is generally increased and detected ectopically in some small nuclei that correspond to prospective oCBs and their siblings (arrows). (D) Pan- mesodermal overexpression of edl leads to a strong decrease of cardiac PntP1 expression while other mesodermal tissues are less affected. (E) The same effect is seen in pntP2 mutants. (F) In spi mutants PntP1 levels are reduced as well, although not as severely as upon loss of pntP2 function. The Tbx20 ortholog Midline contributes to Pnt-dependent repression of svp in the working myocardial lineage Overexpression of pntP2VP16 via tinD-GAL4 leads to significantly educed svp and Doc expression (examples labeled with green asterisks; average number of Svp+ CBs: 20.6 ± 3.0, p=0,00069; accompanied by an ncreased number of Svp- CBs: 83.4 ± 2.6, p=0.00015; n = 7) as compared to normal oCBs (red asterisks). LG: lymph gland, RG: ring gland, FB: fat body. DOI: https://doi.org/10.7554/eLife.32847.021 Figure 6. Edl is required for svp expression. (A) In stage 12 control embryos (lateral view) carrying one copy of svpAE127-lacZ, b-galactosidase is detected in oCBs (arrows) and their sibling OPCs (arrowheads) within the Mef2-labeled mesoderm. (B) Cardiac svp-LacZ expression is strongly reduced in edl mutants (Df(2R)edl-S0520/Exel7157;svpAE127-lacZ/+). (C–E) Odd/svp-LacZ staining in stage 16 embryos. (C) In the control, each hemisegment contains two oCB-related svp-LacZ+ OPCs and two svp-LacZ- OPCs. The total number of OPCs decreases if edl is absent (Df(2R)edl-S0520/edl-L19; svpAE127-lacZ/+) (D) or overexpressed (E), but different OPC subpopulations account for these losses: svp-LacZ+ OPCs (arrowheads) are reduced in edl mutants, svp-LacZ- OPCs in edl overexpressing embryos. (E,F) Pan-mesodermal overexpression of edl leads to a drastic increase in the number of svp- LacZ+/Doc+ cardioblasts (Odd-). Compare F to the control in Figure 3F. (G,H) Mef2/Doc2+3/b-galactosidase staining in svp-lacZ/+ controls (G) and embryos overexpressing constitutively active pntP2VP16 in the dorsal mesoderm (H). Overexpression of pntP2VP16 via tinD-GAL4 leads to significantly reduced svp and Doc expression (examples labeled with green asterisks; average number of Svp+ CBs: 20.6 ± 3.0, p=0,00069; accompanied by an increased number of Svp- CBs: 83.4 ± 2.6, p=0.00015; n = 7) as compared to normal oCBs (red asterisks). LG: lymph gland, RG: ring gland, FB: fat body. DOI: https://doi.org/10.7554/eLife.32847.021 DOI: https://doi.org/10.7554/eLife.32847.021 The following figure supplement is available for figure 6: The following figure supplement is available for figure 6: Figure supplement 1. Epistatic relationship between edl and svp. Figure supplement 1. Epistatic relationship between edl and svp. DOI: https://doi.org/10.7554/eLife.32847.022 DOI: https://doi.org/10.7554/eLife.32847.022 15 of 36 Schwarz et al. eLife 2018;7:e32847. DOI: https://doi.org/10.7554/eLife.32847 Schwarz et al. eLife 2018;7:e32847. DOI: https://doi.org/10.7554/eLife.32847 Research article Developmental Biology Figure 7. PntP1 and Mid are specifically expressed in early gCB progenitors to antagonize oCB fate. (A) Detection of Doc3+2, b-galactosidase and GFP-tagged Pnt (all isoforms) in a pnt-GFP/+; svpAE127-lacZ/+ embryo at the beginning of stage 12 (lateral view). The Tbx20 ortholog Midline contributes to Pnt-dependent repression of svp in the working myocardial lineage (G) Like PntP1, Mid protein is found in gCB progenitors (arrowheads), but not in prospective oCBs (arrows) at the beginning of germ band retraction. (H,I) The cardiac phenotype of mid mutants is characterized by variable expansion of Doc, which largely correlates with ectopic svp expression in CBs (I, Figure 7 continued on next page Schwarz et al. eLife 2018;7:e32847. DOI: https://doi.org/10.7554/eLife.32847 Figure 7 continued normal pattern shown in Figure 3F). (J) Overexpression of mid represses svp expression in H15-labeled cardioblasts (arrowheads indicate a hemisegment with five lacZ-negative nuclei). (K) Combining homozygous mid and edl mutations results in the restoration of oCBs in comparison to edl single mutants (Figure 4D), suggesting that edl normally antagonizes mid function. An additional edl function regarding the total CB number is not rescued by abrogation of mid. (L) Overexpression of mid in the dorsal mesoderm via tinD-GAL4 in a pnt null background converts many of the extra oCBs into gCBs (cf. Figure 5E). DOI: https://doi.org/10.7554/eLife.32847.023 The following figure supplements are available for figure 7: The following figure supplements are available for figure 7: Figure supplement 1. Expression of tin in gCBs is indirectly stabilized by mid via svp repression. DOI: https://doi.org/10.7554/eLife.32847.024 p g Figure supplement 2. Additional data supporting mid function in gCBs. to the reception of early gCB-specific inputs. Consistent with our assumption that this enhancer is also a target of Pnt, very little midE19-GFP activity is detectable in pnt mutants (Figure 8D), reduced activity is observed in embryos with mesodermal edl overexpression (Figure 8E), and expanded activity is seen upon overexpression of PntP1 (Figure 8F; note occasional expansion into CBs with no detectable Tin) or PntP2VP16 (not shown). An observed reduction of midE19-driven GFP levels in many of the retained Tin+ gCBs of rho mutants (Figure 8G) corroborates that EGF signaling feeds into mid activation. The idea that mid is a target of Pnt is further supported by the almost complete elimination of reporter activity upon mutating a single ETS binding motif within the mid180 minimal cardiac enhancer (Figure 9A–D) as well as the strong reduction of endogenous mid transcription in emerging CBs during germ band retraction stages in pnt mutants (Figure 9E–H). After germ band retraction, endogenous mid is activated indepen- dently of pnt in all CBs (Figure 9J) as observed in the wild type (Figure 9I) indicating that distinct mecha- nisms regulate mid transcription in early gCB progenitors and maturing CBs. to the reception of early gCB-specific inputs. Figure 7 continued Consistent with our assumption that this enhancer is also a target of Pnt, very little midE19-GFP activity is detectable in pnt mutants (Figure 8D), reduced activity is observed in embryos with mesodermal edl overexpression (Figure 8E), and expanded activity is seen upon overexpression of PntP1 (Figure 8F; note occasional expansion into CBs with no detectable Tin) or PntP2VP16 (not shown). An observed reduction of midE19-driven GFP levels in many of the retained Tin+ gCBs of rho mutants (Figure 8G) corroborates that EGF signaling feeds into mid activation. The idea that mid is a target of Pnt is further supported by the almost complete elimination of reporter activity upon mutating a single ETS binding motif within the mid180 minimal cardiac enhancer (Figure 9A–D) as well as the strong reduction of endogenous mid transcription in emerging CBs during germ band retraction stages in pnt mutants (Figure 9E–H). After germ band retraction, endogenous mid is activated indepen- dently of pnt in all CBs (Figure 9J) as observed in the wild type (Figure 9I) indicating that distinct mecha- nisms regulate mid transcription in early gCB progenitors and maturing CBs. In sum, our data lead to the conclusion that EGF signaling contributes to gCB specification by at least two distinct mechanisms, Pnt-independent specification of a subset of cardiac progenitors as well as Pnt-dependent inhibition of ostial cardioblast fate. Modulation by Edl is needed to inhibit Pnt-dependent gene activation and thus enable formation of ostial cardioblasts. Research article Developmental Biology Developmental Biology Schwarz et al. eLife 2018;7:e32847. DOI: https://doi.org/10.7554/eLife.32847 Schwarz et al. eLife 2018;7:e32847. DOI: https://doi.org/10.7554/eLife.32847 Schwarz et al. eLife 2018;7:e32847. DOI: https://doi.org/10.7554/eLife.32847 16 of 36 Research article R Discussion The specification and diversification of particular cell types are linked to the establishment of line- age-specific transcriptional programs. The differences in these programs are often prompted by dis- tinct local signaling activities. The cells in the early heart fields of Drosophila acquire their cardiogenic potential by intersecting BMP and Wnt signal activities (Frasch, 1995; Reim and Frasch, 2005; Wu et al., 1995), but cell diversification within this area requires additional regulatory inputs. Previous studies established that progenitors of cardioblasts, pericardial cells and dorsal somatic muscles are selected by RTK/Ras/MAPK signaling, whereas lateral inhibition by Delta/Notch signal- ing activity counteracts this selection in neighboring non-progenitor cells (Carmena et al., 2002; Grigorian et al., 2011; Hartenstein et al., 1992). The progenitors of the definitive cardiogenic mesoderm, which give rise to all cardiac cells except for the somatic muscle lineage-related EPCs, co-express the cardiogenic factors Tin, Doc and Pnr, a unique feature that separates them from other cells (Reim and Frasch, 2005). In addition to limiting the number of progenitors, Notch signal- ing has a second function during Drosophila cardiogenesis that promotes pericardial (or in thoracic segments, hematopoietic) over myocardial fate (Albrecht et al., 2006; Grigorian et al., 2011; Hartenstein et al., 1992; Mandal et al., 2004). Other factors previously reported to impose hetero- geneity in the heart field include the cross-repressive activities of the homeodomain factors Eve and Lbe (Jagla et al., 2002) as well as ectoderm-derived Hedgehog (Hh) signals (Liu et al., 2006; Ponzielli et al., 2002). In segmental subsets of cardioblasts, Hh signaling was proposed to act as a potential activator of svp in prospective oCBs (Ponzielli et al., 2002) but whether these are direct or indirect effects of Hh on these cells has not been ascertained. Based on the findings of our study, we present a novel model of cardioblast diversification that intro- duces EGF signaling activities and lineage-specific modulation of the MAPK effector Pointed by Edl as crucial factors for the specification of generic working myocardial and ostial cell fates (Figure 10). We Schwarz et al. eLife 2018;7:e32847. DOI: https://doi.org/10.7554/eLife.32847 17 of 36 Research article Developmental Biology Figure 8. Characterization of a Pnt-responsive mid enhancer. Discussion (A–C) Expression analysis of the midE19-GFP reporter in the wild type background showing segmental expression in gCB progenitors at stage 12 (A: co-expression of GFP RNA, Mef2 and low levels of Doc) and later in the Tin+/H15+ gCBs (bracket; B: stage 14 stained for GFP protein and Tin; C: stage 16 stained for GFP, Tin and H15 proteins). No or very little reporter expression is detectable in oCBs and their presumed precursors (arrows). (D) Despite an overall increase in CB number, midE19-GFP expression is severely reduced n amorphic pnt mutants. Most of the Tin+/H15+ gCBs (purple nuclei, arrowheads) lack GFP expression. (E) Mesodermal overexpression of edl via how24B-GAL4 also leads to a loss of midE19-GFP in many gCBs. (F) Overexpression of pntP1 via how24B-GAL4 leads to nearly continuous midE19-GFP expression in CBs. In some instances, the reporter is activated even in Tin- CBs (arrows). (G) Loss of rho function, which is expected to cause reduced PntP2 activity, leads to a complete loss of GFP in some of the retained gCBs (arrowheads) and a level reduction in others (arrows). In comparison to pn mutants (D), a higher fraction of gCBs retains substantial GFP expression indicating additional, rho-independent inputs upstream of Pnt. DOI: https://doi.org/10.7554/eLife.32847.026 Research article Developmental Biolog Figure 8. Characterization of a Pnt-responsive mid enhancer. (A–C) Expression analysis of the midE19-GFP reporter in the wild type background showing segmental expression in gCB progenitors at stage 12 (A: co-expression of GFP RNA, Mef2 and low levels of Doc) and later in the Tin+/H15+ gCBs (bracket; B: stage 14 stained for GFP protein and Tin; C: stage 16 stained for GFP, Tin and H15 proteins). No or very little reporter expression is detectable in oCBs and their presumed precursors (arrows). (D) Despite an overall increase in CB number, midE19-GFP expression is severely reduced in amorphic pnt mutants. Most of the Tin+/H15+ gCBs (purple nuclei, arrowheads) lack GFP expression. (E) Mesodermal overexpression of edl via how24B-GAL4 also leads to a loss of midE19-GFP in many gCBs. (F) Overexpression of pntP1 via how24B-GAL4 leads to nearly continuous midE19-GFP expression in CBs. In some instances, the reporter is activated even in Tin- CBs (arrows). (G) Loss of rho function, which is expected to cause reduced PntP2 activity, leads to a complete loss of GFP in some of the retained gCBs (arrowheads) and a level reduction in others (arrows). Discussion In comparison to pnt mutants (D), a higher fraction of gCBs retains substantial GFP expression indicating additional, rho-independent inputs upstream of Pnt. DOI: https://doi.org/10.7554/eLife.32847.026 propose that EGF/MAPK signaling promotes the development of generic working myocardial progeni- tors (red cell in Figure 10) by two mechanisms that differ in their requirement for the ETS protein Pnt: 1. EGF promotes the correct selection and specification of gCB progenitors. This is evident from our loss- and gain-of-function analysis of EGF signaling components. This EGF function is obvi- ously independent of pnt, since pnt null mutants display excessive numbers of CBs (with gCB numbers comparable to the wild type or even increased), a phenotype different from that of 1. EGF promotes the correct selection and specification of gCB progenitors. This is evident from our loss- and gain-of-function analysis of EGF signaling components. This EGF function is obvi- ously independent of pnt, since pnt null mutants display excessive numbers of CBs (with gCB numbers comparable to the wild type or even increased), a phenotype different from that of Schwarz et al. eLife 2018;7:e32847. DOI: https://doi.org/10.7554/eLife.32847 Schwarz et al. eLife 2018;7:e32847. DOI: https://doi.org/10.7554/eLife.32847 Schwarz et al. eLife 2018;7:e32847. DOI: https://doi.org/10.7554/eLife.32847 18 of 36 Research article Developmental Biology igure 9. Additional experimental support for the regulation of mid by the ETS factor Pnt. Expression of GFP RNA (A, stage 13) and protein (C, stage 6) driven by the minimal cardiac mid enhancer, mid180, is less robust than midE19-GFP but shows essentially the same expression pattern. The minimal enhancer contains a single ETS binding motif flanked by two Tin-binding sites (indicated in the scheme below). (B,D) Mutating the ETS-bindi te leads to near-complete abolishment of mid180-GFP expression. (E–J) Analysis of mid mRNA expression in cardiac cells doubly stained with anti- oc3+2 antibody. In the wild type, mid mRNA is first detected in gCB progenitors at early stage 12 (E); its expression begins to expand during germ and retraction (G) until it reaches continuous expression in all CBs at stage 13 (I). By contrast, amorphic pnt mutants show reduced cardiac mid xpression during germ band retraction (F H) Regular uniform mid expression is observed only after germ band retraction (J) igure 9. Additional experimental support for the regulation of mid by the ETS factor Pnt. Expression of GFP RNA (A, stage 13) and protein (C, stage 6) driven by the minimal cardiac mid enhancer, mid180, is less robust than midE19-GFP but shows essentially the same expression pattern. The minimal enhancer contains a single ETS binding motif flanked by two Tin-binding sites (indicated in the scheme below). (B,D) Mutating the ETS-bindi te leads to near-complete abolishment of mid180-GFP expression. (E–J) Analysis of mid mRNA expression in cardiac cells doubly stained with anti- Doc3+2 antibody. In the wild type, mid mRNA is first detected in gCB progenitors at early stage 12 (E); its expression begins to expand during germ and retraction (G) until it reaches continuous expression in all CBs at stage 13 (I). By contrast, amorphic pnt mutants show reduced cardiac mid xpression during germ band retraction (F,H). Regular uniform mid expression is observed only after germ band retraction (J). DOI: https://doi.org/10.7554/eLife.32847.027 Figure 9. Additional experimental support for the regulation of mid by the ETS factor Pnt. Expression of GFP RNA (A, stage 13) and protein (C, stage 16) driven by the minimal cardiac mid enhancer, mid180, is less robust than midE19-GFP but shows essentially the same expression pattern. Schwarz et al. eLife 2018;7:e32847. DOI: https://doi.org/10.7554/eLife.32847 The minimal enhancer contains a single ETS binding motif flanked by two Tin-binding sites (indicated in the scheme below). (B,D) Mutating the ETS-binding site leads to near-complete abolishment of mid180-GFP expression. (E–J) Analysis of mid mRNA expression in cardiac cells doubly stained with anti- Doc3+2 antibody. In the wild type, mid mRNA is first detected in gCB progenitors at early stage 12 (E); its expression begins to expand during germ band retraction (G) until it reaches continuous expression in all CBs at stage 13 (I). By contrast, amorphic pnt mutants show reduced cardiac mid expression during germ band retraction (F,H). Regular uniform mid expression is observed only after germ band retraction (J). DOI: https://doi.org/10.7554/eLife.32847.027 19 of 36 Schwarz et al. eLife 2018;7:e32847. DOI: https://doi.org/10.7554/eLife.32847 Schwarz et al. eLife 2018;7:e32847. DOI: https://doi.org/10.7554/eLife.32847 Research article Developmental Biology Figure 10. Model of regulatory interactions in generic and ostial CB progenitors. Genes activated in a subtype-specific manner in gCB or oCB progenitors are colored in red and green, respectively. Larger font sizes and thicker lines indicate higher levels. Dashed lines indicate presumed regulations. In principle, MAPK can be activated in cardiac progenitors by EGF/EGFR and FGF/Htl signals. Generic cardioblast development depends on EGF-activated MAPK signaling which provides pnt-independent and pnt-dependent functions. The suppression of svp and subsequent regulation of tin and Doc is a pnt-dependent function that is in part mediated by activation of mid in presumptive gCBs. This step is likely to be supported by the gCB-specific expression of constitutive active PntP1. The gCB-specific cascade may require a higher level of MAPK activity to overcome the blockage of PntP2 by Edl. Alternatively or in addition, Edl levels might be differentially regulated in gCBs and oCBs by yet unknown mechanisms. In oCB progenitors, Edl keeps activated PntP2 below a critical threshold leading to absence or delayed onset of expression of oCB fate antagonists such as mid. This in turn permits svp activation by Hox genes and Tin derived from early stages. Presumed transcriptional activators of svp acting downstream of segmental Hh signals in oCB progenitors are not mandatory in this model, although it does not categorically exclude such contributions. Some details and additional interactions have been omitted for clarity. For a more complex version of the model see the corresponding figure supplement. DOI: https://doi.org/10.7554/eLife.32847.028 The following figure supplement is available for figure 10: Figure 10. Model of regulatory interactions in generic and ostial CB progenitors. Genes activated in a subtype-specific manner in gCB or oCB progenitors are colored in red and green, respectively. Larger font sizes and thicker lines indicate higher levels. Dashed lines indicate presumed regulations. In principle, MAPK can be activated in cardiac progenitors by EGF/EGFR and FGF/Htl signals. Generic cardioblast development depends on EGF-activated MAPK signaling which provides pnt-independent and pnt-dependent functions. The suppression of svp and subsequent regulation of tin and Doc is a pnt-dependent function that is in part mediated by activation of mid in presumptive gCBs. This step is likely to be supported by the gCB-specific expression of constitutive active PntP1. The gCB-specific cascade may require a higher level of MAPK activity to overcome the blockage of PntP2 by Edl. Schwarz et al. eLife 2018;7:e32847. DOI: https://doi.org/10.7554/eLife.32847 Schwarz et al. eLife 2018;7:e32847. DOI: https://doi.org/10.7554/eLife.32847 Extended model of regulatory interactions in generic and ostial CB progenitors. DOI: https://doi.org/10.7554/eLife.32847.029 mutants defective in EGF pathway components upstream of Pnt (Alvarez et al., 2003); this study). mutants defective in EGF pathway components upstream of Pnt (Alvarez et al., 2003); this study). 2. EGF signals affect the diversification of CB progenitors by impinging on a PntP2-dependent transcriptional cascade that eventually leads to suppression of Tin- oCB and the adoption of Tin+ gCB fates. This function is mediated by stimulating the gCB progenitor-specific expres- sion of regulatory genes such as mid (depicted in red in Figure 10), which in turn will promote transcription of gCB-specific differentiation genes and/or repression of oCB-specific factors (depicted in green in Figure 10). Schwarz et al. eLife 2018;7:e32847. DOI: https://doi.org/10.7554/eLife.32847 Alternatively or in addition, Edl levels might be differentially regulated in gCBs and oCBs by yet unknown mechanisms. In oCB progenitors, Edl keeps activated PntP2 below a critical threshold leading to absence or delayed onset of expression of oCB fate antagonists such as mid. This in turn permits svp activation by Hox genes and Tin derived from early stages. Presumed transcriptional activators of svp acting downstream of segmental Hh signals in oCB progenitors are not mandatory in this model, although it does not categorically exclude such contributions. Some details and additional interactions have been omitted for clarity. For a more complex version of the model see the corresponding figure supplement. DOI: https://doi.org/10.7554/eLife.32847.028 Figure 10. Model of regulatory interactions in generic and ostial CB progenitors. Genes activated in a subtype-specific manner in gCB or oCB progenitors are colored in red and green, respectively. Larger font sizes and thicker lines indicate higher levels. Dashed lines indicate presumed regulations. In principle, MAPK can be activated in cardiac progenitors by EGF/EGFR and FGF/Htl signals. Generic cardioblast development depends on EGF-activated MAPK signaling which provides pnt-independent and pnt-dependent functions. The suppression of svp and subsequent regulation of tin and Doc is a pnt-dependent function that is in part mediated by activation of mid in presumptive gCBs. This step is likely to be supported by the gCB-specific expression of constitutive active PntP1. The gCB-specific cascade may require a higher level of MAPK activity to overcome the blockage of PntP2 by Edl. Alternatively or in addition, Edl levels might be differentially regulated in gCBs and oCBs by yet unknown mechanisms. In oCB progenitors, Edl keeps activated PntP2 below a critical threshold leading to absence or delayed onset of expression of oCB fate antagonists such as mid. This in turn permits svp activation by Hox genes and Tin derived from early stages. Presumed transcriptional activators of svp acting downstream of segmental Hh signals in oCB progenitors are not mandatory in this model, although it does not categorically exclude such contributions. Some details and additional interactions have been omitted for clarity. For a more complex version of the model see the corresponding figure supplement. DOI: https://doi.org/10.7554/eLife.32847.028 The following figure supplement is available for figure 10: The following figure supplement is available for figure 10: Figure supplement 1. Extended model of regulatory interactions in generic and ostial CB progenitors. DOI: https://doi.org/10.7554/eLife.32847.029 Figure supplement 1. EGF signaling and cardiac progenitor selection According to our data, EGF signals are the major source for MAPK activation and progenitor specifi- cation in the symmetrically dividing progenitors of gCBs and OPCs (and likely also TCPs). By con- trast, EGF signals are dispensable (in high doses even unfavorable) for the development of progenitors of oCBs and their sibling OPCs. Thus, EGF signaling clearly has a lineage-specific func- tion, which is most easily explained by a requirement for progenitor selection and cell fate specifica- tion. This interpretation does not preclude contributions to cell survival (which might depend on differentiation) or lineage-specific divisions (i.e. correct progenitor specification is a prerequisite of the subsequent final division). Notably, in most hemisegments of the analyzed EGF pathway mutants, the number of gCBs is reduced by even numbers and remaining gCB pairs are usually of the same subtype regarding Lbe expression, arguing for a requirement prior to completion of the final mitotic division at the progenitor stage. Since we have only minor evidence for apoptosis and fate conversions into other cell types in EGF-related mutants (minor increase in oCBs, overall reduc- tion of PCs) we propose that many of the missing gCBs are not selected as highly Delta-expressing CB progenitors upon reduced MAPK signaling activity (Carmena et al., 2002; Grigorian et al., 2011; Hartenstein et al., 1992). Instead, they are likely retained by default within a pool of undiffer- entiated dorsal mesoderm cells. Our overexpression studies demonstrate that the timing of EGF signals is crucial for their function in differential progenitor specification. In previous studies, earlier functions of MAPK signaling might have obscured its specific impact on gCBs and OPC subtypes. While early pan-mesodermal activa- tion of MAPK signaling or expression of constitutive active Pnt forms via the twi-GAL4 driver reduces the numbers of all cardiac cells except the Eve+ progenitors (Alvarez et al., 2003; Bidet et al., 2003; Liu et al., 2006; and our own data), later MAPK activation favors formation of the symmetri- cally dividing OPC, TPC and gCB progenitor subpopulations (e.g. as seen in our experiments with tinD-GAL4-driven rho). Schwarz et al. eLife 2018;7:e32847. DOI: https://doi.org/10.7554/eLife.32847 20 of 36 Research article Research article Developmental Biology Since this study focuses mainly on the second, Pnt-dependent cardioblast diversification function, we elucidate the regulatory circuitry within each cardioblast lineage more extensively in the para- graphs further below. Prior to that, we briefly discuss our findings regarding the EGF signaling func- tion during CB progenitor formation. EGF signaling and cardiac progenitor selection We propose that the specification of these progenitors requires the context of the definitive cardiogenic mesoderm, whereas premature MAPK activation in all mesoderm cells negates any pro-cardiogenic effects due to the massive expansion of Eve+ clusters (which are nor- mally the first cells in the heart field to display MAPK and rho activity) at the expense of the cardiac progenitors in the neighboring C14/C16 clusters (Buff et al., 1998; Jagla et al., 2002; Liu et al., 2006; Qian et al., 2005; and our own data not shown). As discussed above, cardioblast formation as such is independent of pnt. How could this be achieved? Growth factor-activated MAPK can also phosphorylate the repressor Yan thereby dimin- ishing its activity as an antagonist of progenitor selection (Halfon et al., 2000; O’Neill et al., 1994; Rebay and Rubin, 1995). Therefore, it is conceivable that MAPK activity in the context of CB pro- genitor selection might be primarily required to eliminate the repressive activity of Yan. This would be consistent with the observed reduction of cardiac cells upon aop/yan hyperactivation (Halfon et al., 2000; this study). In this context, a minor function of Edl could contribute to the robustness of cardiac progenitor selection and thus total cardioblast and pericardial cell numbers by reducing the repressive Yan activity. A novel model for cardioblast diversification connecting EGF signaling ETS protein activity and lineage-specific transcription factor patterns S p ote act ty a d eage spec c t a sc pt o acto patte s Combining previous findings with our new data we have conceived the regulatory model of cardio- blast diversification illustrated in Figure 10. The central element of this model is the differential modulation of Pnt activity in the gCB and oCB progenitors leading to lineage-specific outcomes. Basic features of gene regulation in the gCB lineage Basic features of gene regulation in the gCB lineage We identified mid as a key target gene of Pnt in gCB progenitors based on its early gCB-specific expres- sion, Pnt-dependent transcriptional regulation and its ability to repress the oCB-specific regulator gene svp. Since Svp represses tin expression (Gajewski et al., 2000; Lo and Frasch, 2001), svp suppression 21 of 36 Schwarz et al. eLife 2018;7:e32847. DOI: https://doi.org/10.7554/eLife.32847 Research article Research article Developmental Biology provides an important part of the explanation for the previously reported positive role of Mid in maintain- ing tin expression in gCBs (Qian et al., 2005; Reim et al., 2005). Furthermore, expanded expression of tin in mid svp double mutants argues against the possibility that Mid stimulates tin expression directly. While Tin acts as a repressor of Doc via unknown mechanisms in gCBs, it does not repress svp (Zaffran et al., 2006; Figure 7—figure supplement 2D). On the contrary, at least in the early cardiogenic mesoderm, it acts as an activator of svp in oCB progenitors (Ryan et al., 2007). Thus, in the absence of appropriate repressors such as Mid, svp expression can expand into gCBs. Basic features of gene regulation in the oCB lineage Basic features of gene regulation in the oCB lineage In prospective oCB progenitors, Pnt activity must be kept in check to permit svp expression and thereby tin repression and Doc activity. Fittingly, we identified edl, a gene linked to negative regula- tion of MAPK signaling and cell identity determination in several tissues - including the eye (Yamada et al., 2003) and recently in certain somatic muscle progenitors (Dubois et al., 2016) - as a novel regulator in the context of cardiac cell specification, particularly that of oCB progenitor fate (green cell in Figure 10). This function is reflected by the over-proportional increase of svp-express- ing oCBs in pnt mutants first reported by (Alvarez et al., 2003). A novel model for cardioblast diversification connecting EGF signaling ETS protein activity and lineage-specific transcription factor patterns Our phenotypic analysis demon- strates that Edl is required for svp and Doc gene activity (the latter being due to restriction of tin expression) as well as the restriction of PntP2-dependent PntP1 expression in cardiac progenitors. Molecularly, Edl can modulate the activities of PntP2 as well as Yan (Baker et al., 2001; Qiao et al., 2006; Qiao et al., 2004; Tootle et al., 2003; Vivekanand et al., 2004; Yamada et al., 2003). The comparison of single and double mutant phenotypes, combined with the reproducibility of nearly all aspects of the cardiac pnt phenotype by Edl overexpression, implies that Edl acts primarily by inhib- iting Pnt during cardiac cell diversification, although we cannot fully exclude additional interactions with Yan. Our observations further support the function of Edl as an antagonist of Pnt (first demon- strated in the context of eye and chordotonal organ development; Yamada et al., 2003) and rule out an initially proposed Pnt-stimulating function (Baker et al., 2001). Linkage of MAPK and Pnt activities DOI: https://doi.org/10.7554/eLife.32847 22 of 36 Research article Research article Developmental Biology Developmental Biology be investigated, the sum of our genetic and enhancer data provide strong indications for mid being a direct and functionally critical target of Pnt during cardiac cell diversification. Thus, by regulating Pnt activity, the timing of Mid protein appearance can be controlled. We predict that this timing is linked to its capability to interfere with svp expression, since later presence of Mid in all CB subtypes including oCBs (mediated by other, Pnt-independent mechanisms; see Figure 9F,H,J) does not lead to svp repression. One possible explanation for the co-occurrence of Svp and Mid in oCBs at later stages is that the chromatin structure determining svp gene activity becomes fixed prior to the delayed appearance of Mid protein in these cells. be investigated, the sum of our genetic and enhancer data provide strong indications for mid being a direct and functionally critical target of Pnt during cardiac cell diversification. Thus, by regulating Pnt activity, the timing of Mid protein appearance can be controlled. We predict that this timing is linked to its capability to interfere with svp expression, since later presence of Mid in all CB subtypes including oCBs (mediated by other, Pnt-independent mechanisms; see Figure 9F,H,J) does not lead to svp repression. One possible explanation for the co-occurrence of Svp and Mid in oCBs at later stages is that the chromatin structure determining svp gene activity becomes fixed prior to the delayed appearance of Mid protein in these cells. y pp p Besides pntP1 and mid, there are very likely additional target genes activated by PntP2 and/or PntP1 to execute the differentiation program in generic working myocardial cells. Incomplete conversion of gCBs in mid mutants also calls for the existence of additional repressors that contribute to oCB fate sup- pression. Interestingly, a study investigating Tin target genes found that cardiac target enhancers of Tin are not only enriched for Tin-binding sites but also for a motif highly reminiscent of ETS binding sites, termed ‘cardiac enhancer enriched (CEE) motif’ (with the consensus ATT[TG]CC or GG[CA]AAT in anti- sense orientation) (Jin et al., 2013). Mutation of four CEE sites (one of which overlapping our predicted ETS binding site) in a ca. 600 bp version of the midE19 enhancer nearly abolished reporter activity in that study. Linkage of MAPK and Pnt activities Thus, many of the CEE-containing Tin target enhancers might in fact also be targets of Pnt (poten- tially mediating ETS-dependent activation) or Yan (potentially mediating ETS-dependent repression in the absence of MAPK signals). Therefore, a combination of closely spaced Tin and ETS binding sites might be a key signature in enhancers of working myocardial genes, although additional features must be present in their architecture to distinguish them from Tin+ETS binding site-containing enhancers active in pericardial cells or their progenitors (Halfon et al., 2000). The differences might include elements directly or indirectly regulated by Delta-Notch signaling. Notably, the juxtacrine Notch ligand Delta is upregu- lated in the CB lineage in an MAPK-activity-dependent manner (Grigorian et al., 2011). Hence, it is con- ceivable that Pnt proteins might stimulate Delta transcription in gCBs to control OPC development in a non-autonomous manner. This would explain both, simultaneous mis-specification of gCB progenitors and non-ostial-related OPCs in EGF mutants as well as phenotypic similarities between pnt mutants and mutants for components of the Delta-Notch signaling pathway. However, because of the herein described function of Pnt in suppressing svp transcription and oCB fate, pnt mutants feature an extreme bias in the increase of oCBs that has not been observed in Notch pathway mutants (Albrecht et al., 2006; this work). Research article Linkage of MAPK and Pnt activities Linkage of MAPK and Pnt activities The involvement of Edl also leads to important conclusions regarding the placement of Pnt function within the cardiac gene regulatory network. Based on the phenotypic discrepancies between pnt and other EGF pathway components (gain and loss of CBs, respectively), Alvarez et al. proposed that PntP2 acts independent of MAPK signaling to limit the number of CBs (Alvarez et al., 2003). Since we found that Edl blocks Pnt activity in oCB progenitors, and Edl is thought to antagonize PntP2 mainly by blocking MAPK-dependent phosphorylation (Qiao et al., 2006), we propose that PntP2 acts downstream of MAPK also during cardiogenesis (see Figure 10). This is further supported by our data demonstrating spi-sensitive cardiac expression of PntP1 and the observation that, if timed properly, both EGF and Pnt activities can lead to expanded gCB and reduced oCB populations. However, not all MAPK activities require pnt, which is the case for the pro-cardiogenic activities of EGF. Notably, parallel pnt-dependent and pnt-independent MAPK signaling functions take place also during other processes such as epithelial branching morphogenesis (Cabernard and Affolter, 2005). Special features of Pnt-dependent regulation in working myocardial cells Our model of CB diversification incorporates the observation that the PntP1 isoform is activated spe- cifically in gCB progenitors in a PntP2-dependent and EGF-sensitive fashion. This is reminiscent of the situation in other tissues such as the developing eye where the PntP1 isoform is also activated in a MAPK/PntP2-dependent manner (Gabay et al., 1996; O’Neill et al., 1994; Shwartz et al., 2013). We propose that PntP1 becomes activated at a particular threshold of MAPK/PntP2 activity. This activation marks a point of no return for CB diversification, because PntP1 cannot be inhibited via Edl. The activation of PntP1 also explains why edl overexpression with relatively late acting drivers such as tinD-GAL4 (as used in the edl mutant rescue experiment) does not cause the cardiac pheno- types observed with early pan-mesodermal drivers. Furthermore, depending on enhancer structure, target genes may be either quickly activated by PntP2 alone or require higher levels only achieved upon additional PntP1 buildup (particularly for sustained expression). In case of the mid gene, our model includes both possibilities (Figure 10 and Figure 10—figure supplement 1). Although the exact details of this activation as well as the direct binding of Pnt to particular sites in vivo remain to Schwarz et al. eLife 2018;7:e32847. What is the original signal that discriminates generic and ostial progenitors? A modula- tion of rho expression via Hh signaling, whether direct or indirect, would also be consistent with the phenotype of mutants lacking the function of patched (encoding a negative regulator of Hh signaling activity), in which we observe a strong increase in the gCBs:oCBs ratio (although absolute CB num- bers are highly variable between embryos and alleles; E. Heyland, F. Karama, B. Schwarz and I. Reim, unpublished observations). On the other hand, mutants with diminished Hh pathway activity, including some that were recovered by our EMS screen because of their partial CB losses (i.e. smoothened mutants), do not display a biased reduction of either oCBs or gCBs (E. Heyland, F. Kar- ama, B. Schwarz and I. Reim; unpublished observations). Hence, the regulation of rho and the role of hh during CB diversification await more detailed analysis. Factors that regulate edl expression levels might also determine the outcome of the competition between Edl and Pnt. The edl gene was found to be positively regulated by EGF signaling, and to be a target of Pnt and Yan, and thus was proposed to provide a negative feedback system for EGF inputs (Baker et al., 2001; Leatherbarrow and Halfon, 2009; Vivekanand et al., 2004; Yamada et al., 2003). Our extended model therefore includes regulation by Pnt as a possibility (dashed arrows in Figure 10—figure supplement 1). Nevertheless, additional or alternative inputs need to be considered to explain the strong edl expression in presumptive oCB progenitors with low Pnt activity. Notably, ChIP-on-chip experiments suggest that edl is also targeted by cardiogenic factors (Junion et al., 2012). Furthermore, edl was identified as a positively regulated target of Notch signaling in a Drosophila cell culture system (Krejcı´ et al., 2009). However, observed persis- tent edl expression in Notch pathway mutants argues against positive inputs from Notch during edl regulation in oCB progenitors. The spatio-temporal dynamics and detailed mechanisms that regulate MAPK and edl activities within the cardiogenic mesoderm remain to be investigated in future studies. Such studies may also help to understand lineage decisions in other tissues and species. Edl/Mae-relatives are also present in non-Dipteran insects (e.g. Tribolium; Bucher and Klingler, 2005), echinoderms, and the chordate Ciona. What is the original signal that discriminates generic and ostial progenitors? p oge to s Our work clearly identifies Pnt and Edl as crucial transducers of spatio-temporal inputs during car- diac cell diversification, but open questions remain regarding the initial source for the differential activities. Our model proposes that factors which tilt the balance between PntP2 activity and Edl will have a major impact on CB subtype choice (see Figure 10). Thus, any input that modestly increases MAPK/PntP2 activity within the appropriate window of time would favor gCB fate, whereas factors that have the opposite effect should promote oCB specification. This points to activities that impinge on the highly complex and dynamic expression of rho and/or edl. The Rhomboid protease is a key determinant in the decision of which cells will activate the more broadly expressed EGF Spitz and thus emanate signaling activity. A prime candidate for an instructive cue to anterior-posterior positioning within each segment could be Hh (indicated in the extended model in Figure 10—figure supplement 1), because it was proposed to be an oCB-promoting and rho/MAPK pathway-modulat- ing signal towards the cardiogenic mesoderm in previous studies (Liu et al., 2006; Ponzielli et al., 2002). In these studies, decreased svp expression and reduced numbers of Tin-negative CBs observed in hh mutants and upon overexpression of constitutive repressor forms of the Hh effector Ci were interpreted as signs of Hh-dependent oCB specification, although no converse effects have been reported using constitutive active Ci forms. However, the role of the Hh pathway in CB diversi- fication is not fully understood, mainly due to complications arising from ectodermal Hh functions, primarily in maintaining pro-cardiogenic wg expression (Bejsovec and Martinez Arias, 1991; Park et al., 1996). Furthermore, the effect of Hh on MAPK and rho activities in the dorsal mesoderm was suggested to be positive rather than negative based on an expansion of stage 12 mesodermal rho expression and expanded numbers of cells with activated MAPK upon pan-mesodermal overex- pression of hh (Liu et al., 2006). This would refute a function favoring oCB fate, but it is an Schwarz et al. eLife 2018;7:e32847. DOI: https://doi.org/10.7554/eLife.32847 23 of 36 Research article Research article Developmental Biology Developmental Biology interesting finding in light of our work, which couples rho activity with gCB specification. What is the original signal that discriminates generic and ostial progenitors? Although no clear ortholog of Edl appears to be present in vertebrates, a SAM domain-only isoform of the human Yan-relative TEL2 as well as Drosophila Edl were shown to inhibit transcrip- tional stimulation by the mammalian Pnt orthologs ETS1/ETS2 in cell culture (Gu et al., 2001; Vivekanand and Rebay, 2012). Hence, the restriction of ETS protein activities by protein-protein interactions offers an intriguing mechanism to fine-tune MAPK signaling output in developing tissues of both invertebrates and vertebrates. Materials and methods Key resources table Reagent type (species) or resource Designation Source or reference Identifiers Additional information Genetic reagent (Drosophila melanogaster) S-18a-13b-16b.1 PMID: 24935095 starter stock used for EMS mutagenesis; genotype: y[] w[]; P{RedH-Pelican.org-1-HN18-dsRed, w[+mC]}18a, P{pGD130.tinC-GFP, y[+]}13b, P{RedH-Pelican.HLH54Fb- dsRed, w[+mC]}16b Genetic reagent (D. melanogaster) S-18a-13b-16c.1 PMID: 24935095 starter stock used for EMS mutagenesis; genotype: y[] w[]; P{RedH-Pelican.org-1-HN18-dsRed, w[+mC]}18a, P{pGD130.tinC-GFP, y[+]}13b, P{RedH-Pelican.HLH54Fb- dsRed, w[+mC]}16 c Genetic reagent (D. melanogaster) aop[1] Bloomington Drosophila Stock Center BDSC:3101 Genetic reagent (D. melanogaster) bib[S1538] this paper mutation in S-18a-13b-16c.1 background Genetic reagent (D. melanogaster) Df(2R)edl-S0520 this paper mutation in S-18a-13b-16b.1 background Continued on next page Schwarz et al. eLife 2018;7:e32847. DOI: https://doi.org/10.7554/eLife.32847 24 of 36 Research article Schwarz et al. eLife 2018;7:e32847. DOI: https://doi.org/10.7554/eLife.32847 Schwarz et al. eLife 2018;7:e32847. DOI: https://doi.org/10.7554/eLife.32847 Developmental Biology Reagent type (species) or resource Designation Source or reference Identifiers Additional information Genetic reagent (D. melanogaster) edl[k06602] Bloomington Drosophila Stock Center BDSC:10633; FBal0057093 Genetic reagent (D. melanogaster) Df(2R)edl-L19 Y. Hiromi, PMID: 12874129 FBab0037748 Genetic reagent (D. melanogaster) P{edl.AF1}BS12; P{edl[+]} this paper derived from injection with pCaSpeR4-X18C12-edl_rescue; line # BS12 carries P{edl.AF1} on chromosome 3 Genetic reagent (D. melanogaster) Egfr[f2] Bloomington Drosophila Stock Center BDSC:2768 Genetic reagent (D. melanogaster) Egfr[S0167] this paper mutation in S-18a-13b-16b.1 background Genetic reagent (D. melanogaster) Egfr[S2145] this paper mutation in S-18a-13b-16c.1 background Genetic reagent (D. melanogaster) Egfr[S2307] this paper mutation in S-18a-13b-16c.1 background Genetic reagent (D. melanogaster) Egfr[S2561] this paper mutation in S-18a-13b-16c.1 background Genetic reagent (D. melanogaster) htl[YY262] PMID: 8957001 Genetic reagent (D. melanogaster) mam[S0669] this paper mutation in S-18a-13b-16b.1 background Genetic reagent (D. melanogaster) mam[S4648] this paper mutation in S-18a-13b-16c.1 background Genetic reagent (D. melanogaster) mid[1] Bloomington Drosophila Stock Center BDSC:3086 Genetic reagent (D. melanogaster) mid[S0021] this paper mutation in S-18a-13b-16b.1 background Genetic reagent (D. melanogaster) midE19-GFP M. Frasch; PMID: 23326246 Genetic reagent (D. melanogaster) mid180-GFP this paper insertion in attP2 Genetic reagent (D. melanogaster) mid180-mETS-GFP this paper insertion in attP2 Genetic reagent (D. melanogaster) pnt[MI03880] Bloomington Drosophila Stock Center BDSC:37615 Genetic reagent (D. melanogaster) pnt[D88] Bloomington Drosophila Stock Center BDSC:861 Genetic reagent (D. melanogaster) pyr[18] PMID: 19515694 Genetic reagent (D. melanogaster) pyr[S3547] PMID: 22609944 Genetic reagent (D. melanogaster) rho[7M43] Bloomington Drosophila Stock Center BDSC:1471 Genetic reagent (D. melanogaster) rho[L68] Bloomington Drosophila Stock Center BDSC:9095 Genetic reagent (D. melanogaster) S[S4550] this paper mutation in S-18a-13b-16c.1 background Genetic reagent (D. melanogaster) S[B0453] F. Schnorrer; PMID: 18327265 Genetic reagent (D. melanogaster) spi[S3384] this paper mutation in S-18a-13b-16c.1 background Continued on next page Schwarz et al. eLife 2018;7:e32847. DOI: https://doi.org/10.7554/eLife.32847 Developmental Biology Reagent type (species) or resource Designation Source or reference Identifiers Additional information Genetic reagent (D. melanogaster) spi[1] Bloomington Drosophila Stock Center BDSC:1859; FBal0016005 Genetic reagent (D. melanogaster) svp[AE127]-lacZ Y. Hiromi, PMID: 11404079 Genetic reagent (D. melanogaster) ths[759] PMID: 19515694 Genetic reagent (D. melanogaster) ‘tin-ABD;tin[EC40]’ PMID: 16987868 Genetic reagent (D. melanogaster) UAS-aop.ACT-IIa Bloomington Drosophila Stock Center BDSC:5789 Genetic reagent (D. melanogaster) UAS-edl-X Y. Hiromi, PMID: 12874129 Genetic reagent (D. melanogaster) ‘UAS-Egfr[DN].B-29-77-1; UAS-EgfrDN.B-29-8-1’; 2x EGFR[DN] Bloomington Drosophila Stock Center BDSC:5364 Genetic reagent (D. melanogaster) UAS-mid-B2 PMID: 15922573 Genetic reagent (D. melanogaster) UAS-pntP1-3 Bloomington Drosophila Stock Center BDSC:869 Genetic reagent (D. melanogaster) UAS-pntP2[VP16]2 C. Developmental Biology Schwarz et al. eLife 2018;7:e32847. DOI: https://doi.org/10.7554/eLife.32847 Kla¨mbt; PMID: 11051548 Genetic reagent (D. melanogaster) UAS-p35 Bloomington Drosophila Stock Center BDSC:5073 Genetic reagent (D. melanogaster) UAS-rho[EP3704] Bloomington Drosophila Stock Center BDSC:17276 Genetic reagent (D. melanogaster) UAS-rho(ve.dC) Bloomington Drosophila Stock Center BDSC:8858 Genetic reagent (D. melanogaster) UAS-svp M. Hoch Genetic reagent (D. melanogaster) how[24B]-GAL4; 24B Bloomington Drosophila Stock Center BDSC:1767 Genetic reagent (D. melanogaster) tinCD4-GAL4 M. Frasch; PMID: 11404079 Genetic reagent (D. melanogaster) tinD-GAL4 J. Weiss; PMID: 16221729 Genetic reagent (D. melanogaster) 2xPE-twi-GAL4 Bloomington Drosophila Stock Center BDSC:2517 Genetic reagent (D. melanogaster) Df(2L)Exel6006 Bloomington Drosophila Stock Center BDSC:8000 Genetic reagent (D. melanogaster) Df(2R)BSC25 Bloomington Drosophila Stock Center BDSC:6865 Genetic reagent (D. melanogaster) Df(2R)Exel7157 Bloomington Drosophila Stock Center BDSC:7894 Genetic reagent (D. melanogaster) Df(3R)Exel9012 Bloomington Drosophila Stock Center BDSC:7990 Genetic reagent (D. melanogaster) lbe-GFP Bloomington Drosophila Stock Center BDSC:55822 Genetic reagent (D. melanogaster) pnt-GFP Bloomington Drosophila Stock Center BDSC:42680 Recombinant DNA reagent pCaSpeR4-X18C12- edl_rescue (plasmid) Y. Hiromi, P MID: 12874129 P transformation plasmid for generation of P{edl.AF1} Continued on next page Schwarz et al. eLife 2018;7:e32847. DOI: https://doi.org/10.7554/eLife.32847 Developmental Biology Continued Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody anti-Doc2+3 (guinea pig polyclonal) PMID: 12783790 (1:2000, TSA) Antibody anti-Doc3+2 (guinea pig polyclonal) PMID: 12783790 (1:1000) Antibody anti-H15 (rabbit polyclonal) J. Skeath; PMID: 19013145 (1:2000) Antibody anti-H15 (guinea pig polyclonal) J. Skeath; PMID: 19013145 (1:2000) Antibody anti-Mid (rabbit polyclonal) J. Skeath; PMID: 19013145 (1:250, TSA or 1:1000) Antibody anti-PntP1 (rabbit polyclonal) J. Skeath; PMID: 12756183 (1:250) Antibody anti-Mef2 (rabbit polyclonal) H.T. Nguyen (1:1500) Antibody anti-Odd (rat polyclonal) PMID: 9683745 (1:600, TSA) Antibody anti-Eve (rabbit polyclonal) PMID: 2884106 (1:3000) Antibody anti-Tin (rabbit polyclonal) PMID: 9362473 (1:750) Antibody anti-Zfh1 (rabbit polyclonal) R. Schwarz et al. eLife 2018;7:e32847. DOI: https://doi.org/10.7554/eLife.32847 Lehmann; PMID: 9435286 (1:2000) Antibody anti-dpMAPK (mouse monoclonal) Sigma (1:500, TSA) Antibody anti-Seven-up 5B11 (mouse monoclonal) Developmental Studies Hybridoma Bank (1:20, TSA) Antibody anti-Wg 4D4 (mouse monoclonal) Developmental Studies Hybridoma Bank (1:30, TSA) Antibody anti-b-galactosidase 40- 1a (mouse monoclonal) Developmental Studies Hybridoma Bank (1:50, TSA or 1:20) Antibody anti-b-galactosidase (rabbit polyclonal) Cappel (1:1500) Antibody anti-GFP (rabbit polyclonal) Molecular Probes Molecular Probes:A6455 (1:2000) Antibody anti-GFP (rabbit polyclonal) Rockland Biomol:600-401-215 (1:1000) Antibody anti-GFP 3E6 (mouse monoclonal) Life Technologies Life Technologies:A11120 (1:100, TSA) Antibody anti-cleaved-Caspase-3 Asp175 (rabbit polyclonal) Cell Signaling Technology Cell Signaling Technology:#9661 (1:100, TSA) Antibody sheep anti-Digoxigenin (sheep polyclonal) Roche Roche:11333089001 (1:1000, TSA) Commercial assay or kit VectaStain Elite ABC-HRP kit Vector Laboratories Linaris:PK-6100 Commercial assay or kit tyramide signal amplification (TSA) reagent Cy3 PerkinElmer PerkinElmer: SAT704A001EA Commercial assay or kit tyramide signal amplification (TSA) reagent Fluorescein PerkinElmer PerkinElmer: SAT701001EA Commercial assay or kit TUNEL apoptosis detection kit (Apoptag) Millipore Millipore:S7100 Schwarz et al. eLife 2018;7:e32847. DOI: https://doi.org/10.7554/eLife.32847 Schwarz et al. eLife 2018;7:e32847. DOI: https://doi.org/10.7554/eLife.32847 27 of 36 Research article Research article Developmental Biology Drosophila melanogaster stocks The mutants bibS1538, Df(2R)edl-S0520, EgfrS0167, EgfrS2145, EgfrS2307, EgfrS2561, kuzS3330, kuzS3832, mamS0669, mamS4648, midS0021, midS2961, numbS1342, numbS3992, numbS4439, pyrS3547 (Reim et al., 2012), spiS3384, StarS4550 were recovered from our EMS screen. The lines mid1, UAS-mid-B2, how24B- GAL4, pnrMD237-GAL4, svpAE127-lacZ (a svp mutant in homozygous condition), UAS-svp.I, 2xPE-twi- GAL4, twi-SG24-GAL4, tinD-GAL4, UAS-tin#2, {tin-ABD}T003-1B1; tinEC40, UAS-p35 were as described previously (Reim et al., 2012; Reim et al., 2005; Zaffran et al., 2006). In addition, the fol- lowing strains were used: aop1 = aopIP (Nu¨sslein-Volhard et al., 1984; Rogge et al., 1995), UAS- aop.ACT-IIa (Rebay and Rubin, 1995), bib1 (Lehmann et al., 1983), edlL19 = Df(2R)edl-L19 (edl and some neighboring genes deleted) and UAS-edl-X (both from Y. Hiromi; Yamada et al., 2003), P {lacW}edlk06602 (Baker et al., 2001; To¨ro¨k et al., 1993), Egfrf2 (Clifford and Schu¨pbach, 1994), UAS-EgfrDN.B-29-77-1;UAS-EgfrDN.B-29-8-1 (Buff et al., 1998), htlYY262 (Gisselbrecht et al., 1996), kuze29-4 (Rooke et al., 1996), PBac{lbe-GFP.FPTB}VK00037 (A. Victorsen and K. White), mam8 (Lehmann et al., 1983), mid1 (Buescher et al., 2004), midE19-GFP (Jin et al., 2013; from M. Frasch), pntD88 (Scholz et al., 1993), pntMI03880 (PntP2-specific; harbors a gene-trap cassette with an artificial splice acceptor followed by stop codons upstream of the pntP1 transcription start site; Venken et al., 2011), UAS-pntP2VP16-2 (Halfon et al., 2000; originally from C. Kla¨mbt), UAS-pntP1- 3 and UAS-pntP2-2 (Klaes et al., 1994), PBac{pnt-GFP.FPTB}VK00037 (R. Spokony and K. White; Boisclair Lachance et al., 2014), pyr18 and ths759 (Klingseisen et al., 2009), rho7M43 (Ju¨rgens et al., 1984), rhoL68 (Salzberg et al., 1994), rhoEP3704 (Bidet et al., 2003), UAS-rho(ve.dC) (de Celis et al., 1997), spi1 = spiIIAIIA14 (Nu¨sslein-Volhard et al., 1984), StarB0453 (Chen et al., 2008; from F. Schnorrer), tinCD4-GAL4 (Lo and Frasch, 2001; from M. Frasch), Df(2R)Exel7157, and about 180 additional deficiencies spanning chromosome 2 (except where noted, all stocks available from the Bloomington Stock Center). Flies expressing edl+ from a transgene were generated anew by standard P-element transgenesis using the previously described edl[+t18] rescue construct (named AF1 in Yamada et al., 2003; pro- vided by Y. Hiromi). Line P{edl.AF1}BS12 carrying an insertion on chromosome three was used in this study. Unless noted otherwise, y w or S-18a-13b-16c.1 control (Hollfelder et al., 2014) flies were used as wild type controls. Schwarz et al. eLife 2018;7:e32847. DOI: https://doi.org/10.7554/eLife.32847 Mutant lines were maintained over GFP- or lacZ-containing balancer chromo- somes to allow recognition of homozygous embryos. Flies were raised at 25˚C, except for UAS/ GAL4-driven overexpression at 29˚C. Isolation and mapping of novel EMS mutants Novel EMS-induced mutants were obtained from our screen for embryonic heart and muscle defects and mapped to a particular gene through extensive complementation testing analogous to the pre- viously described procedure (Hollfelder et al., 2014). Many alleles were mapped by unbiased com- plementation tests with a set of chromosome 2 deficiencies and subsequent non-complementation of lethality and embryonic phenotype by previously described alleles. Df(2R)edl-S0520 was mapped by non-complementation of lethality with Df(2R)Exel7157, Df(2R)edl-L19 and Df(2R)ED3636, but the cardiac phenotype was only reproduced in trans with Df(2R)Exel7157, Df(2R)edl-L19 and edlk06602. Novel alleles of Egfr and Star were mapped using a candidate gene approach. Generation of reporter constructs for enhancer analysis Generation of reporter constructs for enhancer analysis The mid180-GFP reporter constructs were generated according to a similar lacZ construct published by Ryu et al. (2011). The forward primer 5’-EcoRI-CGTGCCTCCCACTTCAGGGCGG-3’ and the backward primer 5’-BamHI-TTAATTTCATTTTTCACTCTGCTCACTTGAGATTCCCCTGCTTTGTC TGCGGCATTTCCGCTTCT-3’ were used to amply DNA from y w flies. The predicted ETS binding site matching the antisense sequence of published ETS binding motifs (Halfon et al., 2000; Hollenhorst et al., 2011; underlined) was mutated in mid180-mETS-GFP by replacing the invariable TCC core (bold) with AAA in the backward primer. Amplicons were cloned into EcoRI/BamHI of pH- Stinger-attB (Jin et al., 2013), sequenced and inserted into the attP2 landing site via nos-driven FC31 integrase. Staining procedures Embryo fixations, immunostainings for proteins and RNA in situ hybridizations were carried out essentially as described (Knirr et al., 1999; Reim and Frasch, 2005), except for stainings with anti- dpMAPK, for which the formaldehyde concentration was doubled and embryos were rehydrated from methanol and stained immediately after fixation. VectaStain Elite ABC-HRP kit (Vector Labora- tories) and tyramide signal amplification (TSA, PerkinElmer Inc.) were used for detection of RNA and certain antigens (as indicated). The following antibodies were used: guinea pig anti-Doc2+3 (1:2000, TSA) and anti-Doc3+2 (1:1000) (Reim et al., 2003), rabbit anti-H15/Nmr1 (1:2000), guinea pig anti- H15/Nmr1 (1:2000), rabbit anti-Mid/Nmr2 (early stages: 1:250, TSA; late stages: 1:1000 direct) and rabbit anti-PntP1 (1:250, TSA) (all from J. Skeath; Alvarez et al., 2003; Leal et al., 2009), rabbit anti-Mef2 (1:1500) (from H.T. Nguyen), rat anti-Odd (1:600, TSA) (Kosman et al., 1998), rabbit anti- Eve (1:3000) (Frasch et al., 1987), rabbit anti-Tin (1:750) (Yin et al., 1997) (all from M. Frasch), rabbit anti-Zfh1 (1:2000) (from R. Lehmann; Broihier et al., 1998), mouse anti-dpMAPK (Sigma, 1:500, TSA), rabbit anti-b-galactosidase (Cappel, 1:1500), rabbit anti-GFP (Molecular Probes, 1:2000 and Rockland, 1:1000), mouse anti-GFP 3E6 (Life Technologies, 1:100, TSA), anti-cleaved-Caspase-3 (Asp175, Cell Signaling Technology, 1:100, TSA), sheep anti-Digoxigenin (Roche, 1:1000, TSA), monoclonal mouse antibodies anti-b-galactosidase 40-1a (1:20 direct or 1:50 with TSA), anti-Seven- up 5B11 (1:20, TSA) and anti-Wg 4D4 (1:30, TSA) (all from Developmental Studies Hybridoma Bank, University of Iowa), fluorescent secondary antibodies (1:200) (Jackson ImmunoResearch Laboratories and Abcam), biotinylated secondary antibodies (1:500) and HRP-conjugated anti-rabbit and anti- mouse IgG (1:1000) (Vector Laboratories). TUNEL staining was performed as described (Reim et al., 2003) using the Millipore ApopTag S7100 kit in combination with TSA. Digoxigenin-labeled antisense riboprobes against mid, edl, rho and pntP2 were used for whole mount in situ hybridizations. The mid probe was generated as described previously (Reim et al., 2005). T7 promoter-tagged edl, rho and pntP2 (isoform-specific exons) templates for in vitro tran- scription were generated by PCR (primers edl: CAATCGTGAAAGAGCGAGGGTC, T7-TGACGAG- CAGAACTAAGGACTAGGC, edlintron: GCACCGACGACTCAACTTCCTG, T7-GCTGCGATTGCGA TTACAAACAAG, pnt: CCAGCAGCCACCTCAATTCGGTC, T7-GCGTGCGTCTCGTTGGGGTAATTG, rho: ATGGAGAACTTAACGCAGAATGTAAACG, T7-TTAGGACACTCCCAGGTCG) from DNA of wild-type flies or flies carrying UAS-rho(ve.dC) or UAS-pntP2, respectively. Embryos were mounted in Vectashield (Vector Laboratories). Images were acquired on a Le SP5 II confocal laser scanning microscope and projected using Leica LAS-AF and ImageJ. Molecular analysis of mutations and deletions Several EMS alleles and the unmutagenized S-18a-13b-16c.1 control were analyzed by sequencing of overlapping PCR products covering the coding sequence and splicing sites of the candidate gene as described (Hollfelder et al., 2014). Details about the mutations are provided in Supplementary file 1-Table S1. The area deleted by Df(2R)edl-S0520 and its approximate break points were determined by iterative PCR amplification tests. The insertion of P{lacW}edlk06602 near the edl transcription start site was confirmed by PCR using primers binding to the 5’ P end and adja- cent genomic DNA. Although the integrity of the both P element ends could be confirmed by PCR, no genomic edl sequences expected next to the 3’ P end could be amplified using several primer pairs shown to amplify control DNA. This indicates that P{lacW}edlk06602 is associated with a deletion in edl. Details of the deletion mapping are listed in Supplementary file 2-Table S2. Schwarz et al. eLife 2018;7:e32847. DOI: https://doi.org/10.7554/eLife.32847 28 of 36 Research article Research article Developmental Biology Developmental Biology Additional information Funding Funder Grant reference number Author Deutsche Forschungsge- meinschaft RE 2985/1-1 Ingolf Reim The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. Funding Funder Grant reference number Author Deutsche Forschungsge- meinschaft RE 2985/1-1 Ingolf Reim The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. Author contributions Benjamin Schwarz, Conceptualization, Data curation, Formal analysis, Validation, Investigation, Meth- odology, Writing—original draft, Writing—review and editing; Dominik Hollfelder, Data curation, Investigation, Methodology; Katharina Scharf, Leonie Hartmann, Formal analysis, Investigation, Visu- alization; Ingolf Reim, Conceptualization, Data curation, Formal analysis, Supervision, Funding acqui- sition, Investigation, Visualization, Methodology, Writing—original draft, Project administration, Writing—review and editing Author ORCIDs Ingolf Reim http://orcid.org/0000-0001-8069-5532 Decision letter and Author response Decision letter https://doi.org/10.7554/eLife.32847.034 Author response https://doi.org/10.7554/eLife.32847.035 Supplementary files . Supplementary file 1. Table S1. Alleles with cardioblast patterning defects isolated and/or charac- terized in this study. The table lists the results from the genetic, phenotypic and molecular analysis of the characterized mutants. Indicated nucleotide positions are relative to transcription start site of transcript RA and amino acid positions of protein isoform PA (FB2017_01, released February 14, 2017; D. melanogaster R6.14); * indicates a nonsense mutation, n.d.: not determined. . Supplementary file 2. Table S2. Characterization of edl deletions via PCR. Presence (+) or absence (-) of DNA fragments after PCR reaction including genomic DNA from homozygous S-18a-13b-16c.1 control (WT), Df(2R)edl-S0520, edlk06602 or Df(2R)edl-L19 animals and primer pairs as indicated. CDS: part of coding sequence, TSS: transcription start site, n.d.: not determined. Amplicons are listed in linear order as located on chromosome 2R. * Six additional intronic GEFmeso amplicons were also negative in S0520. . Transparent reporting form . Transparent reporting form DOI: https://doi.org/10.7554/eLife.32847.032 DOI: https://doi.org/10.7554/eLife.32847.032 Data availability Acknowledgements We are grateful to Manfred Frasch for critical reading of the manuscript, Patrick Lo and Christoph Schaub for their contributions to the EMS screen, Edmar Heyland, Emi Vargatoth, Tanja Drechsler and Angela Bruns for technical assistance. We thank Manfred Frasch, Yasushi Hiromi, James Skeath, Hanh Nguyen, Frank Schnorrer, the Bloomington Stock Center and the Developmental Studies Hybridoma Bank (University of Iowa) for providing fly stocks or reagents. 29 of 36 Schwarz et al. eLife 2018;7:e32847. DOI: https://doi.org/10.7554/eLife.32847 Schwarz et al. eLife 2018;7:e32847. DOI: https://doi.org/10.7554/eLife.32847 Research article Research article Developmental Biology Albrecht S, Wang S, Holz A, Bergter A, Paululat A. 2006. The ADAM metalloprotease Kuzbanian is crucial for proper heart formation in Drosophila melanogaster. 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https://openalex.org/W1980432461	https://www.scielo.br/j/bjb/a/dFLP34NGK6SwhzjbbjRpZDv/?lang=en&format=pdf	English	null	Genetic diversity within and between broodstocks of the white shrimp Litopenaeus vannamei (Boone, 1931) (Decapoda, Penaeidae) and its implication for the gene pool conservation	Brazilian Journal of Biology	2,007	cc-by	3,667	Abstract Genetic variation within and between fifteen closed broodstock lines of the Pacific white shrimp Litopenaeus vannamei, reared at different hatcheries in the Brazilian coast, was assessed by RAPD analysis. Fifty two polymorphic loci were iden- tified when a set of five decamer primers was used in PCR. The genetic diversity analysis within lines evidenced genetic variation loss probably related to bottleneck effects and inbreeding. In addition, the genetic divergence values between the different samples appear to reflect the initial founder composition of such stocks, in some cases, sharing a common origin, suggesting a putative importance of interbreeding for the establishment of genetic improvement programs for these brood- stocks. The genetic variation monitoring appears to be helpful to the gene pool conservation of this aquaculture species, mainly if considered its exotic status in Brazil and the current impossibility of new introduction of wild individuals. Keywords: aquaculture, gene pool conservation, genetic variation. Resumo A variação genética existente dentro e entre quinze linhagens fechadas de reprodutores do camarão branco Litopenaeus vannamei, mantidas em diferentes laboratórios de larvicultura na costa brasileira, foi estudada utilizando análises RAPD. Através de um conjunto de cinco iniciadores decâmeros em PCR, foram identificados 52 locos polimórficos. A análise da diversidade genética dentro de cada linhagem evidenciou perda da variação genética provavelmente devida a efeitos de bottleneck e endocruzamento. Em adição, os valores de divergência genética entre as diferentes linhagens parecem refletir a composição inicial de fundação desses estoques, em alguns casos, compartilhando uma origem co- mum, sugerindo uma importância potencial do exo-cruzamento para estabelecer programas de melhoramento genético baseado nessas linhagens reprodutoras. O monitoramento da variação genética será muito útil para a conservação do conjunto gênico desta espécie de aquacultura, especialmente se for considerado seu status exótico no Brasil e que atualmente é impossível a realização de novas introduções de indivíduos selvagens. Palavras-chave: aquacultura, conservação do conjunto gênico, variação genética. Genetic diversity within and between broodstocks of the white shrimp Litopenaeus vannamei (Boone, 1931) (Decapoda, Penaeidae) and its implication for the gene pool conservation Freitas, PD., Calgaro, MR. and Galetti Jr., PM. Departamento de Genética e Evolução, Universidade Federal de São Carlos – UFSCar, Rodovia Washington Luiz, Km 235, CP 676, CEP 13565-905, São Carlos, SP, Brazil e-mail: patdf@iris.ufscar.br Received March 21, 2006 – Accepted April 12, 2007 – Distributed December 1, 2007 (With 2 fi ) Freitas, PD., Calgaro, MR. and Galetti Jr., PM. Departamento de Genética e Evolução, Universidade Federal de São Carlos – UFSCar, Rodovia Washington Luiz, Km 235, CP 676, CEP 13565-905, São Carlos, SP, Brazil e-mail: patdf@iris.ufscar.br Received March 21, 2006 – Accepted April 12, 2007 – Distributed December 1, 2007 Genetic diversity within and between broodstocks of the white shrimp Litopenaeus vannamei (Boone, 1931) (Decapoda, Penaeidae) and its implication for the gene pool conservation Freitas, PD., Calgaro, MR. and Galetti Jr., PM. Departamento de Genética e Evolução, Universidade Federal de São Carlos – UFSCar, Rodovia Washington Luiz, Km 235, CP 676, CEP 13565-905, São Carlos, SP, Brazil e-mail: patdf@iris.ufscar.br Received March 21, 2006 – Accepted April 12, 2007 – Distributed December 1, 2007 (With 2 figures) Genetic diversity within and between broodstocks of the white shrimp Litopenaeus vannamei (Boone, 1931) (Decapoda, Penaeidae) and its implication for the gene pool conservation Freitas, PD., Calgaro, MR. and Galetti Jr., PM. Departamento de Genética e Evolução, Universidade Federal de São Carlos – UFSCar, Rodovia Washington Luiz, Km 235, CP 676, CEP 13565-905, São Carlos, SP, Brazil e-mail: patdf@iris.ufscar.br Received March 21, 2006 – Accepted April 12, 2007 – Distributed December 1, 2007 (With 2 figures) 1. Introduction A long-term shrimp farming in the Brazilian coast can depend of how well we manage our national brood- stocks of Litopenaeus vannamei (Penaeidae), an exotic species from the American Pacific coast. In the past years, Brazilian farmers have quickly increased their shrimp production exclusively based on this shrimp prof- its and the country is currently placed among the major producers of captive shrimp in the Western hemisphere (Rocha et al., 2004). However, the lack of knowledge on the genetic structure of this shrimp could impair farm- ing activity since inbreeding and genetic drift effects can rapidly lead to problems such as disease susceptibility and reduced quality of breeders and post-larvae (Freitas et al., 2001; Freitas and Galetti, 2002). In the earlier years of the L. vannamei farming in Brazil, the broodstocks were formed through the impor- tation of wild animals from Panama, Ecuador, Mexico, and Costa Rica, and reared animals from Venezuela. However, in order to avoid the introduction of exotic pathogens, the importation of wild or reared animals is 939 Braz. J. Biol., 67(4, Suppl.): 939-943, 2007 Freitas, PD., Calgaro, MR. and Galetti Jr., PM. Freitas, PD., Calgaro, MR. and Galetti Jr., PM. seven Brazilian hatcheries was assessed by using RAPD analysis, in order to provide an original genetic data base useful for further breeding programs and for the gene pool conservation of this exotic shrimp in Brazil. not allowed since 1998, and new broodstock lines have been founded in the different Brazilian hatcheries by us- ing their own captive-born animals. The gene pool con- servation of this exotic species seems to be a great chal- lenge. Presently, there are several closed lines at these different hatcheries spread throughout the Brazilian coast. Each hatchery presents broodstocks with peculiar characteristics, related to their different origin and cap- tive conditions. 2.1 Broodstocks, sample collection, and DNA extraction Specimens of fifteen L. vannamei closed broodstock lines owned by seven Brazilian hatchery industries locat- ed at the States of Bahia (Lusomar and Maricultura), Rio Grande do Norte (Aquatec and Emparn), Paraíba (Compar and Aquamaris), and Santa Catarina (Ufsc), were analyzed (Table 1). Pleopod samples from 252 individuals were fixed in 1 mL of 95% ethanol and stored at –20 °C. DNA extraction was performed using a phenol: chloroform: iso- amylic alcohol mix (Sambrook et al., 1989). It is well known that a population can present a unique gene combination and the genetic structure of a set of reproductively isolated populations can mutually diverge (Barker, 1994). Moreover, the genetic variation has been claimed to be the original source to the suc- cessful development of captive stocks and it is believed that genetic variability loss restrains the possibilities of improvement of these animals (Allendorf and Ryman, 1987; Barker, 1994). Thus, an extensive genetic char- acterization of the Brazilian shrimp closed lines can be helpful to the development of breeding programs. 2.2 RAPD-PCR and electrophoresis analysis 2.2 RAPD-PCR and electrophoresis analysis 2.2 RAPD-PCR and electrophoresis analysis The Ready-To-GoTM RAPD Analysis Beads Kit (GE Healthcare Life Sciences) was used following the In the present work, the genetic diversity within and between fifteen L. vannamei broodstocks owned by Table 1. Data on the different studied broodstocks. Generation time (G), number of analyzed animals (N), and within Jac- card’s genetic similarity (SJ). Broodstock Hatchery G Origin N SJ Mar F7 Maricultura F7 Closed line founded with individuals imported from Panama (80%), Ecuador, Mexico, Costa Rica, and Venezuela (20%) 26 0. 692 Mar NHP Maricultura F7 Survived animals derived from Mar F 7 Stock (Maricultura) after contact with NHP bacteria 20 0.683 Luso F0 Lusomar F0 Founded with animals from SECOM and EDUARDO LEMOS Industries 31 0.696 Luso F2 Lusomar F2 Closed line since F0 30 0.720 Emp F0 Emparn F0 Founder animals from TECNARÃO Industry 08 0.699 Aqua F1 (1) Aquatec F1 Founder animals from Panama (F0) plus animals from LUSOMAR and SECOM Industries 20 0.699 Aqua F1 (2) Aquatec F1 Founder animals from Panama (F0) plus animals from SECOM Industry 20 0.705 Aqua F3 Aquatec F3 Closed line since F1 founded with animals from Panama (F0) plus animals from LUSOMAR Industry 18 0.699 Aqua F4 Aquatec F4 Animals from F3 plus animals from CAMANOR Industry 20 0.697 Aquama F0 Aquamaris F0 Nauplius and pos-larvae from AQUALIDER and COMPAR Industries, respectively 10 0.745 Com F0 (1) Compar F0 Animals from ponds of COMPAR Industry initially founded with animals from AQUATEC industry 10 0.688 Com F0 (2) Compar F0 Animals from ponds of COMPAR Industry initially founded with animals from AQUATEC industry 10 0.712 Com F0 (3) Compar F0 Animals from ponds of COMPAR Industry initially founded with animals from AQUATEC industry 10 0.646 Ufsc F0 UFSC F0 Nauplius from MARICULTURA Industry 10 0.712 Ufsc F2 UFSC F2 Closed line since F0 found with animals also from MARICULTURA Industry 10 0.733 Information on the origin of the animals of SECOM, EDUARDO LEMOS, TECNARÃO, and CAMANOR Industries was not available. Braz. J. Biol., 67(4, Suppl.): 939-943, 2007 940 Genetic diversity in shrimp manufacturer’s instructions. Five decamer primers were used after selection from a panel of 38 tested primers (Table 2). 2.2 RAPD-PCR and electrophoresis analysis The reactions were carried out in a PT-100TM thermal cycler (MJ Research) programmed as follows: 4 minutes of hot start at 94 °C, followed by 35 cycles of 1 minute of denaturation at 92 °C, 1.5 minute of hybridi- zation at 37 °C, and 2 minutes of extension at 72 °C, with a final 3 minutes extension at 72 °C. Fifty nanograms of template DNA and 25 pmoles of primer were used in a fi- nal solution volume of 25 µL, containing 0.4 mM of each dNTP, 2.5 µg BSA, 3 mM MgCl2, 30 mM KCl, 10 mM Tris (pH 8.3), milliQ water, AmpliTaqTM DNA polymer- ase, and Stoffel fragment (concentrations not provided by the manufacturer). The amplification products were ana- lyzed in 1.5% agarose gel with 0.5 µg.mL-1 of ethidium bromide (3 hours at 100 V in TBE buffer 1x), under ul- traviolet light. The Edas 290 (Kodak Digital Science TM) system was used for photodocumentation. branches were revealed in the genetic distance dendro- gram (Figure 2). A larger branch (1) joins all brood- stocks owned by Aquatec (Aqua F1 (1), Aqua F1 (2), Aqua F3, Aqua F4), and Compar (Com F0 (1), Com F0 (2), Com F0 (3)). A second branch (2) joins Luso F2 and Luso F0. Aquama F0 is alone in branch 3. Ufsc F0 and Ufsc F2 are joined in branch 4, Emp F0 is alone in branch 5, and finally Mar F7 and Mar NHP are joined in branch 6. 4. Discussion Two main phenomena that are greatly responsible for genetic variation loss in small and isolated breeder popu- lations in captive conditions are the founder effect and in- breeding (Barker, 1994). Breeder selection, the use of cap- tive-reared animals to establish subsequent broodstocks, and the reduced number of breeders (usually 100 dams and 100 sires) commonly used in the hatcheries can fa- vor the decreasing of the genetic diversity levels within a closed broodstock line (e.g. Sbordoni et al., 1986). 2.3 Statistical analysis Fragments of high reproducibility were selected for the statistical analyses, and binary matrixes based on the presence (1) or absence (0) of each fragment were con- structed. All statistical tools were based on the analysis of diploid data for dominant molecular data (Yeh et al., 1999). Jaccard’s genetic similarity coefficient (Jaccard, 1901) was obtained using the software NTSYS-pc ver- sion 1.80 (Rohlf, 1993). Within allele frequencies (Nei, 1987) and genetic distance (Nei, 1972) between the broodstocks were calculated using the software Popgene version 1.31 (Yeh et al., 1999). Allele frequency diver- gence was tested based on Chi-square analysis (P < 0.05) and homogeneity test between samples. A genetic dis- tance dendrogram based on the UPGMA grouping meth- od was generated (Sneath and Sokal, 1973). Although there is no available information on the ge- netic variation of wild L. vannamei populations by simi- Primer 2 Primer 3 Primer 4 Primer 5 Primer 6 Figure 1. RAPD reproducibility assay in agarose gel. The standardized RAPD reaction made in three distinct times with a single DNA sample using primers 2, 3, 4, 5, and 6. Figure 1. RAPD reproducibility assay in agarose gel. The standardized RAPD reaction made in three distinct times with a single DNA sample using primers 2, 3, 4, 5, and 6. 3. Results Figure 2. Dendrogram based on the genetic distance for the fifteen studied stocks. Branches are indicated by Arabic nu- merals (1-6). 1 2 3 4 5 6 Aqua F1 (1) Aqua F1 (2) Aqua F3 Aqua F4 Aquama F0 Com F0 (3) Com F0 (1) Com F0 (2) Luso F2 Luso F0 Ufsc F0 Ufsc F2 Emp F0 Mar F7 Mar NHP 1 2 3 4 5 6 Aqua F1 (1) Aqua F1 (2) Aqua F3 Aqua F4 Aquama F0 Com F0 (3) Com F0 (1) Com F0 (2) Luso F2 Luso F0 Ufsc F0 Ufsc F2 Emp F0 Mar F7 Mar NHP The five selected primers gave highly reproducible patterns (Figure 1), and a total of 52 polymorphic frag- ments were scored. The Jaccard’s genetic similarity (Sj) ranged from 64.6 to 74.5% (Table 1). 1 Allele frequencies were quite variable between sam- ples, presenting significant differences (P < 0.05) for most of the analyzed loci (data not shown). The small- est genetic distance was observed between Luso F0 and Luso F2 (0.0302), while the largest one was identified between Mar NHP and Emp F0 (0.1514) (Table 3). Mar F7 and Mar NHP were both the most divergent lines when comparing to the remaining ones. Six major Table 2. Decamer primers used in the RAPD reactions. Oligonucleotides Sequence (5’ - 3’) 2 (GE Healthcare Life Sciences) GGTGCGGGAA 3 (GE Healthcare Life Sciences) GTTTCGCTCC 4 (GE Healthcare Life Sciences) GTAGACCCGT 5 (GE Healthcare Life Sciences) AAGAGCCCGT 6 (GE Healthcare Life Sciences) AACGCGCAAC Table 2. Decamer primers used in the RAPD reactions. Table 2. Decamer primers used in the RAPD reactions. Figure 2. Dendrogram based on the genetic distance for the fifteen studied stocks. Branches are indicated by Arabic nu- merals (1-6). Braz. J. Biol., 67(4, Suppl.): 939-943, 2007 941 Freitas, PD., Calgaro, MR. and Galetti Jr., PM. ua 2) Aqua F3 Aqua F4 Aquama F0 Com F0 (1) Com F0 (2) Com F0 (3) Ufsc F0 Ufsc F2 Braz. J. Biol., 67(4, Suppl.): 939-943, 2007 References ABCC, 2002. O Agronegócio do camarão marinho cultivado. Recife, Associação Brasileira de Criadores de Camarão, 20p. ALLENDORF, FW. and RYMAN, N., 1987. Genetic management of hatchery stocks. In RYMAN, N. and UTTER, F. (eds.), Population Genetics and Fishery Management. Seattle, University of Washington Press, p. 141-159. BARKER, JSF., 1994. Animal breeding and conservation genetics. In Loeschcke, V., TOMIUK, J. and JAIN, SK. (eds.). Conservation Genetics. Switzerland, Birkhäuser Verlag Basel, p. 382-395. FREITAS, PD., CALGARO, MR., DE FRANCISCO, AK. and GALETTI JR., PM., 2001. Um pouco da genética dos nossos plantéis de reprodutores de Litopenaeus vannamei. Rev. ABCC, vol. 3, no. 1, p. 20-24. There is a trend for the clustering among lines owned by a single hatchery, resulting on distinct branches in the genetic distance dendrogram. The exception was observed in the branch #1 joining broodstocks owned by Aquatec and Compar. It is registered, however, that breeders from Aquatec lines were used to establish both Compar broodstocks studied here, explaining their more strict genetic relationship. The remaining studied brood- stocks were separated in different branches following their hatchery origin, reflecting the usual practice to begin a new line in each hatchery with genetic material present in their own living broodstocks. Thus, smaller genetic distance must be expected between broodstocks of a determined hatchery. FREITAS, PD. and GALETTI JR., PM., 2002. PCR-based VNTR core sequence analysis for inferring genetic diversity in the shrimp Litopenaeus vannamei. Genet. Mol. Biol., vol. 25, no. 4, p. 431-344. JACCARD, P., 1901. Estude comparative de la distribution florale dans une portion des Alpes et des Jura. Bull. Soc. Vaudoise Sci. Nat., vol. 37, p. 547-579. NEI, M., 1972. Genetic distance between population. Am. Nat., vol. 106, no. 50, p. 283-292. -, 1987. Molecular Evolutionary Genetics. New York, Columbia University Press, 512p. Despite of the absence of stock-specific markers, the established genetic relationships provide helpful information to compose new broodstocks. Currently in Brazil, there is a strong technological tendency focused on genetic improvement programs of L. vannamei, with the development of select lines for growth and pathogen- free/resistant individuals (ABCC, 2002). The utilization of genetically unrelated individuals as breeders can in- crease the levels of genetic variation within new stocks, mitigating possible negative effects caused by genetic drift and/or inbreeding (Thorpe et al., 2000). ROCHA, IP., RODRIGUES, J. and AMORIM, L., 2004. A carcinicultura brasileira em 2003. Recife, ABCC. ROHLF, FJ., 1993. enetic distance between the broodstocks. Mar F7 Mar NHP Luso F0 Luso F2 Emp F0 Aqua F1 (1) Aqua F1 (2) Aqua F3 Aqua F4 Aquama F0 Com F0 (1) Com F0 (2) Com F0 (3) Ufsc F0 Ufsc F2 The lines were founded by gene pools of different origin (Table 1) and the hatcheries have distinct broodstock management, such as different number of breeders in each spawning, usually ranging from 50 to 100 couples. In addition to the genetic drift due to using few breeders, the inbreeding that occurred along the subsequent generations and the selection pro- moted to distinct captive condition (in special weather and diseases) can also contribute for the significant allele divergence observed among the studied broodstocks. lar RAPD analysis, our results suggest that a significant genetic variation loss occurred in the studied broodstock lines and could be detect in subsequent generations of some closed lines. For instance, Sj values are slightly in- creased on Luso F0 to Luso F2 and Ufsc F0 to Ufsc F2. However, a strict correlation between generation time and genetic variation in overall was not found. Some brood- stocks (e.g. Aquama F0) at initial generation showed a reduced genetic diversity compared to other older lines (e.g. Mar F7). At least two explanations appear to ac- count for this finding. The lines were founded by gene pools of different origin (Table 1) and the hatcheries have distinct broodstock management, such as different number of breeders in each spawning, usually ranging from 50 to 100 couples. In addition to the genetic drift due to using few breeders, the inbreeding that occurred along the subsequent generations and the selection pro- moted to distinct captive condition (in special weather and diseases) can also contribute for the significant allele divergence observed among the studied broodstocks. enetic distance between the broodstocks. Mar F7 Mar NHP Luso F0 Luso F2 Emp F0 Aqua F1 (1) Aqua F1 (2) Aqua F3 Aqua F4 Aquama F0 Com F0 (1) Com F0 (2) Com F0 (3) Ufsc F0 Ufsc F2 Table 3. Genetic distance between the broodstocks. Mar F7 Mar NHP Luso F0 Luso F2 Emp F0 Aqua F1 (1) Aqua F1 (2) Aqua F3 Aqua F4 Aquama F0 Com F0 (1) Com F0 (2) Com F0 (3) Ufsc F0 Ufsc F2 Mar F7 - Mar NHP 0.0555 - Luso F0 0.0757 0.0528 - Luso F2 0.0891 0.0993 0.0302 - Emp F0 0.1038 0.1514 0.0830 0.0708 - Aqua F1 (1) 0.1178 0.0967 0.0501 0.0800 0.1021 - Aqua F1 (2) 0.0915 0.0973 0.0472 0.0533 0.0752 0.0428 - Aqua F3 0.1407 0.936 0.735 0.984 0.1273 0.0343 0.0547 - Aqua F4 0.1060 0.1044 0.0549 0.0780 0.0850 0.0591 0.0389 0.0602 - Aquama F0 0.1252 0.1168 0.1063 0.1120 0.1206 0.0652 0.0635 0.0822 0.0925 - Com F0 (1) 0.0904 0.0660 0.0504 0.0671 0.0818 0.0428 0.0330 0.0455 0.0589 0.0506 - Com F0 (2) 0.0961 0 0924 0.0475 0.0623 0.0666 0.0579 0.0623 0.0804 0.0506 0.0871 0.0372 - Com F0 (3) 0.0918 0.0598 0.0386 0.0738 0.0986 0.0335 0.0580 0.0564 0.0684 0.0826 0.0332 0.0477 - Ufsc F0 0.1083 0.1090 0.0789 0.1111 0.1235 0.0882 0.0820 0.0886 0.0485 0.0678 0.0538 0.0627 0.0835 - Ufsc F2 0.1440 0.1149 0.0975 0.1025 0.1254 0.0890 0.0969 0.0823 0.0770 0.0654 0.0592 0.0572 0.0951 0.0420 - Emp F0 Aqua F1 (1) Aqua F1 (2) Aqua F3 Aqua F4 Aquama F0 Com F0 (1) Com F0 (2) Com F0 (3) 942 Braz. J. Biol., 67(4, Suppl.): 939-943, 2007 Genetic diversity in shrimp Acknowledgments — This research was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and Associação Brasileira de Criadores de Camarão (ABCC). The authors thank the shrimp hatchery industry for the animal samples and useful information about the studied stocks. lar RAPD analysis, our results suggest that a significant genetic variation loss occurred in the studied broodstock lines and could be detect in subsequent generations of some closed lines. For instance, Sj values are slightly in- creased on Luso F0 to Luso F2 and Ufsc F0 to Ufsc F2. However, a strict correlation between generation time and genetic variation in overall was not found. Some brood- stocks (e.g. Aquama F0) at initial generation showed a reduced genetic diversity compared to other older lines (e.g. Mar F7). At least two explanations appear to ac- count for this finding. References NTSYS-pc: numerical taxonomy and multivariate analysis system, version 1.80. Exeter software. Setauket, New York. SAMBROOK, J., FRITSCH, EF. and MANIATIS, T., 1989. Molecular cloning: A Laboratory Manual. 2nd ed. New York, Cold Spring Harbor. SBORDONI, V., DE MATTHAEIS, M., COBOLLI- SBORDONI, M., LA ROSA, G. and MATTOCCIA, M., 1986. Bottleneck effects and the depression of genetic variability in hatchery stocks of Penaeus japonicus (Crustacea, Decapoda). Aquaculture, vol. 57, no. 1- 4, p. 239-251. Thus, the genetic relationships estimated here among fifteen broodstock lines seem to be an interesting tool for the development of efficient management programs for these animals. In addition, the results suggested that genetic variation monitoring might be very useful to the gene pool conservation currently available for the Brazilian aquaculture industry, mainly if considered the exotic status of this species and the current impossibility of new introductions of wild individuals. Further stud- ies using other nuclear (microsatellites) and mitochon- drial molecular markers could contribute to extend the knowledge of the overall genetic variation of the national broodstocks of L. vannamei. SNEATH, PA. and SOKAL, RR., 1973. Numerical Taxonomy. San Francisco, Freeman, 573p. THORPE, JP., SOLÉ-CAVA, AM. and WATTS, PC., 2000. Exploited marine invertebrates: genetics and fisheries. Hydrobiologia, vol. 420, no. 1, p. 165-184. YEH, F., YANG, R. and BOYLE, T., 1999. Popgene, version 1.31. Microsoft window-based freeware for population genetic analysis. University of Alberta, Edmonton. 943 Braz. J. Biol., 67(4, Suppl.): 939-943, 2007
https://openalex.org/W2521638074	https://infoscience.epfl.ch/record/274405/files/ncomms12789.pdf	English	null	Real-time visualization of conformational changes within single MloK1 cyclic nucleotide-modulated channels	Nature communications	2,016	cc-by	10,674	ARTICLE Received 11 Feb 2016 \| Accepted 1 Aug 2016 \| Published 20 Sep 2016 1 INSERM U1006, Aix-Marseille Universite´, Parc Scientiﬁque et Technologique de Luminy, 163 Avenue de Luminy, Marseille 13009, France. 2 Center for Cellular Imaging and NanoAnalytics, Biozentrum, University of Basel, Mattenstrasse 26, Basel CH-4058, Switzerland. 3 Departments of Anesthesiology, Physiology and Biophysics, and Biochemistry, Weill Cornell Medical College, 1300 York Avenue, New York, New York 10065, USA. Correspondence and requests for materials should be addressed to C.M.N. (email: crn2002@med.cornell.edu) or S.S. (email: simon.scheuring@inserm.fr). URE COMMUNICATIONS \| 7:12789 \| DOI: 10.1038/ncomms12789 \| www.nature.com/naturecommunications Results MloK1 channels display blinking behaviour. HS-AFM was used to image dynamics of ligand-induced structural changes in MloK1. For this, 2D crystals of wild-type MloK1, grown in the presence of cAMP15, were used. Large membranes with densely packed channels were imaged in 100 mM cAMP. High-resolution movies revealed individual channels as windmill-like structures formed by four clearly distinguishable CNBDs emerging 1.6±0.1 nm from the membrane (Fig. 1d). The structure and dimensions were in agreement with results obtained by conventional AFM15 and earlier work13; however, the fast HS-AFM imaging acquisition in the sub-second range (down to 80 ms) revealed that about 10% of the molecules dynamically switched between two conformations of different heights. These changes would not have been observable with conventional AFM techniques due to their low time resolution. A representative membrane area shows several individual molecules blinking (Fig. 2a, left; from an experiment performed in 10 nM cAMP) and switching back and forth between two height levels over time (Fig. 2a, right). In an attempt to measure the dwell-times in the up- and down-states, we monitored blinking molecules with scan rates between 80–800 ms per frame. Members of the family include the cyclic nucleotide-gated (CNG) and hyperpolarization-activated and cyclic nucleotide- gated (HCN) channels5,7. Both channel subfamilies belong to the S4 superfamily of voltage-gated cation channels that form tetramers whose subunits are aligned around a central pore. Each subunit consists of six transmembrane helices. The ﬁrst four form the voltage sensor (S1–S4) and the last two form the pore (S5–S6) (Fig. 1a). The CNBD is C-terminally connected to S6 via a C-linker, a highly conserved domain within the family. CNG and HCN channels have been studied extensively with electrophysiological methods, while structural approaches were not very successful so far. A prokaryotic homologue from the bacterium Mesorhizobium loti, MloK1, proved to be the best platform to study the structure- function relationship of CNM channels8. MloK1 is the only CNM channel for which an X-ray structure of the transmembrane region is available9. MloK1 also has X-ray and nuclear magnetic resonance structures of both liganded and unliganded isolated CNBDs, respectively10–12. Electron microscopy (EM) studies of full-length MloK1 (with and without ligand) as single particles and in two-dimensional (2D) crystals resulted in three-dimensional structures at 7–10 Å resolution13–15 that have been used for atomic modelling (Fig. 1b,c). ARTICLE ARTICLE I I on channels are integral membrane proteins facilitating ion ﬂux across cell membranes, thereby regulating signal pathways for physiological processes1. The activity of ion channels can be controlled by stimuli such as voltage, temperature, pH, mechanical stress and small signalling molecules such as Ca2 þ and cyclic nucleotides2. Cyclic nucleotide-modulated (CNM) ion channels are key players throughout the entire nervous system as they regulate certain modes of signal transduction3,4 and play a role in neuronal excitability in brain and pacemaking in heart cells5,6. They detect levels of intracellular cyclic AMP or GMP (cAMP or cGMP) by direct binding to a specialized cyclic nucleotide-binding domain (CNBD). This chemical information, that is, ligand binding, is translated into an electrical response by opening of the channel pore allowing ion ﬂow across the membrane. molecules gradually increase over time. Hence, we reasoned that the blinking process that we were able to capture using HS-AFM is speciﬁc to the unliganded CNBDs within a tetramer. The gradually increasing heights correspond to the gradually increasing number of unliganded, and hence blinking, subunits within the tetramer, during ligand depletion. We propose that upon ligand binding the channel switches from a set of ﬂexible conformations created by the highly dynamic unliganded CNBDs to an ordered conformation with stable, liganded CNBDs, and that these conformations are important for gating. Results A gating model was proposed, where the binding of ligand causes a movement of the CNBDs towards the membrane15. A similar movement, albeit larger in amplitude, was observed by conventional atomic force microscopy (AFM)15,16. All these techniques provided static views of these channels: full-length channels at medium resolution, and channel fragments at angstrom resolution. CNBDs a N Topology Structure top view 2 4 6 CNBD CNBDs Out In b c HS-AFM image d Out In Structure side view 1 3 5 Figure 1 \| Architecture of MloK1. (a) Topology of one subunit with six transmembrane segments (orange); the C-terminus contains the CNBD (yellow). The voltage sensor (S1–4) and the pore (S5–6) are in the transmembrane region. Atomic model based on the cryo-EM MloK1 structure (PDB 4CHV, ref. 15), in (b) side and (c) top views, respectively. The CNBDs arrange in a wind mill-like fashion (yellow outlines). (d) High- resolution HS-AFM topograph of MloK1 in 2D crystals in presence of 100 mM cAMP. Scale bar: 15 nm. Full colour scale: 2 nm. a N Topology 2 4 6 CNBD Out In 1 3 5 b a CNBDs Out In b Structure side view Structure side view g g We used high-speed AFM (HS-AFM)17 to explore the dynamics of MloK1 channels during gating and to study in real-time and with high resolution the conformational changes of MloK1 upon ligand binding/unbinding. HS-AFM allows the observation of single molecules with high lateral and temporal resolution (B1 nm and B100 ms, respectively) under near- physiological conditions (that is, in buffer solution, at ambient temperature and pressure, without labelling, staining or ﬁxing procedures) (Fig. 1d), ideal for monitoring conformational changes without the need for large ﬂuorescent labels that may prevent free protein motion18–21. We exploited the large difference in protrusion heights between liganded and unliganded MloK1 channels, previously also detected by conventional AFM15,16, to monitor by HS-AFM in real time the dynamics of single MloK1 channels upon ligand depletion. We show here that MloK1 channels, as well as individual subunits within a tetramer, can switch back and forth (blink) between conformational states with different heights protruding from the membrane. At saturating cAMP, where most channels are fully liganded, the molecules are mainly in a down-state with low height, close to the membrane, and the incidence of blinking is low. Real-time visualization of conformational changes within single MloK1 cyclic nucleotide-modulated channels Martina Rangl1, Atsushi Miyagi1, Julia Kowal2, Henning Stahlberg2, Crina M. Nimigean Martina Rangl1, Atsushi Miyagi1, Julia Kowal2, Henning Stahlberg2, Crina M. Nimigean1,3 & Simon Scheuring1 Eukaryotic cyclic nucleotide-modulated (CNM) ion channels perform various physiological roles by opening in response to cyclic nucleotides binding to a specialized cyclic nucleotide-binding domain. Despite progress in structure-function analysis, the conformational rearrangements underlying the gating of these channels are still unknown. Here, we image ligand-induced conformational changes in single CNM channels from Mesorhizobium loti (MloK1) in real-time, using high-speed atomic force microscopy. In the presence of cAMP, most channels are in a stable conformation, but a few molecules dynamically switch back and forth (blink) between at least two conformations with different heights. Upon cAMP depletion, more channels start blinking, with blinking heights increasing over time, suggestive of slow, progressive loss of ligands from the tetramer. We propose that during gating, MloK1 transitions from a set of mobile conformations in the absence to a stable conformation in the presence of ligand and that these conformations are central for gating the pore. 1 INSERM U1006, Aix-Marseille Universite´, Parc Scientiﬁque et Technologique de Luminy, 163 Avenue de Luminy, Marseille 13009, France. 2 Center for Cellular Imaging and NanoAnalytics, Biozentrum, University of Basel, Mattenstrasse 26, Basel CH-4058, Switzerland. 3 Departments of Anesthesiology, Physiology and Biophysics, and Biochemistry, Weill Cornell Medical College, 1300 York Avenue, New York, New York 10065, USA. Correspondence and requests for materials should be addressed to C.M.N. (email: crn2002@med.cornell.edu) or S.S. (email: simon.scheuring@inserm.fr). 1 NATURE COMMUNICATIONS \| 7:12789 \| DOI: 10.1038/ncomms12789 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12789 NATURE COMMUNICATIONS \| 7:12789 \| DOI: 10.1038/ncomms12789 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12789 On the right are the corresponding height proﬁles along the centre axis of the outlines in each frame on the left. During blinking the CNBDs undergo a height change of B1 nm. Scale bar: 20 nm. Full colour scale: 2.5 nm. (b) Cartoon of the blinking process: unliganded CNBDs alternate between down- and up-states. The kymograph (lower) plots the height evolution over 3 min for the eight MloK1 molecules highlighted in a. Colour display is identical. (c) Height histograms of the non-blinking (grey) and blinking (red) molecule from the kymograph in b (arrows). While the non-blinking channels show a one-peak distribution, the blinking channels display a broad height histogram. (d) High-resolution HS-AFM image frames showing an example of individual blinking subunits (arrows) within an MloK1 tetramer. Scale bar: 10 nm. Full colour scale: 2 nm. Buffer contains 10 nM cAMP for a–c, and 100 mM cAMP for d. 3.5 s 11.2 s 14.2 s 16.5 s 21.8 s 24.8 s 7.1 s 23.6 s 7.7 s 15.3 s 19.5 s 20.7 s d Height (nm) d Figure 2 \| MloK1 dynamically changes protrusion height during HS-AFM imaging. (a) Representative HS-AFM movie frames of the same MloK1- containing membrane. Arrows 1 and 2 highlight two individual molecules continuously blinking between two heights. On the right are the corresponding height proﬁles along the centre axis of the outlines in each frame on the left. During blinking the CNBDs undergo a height change of B1 nm. Scale bar: 20 nm. Full colour scale: 2.5 nm. (b) Cartoon of the blinking process: unliganded CNBDs alternate between down- and up-states. The kymograph (lower) plots the height evolution over 3 min for the eight MloK1 molecules highlighted in a. Colour display is identical. (c) Height histograms of the non-blinking (grey) and blinking (red) molecule from the kymograph in b (arrows). While the non-blinking channels show a one-peak distribution, the blinking channels display a broad height histogram. (d) High-resolution HS-AFM image frames showing an example of individual blinking subunits (arrows) within an MloK1 tetramer. Scale bar: 10 nm. Full colour scale: 2 nm. Buffer contains 10 nM cAMP for a–c, and 100 mM cAMP for d. visualize blinking of single CNBDs in a channel tetramer (Fig. 2d and Supplementary Movie 1) suggesting that each subunit can undergo these conformational changes individually. Results Upon lowering the cAMP concentration, the number and height of blinking CNBDs Structure top view c c d HS-AFM image d Structure top view CNBDs Figure 1 \| Architecture of MloK1. (a) Topology of one subunit with six transmembrane segments (orange); the C-terminus contains the CNBD (yellow). The voltage sensor (S1–4) and the pore (S5–6) are in the transmembrane region. Atomic model based on the cryo-EM MloK1 structure (PDB 4CHV, ref. 15), in (b) side and (c) top views, respectively. The CNBDs arrange in a wind mill-like fashion (yellow outlines). (d) High- resolution HS-AFM topograph of MloK1 in 2D crystals in presence of 100 mM cAMP. Scale bar: 15 nm. Full colour scale: 2 nm. Figure 1 \| Architecture of MloK1. (a) Topology of one subunit with six transmembrane segments (orange); the C-terminus contains the CNBD (yellow). The voltage sensor (S1–4) and the pore (S5–6) are in the transmembrane region. Atomic model based on the cryo-EM MloK1 structure (PDB 4CHV, ref. 15), in (b) side and (c) top views, respectively The CNBDs arrange in a wind mill-like fashion (yellow outlines). (d) High resolution HS-AFM topograph of MloK1 in 2D crystals in presence of 100 mM cAMP. Scale bar: 15 nm. Full colour scale: 2 nm. 2 NATURE COMMUNICATIONS \| 7:12789 \| DOI: 10.1038/ncomms12789 \| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| 7:12789 \| DOI: 10.1038/ncomms12789 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12789 NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12789 a b c 3 min 3.5 s 11.2 s 14.2 s 16.5 s 21.8 s 24.8 s 7.1 s 23.6 s 7.7 s 15.3 s 19.5 s 20.7 s d Blinking Down 50 Count 100 Stable down state molecule Blinking molecule 0 Height (nm) 0 1 2 3 4 1 nm 1 2 6.1 s 1 2 1 nm 8.2 s 1 nm 20.8 s 1 nm 25.7 s 1 nm 50.9 s 1 nm 51.6 s Figure 2 \| MloK1 dynamically changes protrusion height during HS-AFM imaging. (a) Representative HS-AFM movie frames of the same MloK1- containing membrane. Arrows 1 and 2 highlight two individual molecules continuously blinking between two heights. On the right are the corresponding height proﬁles along the centre axis of the outlines in each frame on the left. During blinking the CNBDs undergo a height change of B1 nm. Scale bar: 20 nm. Full colour scale: 2.5 nm. (b) Cartoon of the blinking process: unliganded CNBDs alternate between down- and up-states. The kymograph (lower) plots the height evolution over 3 min for the eight MloK1 molecules highlighted in a. Colour display is identical. (c) Height histograms of the non-blinking (grey) and blinking (red) molecule from the kymograph in b (arrows). While the non-blinking channels show a one-peak distribution, the blinking channels display a broad height histogram. (d) High-resolution HS-AFM image frames showing an example of individual blinking subunits (arrows) within an MloK1 tetramer. Scale bar: 10 nm. Full colour scale: 2 nm. Buffer contains 10 nM cAMP for a–c, and 100 mM cAMP for d. a 1 nm 1 2 6.1 s 1 2 1 nm 8.2 s 1 nm 20.8 s 1 nm 25.7 s 1 nm 50.9 s 1 nm 51.6 s b c 3 min Blinking Down b a Blinking c c 3 min 50 Count 100 Stable down state molecule Blinking molecule 0 Height (nm) 0 1 2 3 4 Stable down state molecule Blinking molecule Stable down state molecule Blinking molecule Count 3.5 s 11.2 s 14.2 s 16.5 s 21.8 s 24.8 s 7.1 s 23.6 s 7.7 s 15.3 s 19.5 s 20.7 s d Height (nm) Figure 2 \| MloK1 dynamically changes protrusion height during HS-AFM imaging. (a) Representative HS-AFM movie frames of the same MloK1- containing membrane. Arrows 1 and 2 highlight two individual molecules continuously blinking between two heights. ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12789 lose their ligands. We chose 10 nM instead of 0 nM cAMP because we wished to preserve a few molecules in an ordered, liganded conformation in the imaged membrane, as an important technical quality control. lose their ligands. We chose 10 nM instead of 0 nM cAMP because we wished to preserve a few molecules in an ordered, liganded conformation in the imaged membrane, as an important technical quality control. with Fig. 3c) of blinking molecules. In addition, the height of the non-blinking molecules in the vicinity of blinking ones remained constant over time. This strongly indicated that the increase in blinking over time was related to a gradual loss of cAMP from the CNBD binding pocket at low ligand concentration. q y As reported previously, complete removal of cAMP from the CNBDs of full-length MloK1 in dense reconstitutions is a slow process (hours to days) reﬂecting a slow off-rate of the ligand from the binding site11,15,22 Accordingly, the molecules were repeatedly imaged by HS-AFM over 90 min in 10 nM cAMP to capture their development over time (Fig. 3a and Supplementary Movie 2). After 30 min of incubation in low cAMP, the majority of the MloK1 molecules observed were still in the down-state, presumably cAMP-bound, and only 9±1% of the molecules were blinking (Fig. 3a, upper), and therefore no difference was observed from the high-cAMP condition. However, after re-imaging the same patch after 600 (Fig. 3a, middle) and then 900 (Fig. 3a, lower panel), the number of blinking MloK1 molecules steadily increased from B10 to B25% (Fig. 3b). This is illustrated in time-averaged topography maps (Fig. 3a, centre panels), in which blinking molecules appear as brighter, often blurry spots. The blinking is even more apparent in s.d. maps over all movie frames: the brighter the area, the larger the height variability, indicating blinking (Fig. 3a, right). This increase in height variability (blinking) over time in the imaged area is highlighted in the s.d. histograms (Fig. 3c). Interestingly, the exit of ligand from the binding pockets appeared to be helped to some extent by the energy input of the HS-AFM tip. Molecules in membrane patches that were not imaged, maintained their low heights and windmill-like appearance after 2 h incubation in 10 nM cAMP (Fig. ARTICLE 3g), while repeatedly imaging a different region of the same membrane after the same amount of time in the same conditions led to an increase in molecule heights and blinking, as shown above (Fig. 3). Again, this did not happen in high-cAMP conditions, directly correlating the increase in blinking upon tip interaction with loss of cAMP (Fig. 3d). Binding of cAMP to ligand-free MloK1 channels is slow. To understand the association process of cAMP to MloK1 channels that have lost the ligand, we monitored an identical membrane over B4 h at different ligand concentrations. We ﬁrst imaged the channels in presence of 100 mM cAMP for B60 min (Fig. 4a, upper), after which the ligand was washed out, using a buffer exchange system23, and the channels were imaged several times in 10 nM cAMP for an additional hour. We stopped imaging in low-cAMP before all molecules lost their ligand, as evidenced by lack of blinking and maintained low height in at least half of the molecules (Fig. 4a, middle). At this point, we reintroduced cAMP (100 mM) into the buffer. The reintroduction of cAMP stopped the increase in the number of blinking molecules, lead to a decrease in blinking activity and a few molecules even reverted to The increase in the number of blinking molecules in low cAMP is not due to a systematic error from the scanning process or sample deterioration by the AFM tip because membranes imaged in 100 mM cAMP over the same period of time (Fig. 3d and Supplementary Movie 3) did not change in either the number (compare Fig. 3e with Fig. 3b and Supplementary Movie 2 with Supplementary Movie 3) or height variability (compare Fig. 3f c b e f CNBDs up (%) Incubation (min) 30 60 90 0 10 20 30 50 100 150 0.2 0.4 0.6 0.8 s.d. (nm) Count CNBDs up (%) Incubation (min) 30 60 90 0 10 20 30 30 min 60 min 90 min 50 100 150 0.2 0.4 0.6 0.8 s.d. (nm) Count 30 min 60 min 90 min 0.01 µM cAMP Single frame Average height s.d. s.d. 100 µM cAMP Single frame Average height Position change 0.01 µM cAMP a d g Figure 3 \| The number of blinking MloK1 channels increases over time at low ligand concentration. (a) Representative membrane imaged after 30 (upper), 60 (centre) and 90 (lower) minutes in 10 nM cAMP. ARTICLE (a) Represent Figure 3 \| The number of blinking MloK1 channels increases over time at low ligand concentration. (a) Representative membrane imaged after 30 (upper), 60 (centre) and 90 (lower) minutes in 10 nM cAMP. For each condition, a single HS-AFM frame (left), the average height (middle) and the s.d. map (right) of the complete movie are shown. Blinking channels are highlighted by circles. While the single frame only shows MloK1s with elevated CNBDs at a given time, the average height-map displays all molecules with elevated heights throughout the movie, and the s.d.-map highlights the pixels changing over time, a hallmark of blinking. (b) The percentage of blinking channels in 10 nM cAMP plotted as a function of incubation time (mean±s.d.). (c) Histograms of the s.d. of all pixel values in each movie frame from a membrane area of 12,750 nm2 (15,000 pixels) and B500 frames at 10 nM cAMP after 30 (red), 60 (yellow) and 90 (blue) minutes. An increase in the s.d. reﬂects increased blinking. (d–f) as described in a–c, but in the presence of 100 mM cAMP. In contrast to the low-cAMP condition, the number and intensity of blinking molecules stays virtually identical over time. (g) The height increase of the CNBD in low cAMP requires an input of energy provided by the AFM tip. The imaged membrane area shows an increasing number of blinking molecules (upper), while most MloK channels are in cAMP-bound conformation in the untouched membrane area (lower). The full colour scales and scale bars are identical for all AFM images and correspond to 3 nm (z) and 30 nm (x/y). Figure 3 \| The number of blinking MloK1 channels increases over time at low ligand concentration. (a) Representative membrane imaged after 30 (upper), 60 (centre) and 90 (lower) minutes in 10 nM cAMP. For each condition, a single HS-AFM frame (left), the average height (middle) and the s.d. map (right) of the complete movie are shown. Blinking channels are highlighted by circles. While the single frame only shows MloK1s with elevated CNBDs at a given time, the average height-map displays all molecules with elevated heights throughout the movie, and the s.d.-map highlights the pixels changing over time, a hallmark of blinking. (b) The percentage of blinking channels in 10 nM cAMP plotted as a function of incubation time (mean±s.d.). (c) Histograms of the s.d. ARTICLE For each condition, a single HS-AFM frame (left), the average height (middle) and the s.d. map (right) of the complete movie are shown. Blinking channels are highlighted by circles. While the single frame only shows MloK1s with elevated CNBDs at a given time, the average height-map displays all molecules with elevated heights throughout the movie, and the s.d.-map highlights the pixels changing over time, a hallmark of blinking. (b) The percentage of blinking channels in 10 nM cAMP plotted as a function of incubation time (mean±s.d.). (c) Histograms of the s.d. of all pixel values in each movie frame from a membrane area of 12,750 nm2 (15,000 pixels) and B500 frames at 10 nM cAMP after 30 (red), 60 (yellow) and 90 (blue) minutes. An increase in the s.d. reﬂects increased blinking. (d–f) as described in a–c, but in the presence of 100 mM cAMP. In contrast to the low-cAMP condition, the number and intensity of blinking molecules stays virtually identical over time. (g) The height increase of the CNBD in low cAMP requires an input of energy provided by the AFM tip. The imaged membrane area shows an increasing number of blinking molecules (upper), while most MloK channels are in cAMP-bound conformation in the untouched membrane area (lower). The full colour scales and scale bars are identical for all AFM images and correspond to 3 nm (z) and 30 nm (x/y). 4 NATURE COMMUNICATIONS \| 7 12789 \| DOI 10 1038/ 12789 \| t / t i ti e f 0.8 CNBDs up (%) Incubation (min) 30 60 90 0 10 20 30 30 min 60 min 90 min 50 100 150 0.2 0.4 0.6 0.8 s.d. (nm) Count 30 min 60 min 90 min s.d. 100 µM cAMP Single frame Average height Position change 0.01 µM cAMP d g c b CNBDs up (%) Incubation (min) 30 60 90 0 10 20 30 50 100 150 0.2 0.4 0.6 0. s.d. (nm) Count 0.01 µM cAMP Single frame Average height s.d. a 0.01 µM cAMP 100 µM cAMP Average height d g a Position change 30 min c b CNBDs up (%) Incubation (min) 30 60 90 0 10 20 30 50 100 150 0.2 0.4 0.6 0.8 s.d. (nm) Count f b ) e c Figure 3 \| The number of blinking MloK1 channels increases over time at low ligand concentration. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12789 However, blinking molecules had a height-change rate of typically 1–2 frames even at the fastest scan rates, suggesting that the dwell- times of the blinking states are faster than the time resolution of B10 s 1. This was the case over a wide range of cAMP concentrations (10 nM–1 mM). The blinking (Fig. 2b, top) can be captured in kymographs over extended imaging periods, illustrating the reversible nature of this conformational change in the channels (Fig. 2b, bottom). The blinking state illustrated here is reminiscent of the ill-deﬁned unliganded MloK1 channels, previously reported by conventional AFM15,16, but its height is dynamically changing. The up-down height switch of individual molecules (Fig. 2b) is reﬂected in a drastic broadening of the height distribution of blinking molecules, which was used as criterion for discerning blinking from non-blinking channels (Fig. 2c). As the blinking CNBDs ﬂuctuate in height, high-resolution imaging at the submolecular level is hard to achieve. However, in rare cases, we were able to The number of blinking MloK1 channels increases in low cAMP. Since MloK1 CNBDs imaged by AFM in ligand-free conditions display an increased height, which is in agreement with the unliganded EM structure showing a detachment of the CNBD from the membrane15, we hypothesized that the observed back and forth height changes seen in the blinking molecules are related to ligand dissociation from the channels. In this case, lowering the cAMP concentration should lead to more channels losing their ligands and an increase in the number of blinking molecules. Hence, we analysed MloK1 blinking at 10 nM cAMP, a concentration about ten times lower than the reported afﬁnity (KD B80 nM; ref. 22), and where most molecules are expected to NATURE COMMUNICATIONS \| 7:12789 \| DOI: 10.1038/ncomms12789 \| www.nature.com/naturecommunications 3 NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12789 As a control, an identical non-blinking molecule in the same membrane area showed no height increases over time (1.6±0.4 nm after 600 and 1.6±0.3 nm after 900; Fig. 5b) highlighting that the above-described gradual height increase is speciﬁc to the subset of blinking molecules and is not a degradation of the whole membrane. As a second control, a similar analysis of the blinking molecules in 100 mM cAMP, revealed that similar prolonged incubations and scanning did not lead to the gradual increase in heights as observed in 10 nM cAMP (compare Fig. 5a,c). a Single frame Average height s.d. Exchange to 0.01 µM cAMP 60 min in 100 µM 50 min in 10 nM 40 min in 100 µM Re-addition of 100 µM cAMP Imaging in 100 µM cAMP a Altogether, the gradual height increase in blinking molecules occurs only in the low ligand concentrations (Fig. 5d), and not when saturating cAMP is provided (Fig. 5e). Accordingly the gradual height increase is consistent with a progressive, one-by-one cAMP dissociation from individual CNBDs within a tetramer over time. Thus, in an ideal situation a direct classiﬁcation of molecules with 0, 1, 2, 3 or 4 CNBDs unbound should be possible using a height threshold approach. However, in this case, the contribution of an individual subunit to the height increase is too small and the variability of each state is too large precluding such an analysis (see Fig. 5). b c 20 0.2 0.3 0.4 0.6 s.d. (nm) Count 40 60 0.5 1 2 0.1 0.2 0.3 0.4 s.d. (nm) 3 4 100 µM 10 µM 100 µM Count (k) b c Hence, in order to correlate the height values with ligand loss, the average height for each blinking molecule (a measure of how many cAMP molecules have unbound from a tetramer) was plotted as a function of its s.d. (a measure of the ‘blinking activity’) (Fig. 5f). We found that molecules displayed a trend where larger heights corresponded to increased blinking activity up to a height of B2.5 nm (Fig. 5f, cluster 1). Molecules with further height increase to B3.1 nm displayed high blinking activity (cluster 2). We interpret cluster 1 to represent mainly tetramers that lost one or two ligands, while the molecules in cluster 2 comprised channels that approached the fully unliganded state. The few blinking molecules in the movies acquired in 100 mM cAMP populated cluster 1 (Fig. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12789 5g) suggesting that in saturating cAMP the majority of the CNBDs remain liganded. The detection of single CNBDs blinking independently within a tetramer (Fig. 2d and Supplementary Movie 1) lends strong support to the above- described hypothesis that the gradual height increase of the entire channel indeed represents a successive loss of ligands from each subunit in the tetramer. Interestingly, it appears that if one CNBD loses its ligand and starts blinking, then other subunits within the same tetramer are favoured to also lose their ligand and start blinking, suggesting that there is some crosstalk within the tetramer. This is evident from the multiple subunits blinking within a molecule in Fig. 2d, as well as in the trend of the height increase over time within the same molecules upon ligand depletion while neighbouring tetramers are not affected (Fig. 5a,d). Figure 4 \| Reversibility of CNBD blinking. (a) An identical membrane Figure 4 \| Reversibility of CNBD blinking. (a) An identical membrane imaged in presence of 100 mM cAMP (upper), then in 10nM cAMP (middle), and after re-addition of 100 mM cAMP (lower). Single HS-AFM frames (left), average height topographies (centre) and the s.d. maps (right) of the movies are shown. Scale bar: 50nm. Full colour scale: 3 nm. (b) Histograms of the s.d. of each original movie frame (n ¼ 200) consisting of 30,000 pixel at 100 mM cAMP (black), 10nM cAMP (blue), and after re-introduction to 100 mM cAMP (red). An increase in the s.d. distributions reﬂects increasing height distributions. (c) Histograms of the s.d. maps of the movies (a, right). The more blinking molecules, the higher the height variability. Figure 4 \| Reversibility of CNBD blinking. (a) An identical membrane imaged in presence of 100 mM cAMP (upper), then in 10nM cAMP (middle), and after re-addition of 100 mM cAMP (lower). Single HS-AFM frames (left), average height topographies (centre) and the s.d. maps (right) of the movies are shown. Scale bar: 50nm. Full colour scale: 3 nm. (b) Histograms of the s.d. of each original movie frame (n ¼ 200) consisting of 30,000 pixel at 100 mM cAMP (black), 10nM cAMP (blue), and after re-introduction to 100 mM cAMP (red). An increase in the s.d. distributions reﬂects increasing height distributions. (c) Histograms of the s.d. maps of the movies (a, right). The more blinking molecules, the higher the height variability. their initial lower height (Fig. 4a, bottom panel). NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12789 NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12789 b a Single frame Average height s.d. Exchange to 0.01 µM cAMP 60 min in 100 µM 50 min in 10 nM 40 min in 100 µM Re-addition of 100 µM cAMP c 20 0.2 0.3 0.4 0.6 s.d. (nm) Count 40 60 0.5 1 2 0.1 0.2 0.3 0.4 s.d. (nm) 3 4 100 µM 10 µM 100 µM Count (k) Imaging in 100 µM cAMP Figure 4 \| Reversibility of CNBD blinking. (a) An identical membrane imaged in presence of 100 mM cAMP (upper), then in 10nM cAMP (middle), and after re-addition of 100 mM cAMP (lower). Single HS-AFM frames (left), average height topographies (centre) and the s.d. maps (right) of the movies are shown. Scale bar: 50nm. Full colour scale: 3 nm. (b) Histograms of the s.d. of each original movie frame (n ¼ 200) consisting of 30,000 pixel at 100 mM cAMP (black), 10nM cAMP (blue), and after re-introduction to 100 mM cAMP (red). An increase in the s.d. distributions reﬂects increasing height distributions. (c) Histograms of the s.d. maps of the movies (a, right). The more blinking molecules, the higher the height variability. a Single frame Average height s.d. 60 min in 100 µM Imaging in 100 µM cAMP ligand, because in overview images the HS-AFM measures an average height over all four CNBDs. For instance, if only one CNBD in a tetramer would have lost its ligand, then the overall blinking height of that channel would be lower than if all four CNBDs would have lost the ligand and switch between up and down states. We thus performed a height analysis of individual blinking molecules. For this, single molecules were tracked within subsequent movies at different incubation times and their heights were compared. The non-blinking, stable molecules, showed an average protrusion height of 1.6±0.2 nm and ﬂuctuated just slightly after 30min incubation in 10 nM cAMP (Fig. 5a, grey trace). In the same conditions, the blinking molecules show larger ﬂuctuations and reach a higher height level of 2.0±0.3 nm (illustrated by the molecule tracked in Fig. 5a, red trace). The identical blinking molecule tracked after 600 and then 900 displayed progressively higher heights of 2.5±0.4 nm and 2.8±0.4 nm, respectively (Fig. 5a, yellow and blue traces, respectively), where the blinking height reached a plateau. ARTICLE ARTICLE NATURE COMMUNICATIONS \| 7:12789 \| DOI: 10.1038/ncomms12789 \| www.nature.com/naturecommunications ARTICLE of all pixel values in each movie frame from a membrane area of 12,750 nm2 (15,000 pixels) and B500 frames at 10 nM cAMP after 30 (red), 60 (yellow) and 90 (blue) minutes. An increase in the s.d. reﬂects increased blinking. (d–f) as described in a–c, but in the presence of 100 mM cAMP. In contrast to the low-cAMP condition, the number and intensity of blinking molecules stays virtually identical over time. (g) The height increase of the CNBD in low cAMP requires an input of energy provided by the AFM tip. The imaged membrane area shows an increasing number of blinking molecules (upper), while most MloK channels are in cAMP-bound conformation in the untouched membrane area (lower). The full colour scales and scale bars are identical for all AFM images and correspond to 3 nm (z) and 30 nm (x/y). NATURE COMMUNICATIONS \| 7:12789 \| DOI: 10.1038/ncomms12789 \| www.nature.com/naturecommunications 4 4 Discussion We captured the dynamics of the conformational changes of MloK1 channels upon cAMP unbinding using HS-AFM. As MloK1 loses ligands from its CNBDs, the molecule starts ‘blinking’, that is, the topography height of the CNBDs is switching back and forth between at least two levels. With time, the process tends towards higher heights that correspond to an increasing number of ligands unbound from the CNBDs, until all ligands have dissociated and all four CNBDs within a tetramer are blinking. The fully liganded and unliganded MloK1 states have been previously characterized with EM and AFM15,16 by presenting static snapshots of supposedly two distinct states. Our real-time imaging study provides an analysis of the conformational changes undergone in MloK1 as single molecules transit between several height regimes in the process of losing all four ligands over time at sub-KD cAMP concentration and show that the process is more complex than what a two-state model would propose. d e 1 3 4 2 0 1k 2k Count 0 1k 2k Count Height (nm) 1 3 4 2 Height (nm) 100 µM cAMP 0.01 µM cAMP e 0 1k 2k Count 1 3 4 2 Height (nm) 100 µM cAMP d 1 3 4 2 0 1k 2k Count Height (nm) 0.01 µM cAMP d e f 1 4 3 2 Average height (nm) 0.3 0.4 0.5 Height s.d. (nm) 0.2 c1 c2 0.6 g 1 4 3 2 Average height (nm) 0.3 0.4 0.5 Height s.d. (nm) 0.2 0.6 c1 c2 g p p The blinking process may represent a conformational change directly reﬂecting ligand binding and unbinding from a CNBD, where the bound state is represented by a down- and the unbound state by an up-conformation. Alternatively, it may represent an equilibrium process that occurs within each unliganded subunit where the subunit continuously switches between down- and up-conformations when the ligand is lost. Our results favour the latter explanation for the following reasons. First, in the presence of saturating ligand, the same molecule, and in some cases the same subunit, is observed to continuously blink over extended imaging periods. This is consistent with an unliganded molecule oscillating between conformations, as it is highly improbable that the same exact molecule continuously binds and unbinds ligand over an extended period of time. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12789 (g) correlation between average height and height s.d., at 100 mM cAMP. Mean±s.d. of non-blinking molecules (n ¼ 15) is shown as a grey square. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12789 NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12789 g f Height (nm) 1 4 3 2 Average height (nm) 1 4 3 2 Average height (nm) 0.3 0.4 0.5 Height s.d. (nm) 0.2 c1 c2 0.6 0.3 0.4 0.5 Height s.d. (nm) 0.2 0.6 c1 c2 d e 1 3 4 2 0 1k 2k Count 0 1k 2k Count Height (nm) 1 3 4 2 Height (nm) 100 µM cAMP b c a 1 2 3 4 5 Height (nm) 1 2 3 4 5 Height (nm) 1 2 3 4 5 Blinking molecule in 0.01 µM cAMP Blinking molecule in 100 µM cAMP 0.01 µM cAMP Non-blinking molecule in 0.01 µM cAMP 3.3 nm 1.7 nm Figure 5 \| MloK1 blinking heights increase over time in low cAMP. Height (nm) d e 1 3 4 2 0 1k 2k Count 0 1k 2k Count Height (nm) 1 3 4 2 Height (nm) 100 µM cAMP b c a 1 2 3 4 5 Height (nm) 1 2 3 4 5 Height (nm) 1 2 3 4 5 Blinking molecule in 0.01 µM cAMP Blinking molecule in 100 µM cAMP 0.01 µM cAMP Non-blinking molecule in 0.01 µM cAMP 3.3 nm 1.7 nm Height (nm) b c a 1 2 3 4 5 Height (nm) 1 2 3 4 5 Height (nm) 1 2 3 4 5 Blinking molecule in 0.01 µM cAMP Blinking molecule in 100 µM cAMP Non-blinking molecule in 0.01 µM cAMP 3.3 nm 1.7 nm as all of their CNBDs are now devoid of ligand. To investigate this, we imaged an MloK1 membrane patch that was dialyzed over 2 weeks in cAMP-free buffer, conditions shown previously to lead to the complete removal of cAMP from the sample15, and we indeed observed individual MloK1 channels blinking (Supplementary Fig. 1). We could not quantify this effect as the resolution of individual molecules in these conditions is low, as previously reported15,16. The alternating head-to-tail packing of the MloK1 tetrameric channels in the membranes leads to channel CNBDs facing the underlying mica support in the HS-AFM, so that the blinking of these molecules will result in instability of the entire membrane. This instability leads to the observed resolution loss in this experiment and explains the somewhat lower resolution of HS-AFM imaging under low cAMP conditions. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12789 a g f Height (nm) 1 4 3 2 Average height (nm) 1 4 3 2 Average height (nm) 0.3 0.4 0.5 Height s.d. (nm) 0.2 c1 c2 0.6 0.3 0.4 0.5 Height s.d. (nm) 0.2 0.6 c1 c2 d e 1 3 4 2 0 1k 2k Count 0 1k 2k Count Height (nm) 1 3 4 2 Height (nm) 100 µM cAMP b c 1 2 3 4 5 Height (nm) 1 2 3 4 5 Height (nm) 1 2 3 4 5 Blinking molecule in 0.01 µM cAMP Blinking molecule in 100 µM cAMP 0.01 µM cAMP Non-blinking molecule in 0.01 µM cAMP 3.3 nm 1.7 nm Figure 5 \| MloK1 blinking heights increase over time in low cAMP. (a) Height versus time representative trace of an individual channel after incubation for 30 (red), 60 (yellow) and 90 (blue) minutes in 10 nM cAMP. A non-blinking channel from the same patch is shown in grey. Over time the molecule traverses several height levels. Cartoons representing liganded and unliganded MloK1 are shown. (b) Height versus time representative trace of a non-blinking channel after 30, 60 and 90 min in 10 nM cAMP (same colour scheme as in a). In contrast to the blinking molecule, no progressive height increase could be detected. (c) Height versus time representative trace of a blinking channel at 100 mM cAMP after 30, 60 and 90 min (same colour scheme as in a). The molecule starts blinking at the second scanning round (60 min) and keeps blinking at the same height level throughout the entire experiment. Scale bar: 10 s (a–c). (d,e) Histograms of MloK1 heights after 30, 60 and 90 min incubation in 10 nM cAMP (d) or100mM cAMP (e), respectively. Colour code is as in a. As a reference, the average height (solid line) and s.d. (dashed line) of non-blinking down-state molecules (grey, n ¼ 18 (10 nM cAMP) and n ¼ 15 (100 mM cAMP) molecules) are shown. (f) Correlation between average height and height s.d. for each molecule at 10 nM cAMP. Colour code as above. Each cross in the distribution is the average height of a single molecule plotted against the s.d. from all 500 frames. Mean±s.d. of non-blinking molecules (n ¼ 18) are shown as a grey square. Blinking molecules pass through different height regimes (clusters c1 and c2, outlined by large circles). NATURE COMMUNICATIONS \| 7:12789 \| DOI: 10.1038/ncomms12789 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12789 The s.d. of height values of each individual frame (Fig. 4b) and the pixel-by-pixel s.d. along the time axis (Fig. 4c) indicate that reintroduction of cAMP led to fewer molecules blinking and to lower heights. However, full recovery of the windmill-like structures of the molecules could not be achieved even after 1 h. This suggests that recovery, which includes ligand binding to all four subunits and rearrangement to the windmill structure, is a slow process, of the same order as the ligand unbinding from the CNBD15. MloK1 channels blink to higher heights over time in low cAMP. If the blinking process in a channel is due to ligand loss from individual CNBDs, then the height to which channels blink will depend on how many CNBDs within the tetramer have lost their All of the above experiments predict that in the complete absence of ligands, MloK1 channels will blink continuously, 5 ARTICLE Discussion Second, the reported cAMP unbinding rate from the CNBD in the full-length MloK1 is about 0.2 s 1 on average24, which is much slower than the blinking frequency measured at our highest imaging rates (410 s 1). Third, the blinking rates do not depend on the cAMP concentration, which they should if the on-rate depended on the ligand abundance in the environment. Fourth, if a blinking subunit is the result of unliganding its CNBD, then a fully unliganded channel should be a mobile unit consisting of four blinking subunits. In contrast, if each blinking event represented binding and unbinding of ligand, then a fully unliganded channel would have a non-blinking, stable and resolvable conformation with higher height. The former is supported by our ﬁndings: the measured height s.d. is high for unliganded channels and fully unliganded channels remain unresolved and blink. 3 Average height (nm) Figure 5 \| MloK1 blinking heights increase over time in low cAMP. Figure 5 \| MloK1 blinking heights increase over time in low cAMP. (a) Height versus time representative trace of an individual channel after incubation for 30 (red), 60 (yellow) and 90 (blue) minutes in 10 nM cAMP. A non-blinking channel from the same patch is shown in grey. Over time the molecule traverses several height levels. Cartoons representing liganded and unliganded MloK1 are shown. (b) Height versus time representative trace of a non-blinking channel after 30, 60 and 90 min in 10 nM cAMP (same colour scheme as in a). In contrast to the blinking molecule, no progressive height increase could be detected. (c) Height versus time representative trace of a blinking channel at 100 mM cAMP after 30, 60 and 90 min (same colour scheme as in a). The molecule starts blinking at the second scanning round (60 min) and keeps blinking at the same height level throughout the entire experiment. Scale bar: 10 s (a–c). (d,e) Histograms of MloK1 heights after 30, 60 and 90 min incubation in 10 nM cAMP (d) or100mM cAMP (e), respectively. Colour code is as in a. As a reference, the average height (solid line) and s.d. (dashed line) of non-blinking down-state molecules (grey, n ¼ 18 (10 nM cAMP) and n ¼ 15 (100 mM cAMP) molecules) are shown. (f) Correlation between average height and height s.d. for each molecule at 10 nM cAMP. Colour code as above. Discussion If only three binding-sites are occupied with cAMP, the MloK1 channel starts to blink as the empty CNBD starts ﬂuctuating between up- and down-conformations. During extended immersion in low-cAMP, the tetramer then loses gradually all four ligands from the binding pockets, all CNBDs start blinking independently leading to a highly dynamic tetramer (Supplementary Movie 4). Multiple studies, as well as our work, report that the cAMP- CNBD interaction is so strong in MloK1 that only long dialysis (over several days) and lower-afﬁnity CNBD mutants allow cAMP removal11,15,22. In contrast, kinetic rates signiﬁcantly faster (milliseconds to seconds) were shown in stopped-ﬂow experiments using detergent-solubilized MloK124. In our experiments however, the channels are in a lipid bilayer and densely packed in 2D crystals, which may account for the differences in the rates. Furthermore, within an immobilized membrane adsorbed to the support in the HS-AFM, ligands unbinding from CNBDs may well rebind to it before being able to escape into bulk, leading to an apparent decrease in the off-rate. p g pp We found that HS-AFM imaging can favour cAMP removal from the CNBD and we hypothesized that the HS-AFM tip tapping lowers slightly the energy barrier for ligand dissociation so that a few more molecules on average lose their ligand. To estimate the required external energy, we considered typical HS-AFM condi- tions (that is, 100 pNnm 1 spring constant, 600 kHz resonance frequency, 1 nm free oscillation amplitude, and 0.9 nm setpoint amplitude) yielding in an impact force on the sample in the 10 18 Ns range per tap, which corresponds to about 1–2 kBT (refs 18,26). It is noteworthy that under the same imaging conditions but with saturating cAMP, the cAMP-bound CNBDs can be imaged faithfully over extended periods of time, suggesting that the tip does not damage the sample. The same holds true for numerous other proteins we and others have studied before18–21,27,28. Alternatively, the stirring of the solution layers around the protein through the HS-AFM tip scanning might account for a faster ligand-release rate than molecules that are not scanned. In conclusion, we show that MloK1 channels can blink in both presence and absence of cAMP. The blinking and accompanying height increase is cAMP-dependent and associated with structural changes occurring in the CNBDs upon ligand unbinding. Discussion Each cross in the distribution is the average height of a single molecule plotted against the s.d. from all 500 frames. Mean±s.d. of non-blinking molecules (n ¼ 18) are shown as a grey square. Blinking molecules pass through different height regimes (clusters c1 and c2, outlined by large circles). (g) correlation between average height and height s.d., at 100 mM cAMP. Mean±s.d. of non-blinking molecules (n ¼ 15) is shown as a grey square. A variable channel height caused by continuous subunit blinking in unliganded CNBDs offers an explanation for the discrepancy between the amplitudes of the CNBD movements NATURE COMMUNICATIONS \| 7:12789 \| DOI: 10.1038/ncomms12789 \| www.nature.com/naturecommunications 6 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12789 the low height, low blinking, and windmill-like appearance of the molecules, which does not happen in our experiments. We propose that instead of a simple bimolecular reaction where the CNBD binds ligand, the CNBDs in the tetramer can exist in two conformations, with an open lid, allowing free access of ligand to the binding pocket, or with a closed lid, sterically preventing ligand entrance or exit (Supplementary Fig. 2a). In support of this model, in the existing crystal structures the CNBD binding pocket is formed by a b-roll and a phosphate-binding cassette covered by an a-helical lid, the C-helix, which has been shown to adopt different conformations (Supplementary Fig. 2b)10–12. We propose that the lid-closed conformations, both ligand-bound and ligand-free, have long dwell-times in the tetramer so that ligand exit and entry are sterically hindered (thick arrows in Supplementary Fig. 2a). Thus, it is likely that during our experiments, we mostly image these long-lived closed-lid conformations, both liganded and unliganded. This steric hindrance can explain why it takes days to empty the MloK1 CNBDs of ligand when bathed in low ligand concentration15, and why supplying ligand to ligand-free channels does not lead to immediate recovery (on the time scale of hours for our experiment). We propose that tapping with the AFM tip provides energy to favour the opening of the lid and thus speeds up this rate-limiting step in the channel, allowing faster emptying of the CNBDs (minutes as opposed to days). Discussion The model also offers an explanation for the rare blinking events that nevertheless occur repeatedly within the same tetramer in the presence of cAMP; in the rare events that the lid closes without a ligand bound in high- cAMP conditions, it starts blinking, and the probability for re-binding is low since the lid-opening is the rate-limiting step. upon ligand removal observed with EM and conventional AFM15,16. In the EM structure, the CNBDs shifted only 3 Å away from the membrane, while the AFM experiments suggested a height increase of B15 Å between the liganded and unliganded states. Since the EM structure is an average over hundreds of thousands of individual molecules in the 2D crystal, molecules with different heights would be averaged together in the crystal to yield an average height lower than the maximum height observed with direct single molecule HS-AFM measurements. g The imaging resolution decreases dramatically as liganded channels become unliganded. The loss of resolution precluded in most cases the visualization of individual subunits in the tetramer upon ligand dissociation, consistent with the previously reported ﬂexibility of unliganded CNBDs9–12. However, in the highest resolution HS-AFM movies available, blinking of individual subunits within an MloK1 tetramer is observed, strongly supporting the hypothesis that the height increase upon ligand removal corresponds to individual subunits within the tetramer losing their ligands in succession. The gradual increase in measured heights indicates that the ligands unbind successively in low-cAMP condition, suggesting low cooperativity in ligand unbinding, as it has been previously suggested22,25. The observation of individual subunits within a tetramer blinking independently of the rest of the tetramer over extended periods of time corroborates this interpretation. However, the observation that blinking in one subunit favours blinking in a neighbouring subunit within the same channel suggests that there is some crosstalk between subunits within a tetramer such that unbinding of cAMP from one CNBD tends to increase the probability of ligand loss in the other CNBDs of the same molecule. All our observations indicate that the transition from a fully liganded to an unliganded MloK1 channel occurs gradually and can be described as following: in the fully liganded channel, the CNBDs are arranged close to the membrane and likely make contact with the membrane and/or the voltage sensor domain15, giving it the observed ordered windmill-like structure we image in high-cAMP. Discussion This transition from a channel with highly mobile unliganded CNBDs to an ordered, windmill-like arrangement of CNBDs upon ligand binding has likely a major role in the gating of MloK1 channels and of other CNG channels within the same family. We demonstrate here the unique capability of HS-AFM to study dynamic processes at the single unlabelled membrane protein level for the characterization of protein–ligand interactions and conformational changes that would be difﬁcult to impossible to investigate with any other technique. NATURE COMMUNICATIONS \| 7:12789 \| DOI: 10.1038/ncomms12789 \| www.nature.com/naturecommunications ARTICLE A high-speed atomic force microscope for studying biological macromolecules. Proc. Natl Acad. Sci. USA 98, 12468–12472 (2001). 18. Ando, T., Uchihashi, T. & Scheuring, S. Filming biomolecular processes by high-speed atomic force microscopy. Chem. Rev. 114, 3120–3188 (2014). 19. Casuso, I. et al. Characterization of the motion of membrane proteins using high-speed atomic force microscopy. Nat. Nanotechnol. 7, 525–529 (2012). HS-AFM imaging. A 1mm diameter muscovite mica plate was glued on a HS-AFM glass rod sample holder and mounted on the scanner. MloK1 2D crystals were adsorbed on freshly cleaved mica for 20 min. Subsequently the sample was rinsed with imaging buffer (10 mM Tris, pH 7.4, 150 mM KCl) containing 100 mM cAMP. To change the cAMP concentration, the buffer solution in the imaging pool was washed with ﬁve volumes of imaging buffer containing 10 nM cAMP, using an integrated constant-pressure and constant-ﬂow pump system23 (Harvard Instruments, USA). HS-AFM17,18 (RIBM, Japan) was operated in oscillating mode, equipped with ultra short cantilevers of 8 mm in length (USC, NanoWorld, Switzerland) with a spring constant of 0.15 N m 1, a resonance frequency of B600 kHz and a quality factor of B1.5 in buffer. The applied force to the sample was minimized by adjusting the free amplitude to B10 Å and the imaging amplitude setpoint to B90% (B9 Å) of the free amplitude (the force F can be estimated following F ¼ {kc[(1 a)(A02 AS2)]1/2}/Qc, where a is the ratio of amplitude reduction by frequency shift to total amplitude reduction (typically a ¼ 0.5), kc ¼ 0.15 N m 1 is the cantilever spring constant, and Qc ¼ 1.5 is the cantilever quality factor in liquid). Membrane areas of 150 150 nm2 to 250 250 nm2 were imaged, using 200 200 or 300 300 pixels per frame, respectively, with scan speeds between 300 and 600 ms per frame. 20. Preiner, J. et al. IgGs are made for walking on bacterial and viral surfaces. Nat. Commun. 5, 4394 (2014). 21. Chiaruttini, N. et al. Relaxation of loaded ESCRT-III spiral springs drives membrane deformation. Cell 163, 866–879 (2015). 22. Cukkemane, A. et al. Subunits act independently in a cyclic nucleotide- activated K( þ ) channel. EMBO Rep. 8, 749–755 (2007). 23. Miyagi, A., Chipot, C., Rangl, M. & Scheuring, S. High-speed atomic force microscopy shows that annexin V stabilizes membranes on the second 23. ARTICLE Miyagi, A., Chipot, C., Rangl, M. & Scheuring, S. High-speed atomic force microscopy shows that annexin V stabilizes membranes on the second timescale. Nat. Nanotechnol. doi:10.1038/nnano.2016.89 (2016). timescale. Nat. Nanotechnol. doi:10.1038/nnano.2016.89 (20 24. Peuker, S. et al. Kinetics of ligand-receptor interaction reveals an induced-ﬁt mode of binding in a cyclic nucleotide-activated protein. Biophys. J. 104, 63–74 (2013). 25. Nimigean, C. M. & Pagel, M. D. Ligand binding and activation in a prokaryotic cyclic nucleotide-modulated channel. J. Mol. Biol. 371, 1325–1337 (2007). 26. Guzman, H. V., Perrino, A. P. & Garcia, R. Peak forces in high-resolution imaging of soft matter in liquid. ACS Nano 7, 3198–3204 (2013). 27. Kodera, N., Yamamoto, D., Ishikawa, R. & Ando, T. Video imaging of walking myosin V by high-speed atomic force microscopy. Nature 468, 72–76 (2010). 28. Uchihashi, T., Iino, R., Ando, T. & Noji, H. High-speed atomic force microscopy reveals rotary catalysis of rotorless F1-ATPase. Science 333, 755–758 (2011). Data analysis. HS-AFM images were ﬁrst-order ﬂattened and contrast adjusted using laboratory-made routines in Igor Pro software (WaveMetrics, Lake Oswego, OR, USA). The movies were then drift corrected, by means of frame-to-frame cross-correlation, using a lab-made image analysis software plug-in for ImageJ29,30. Average topography and s.d. maps of the movies were calculated using the standard measurement tools in ImageJ. Protrusion heights (subtracting the minimum pixel value from the maximum pixel value), height averages and s.d.s were calculated of each single molecule, in a box of 20 20 nm2 around the centre of weight of the molecule in each frame. For each condition 2 or more membrane patches were analysed with movies consisting ofB500 frames each. 29. Fechner, P. et al. Structural information, resolution, and noise in high-resolution atomic force microscopy topographs. Biophys. J. 96, 3822–3831 (2009). 30. Husain, M., Boudier, T., Paul-Gilloteaux, P., Casuso, I. & Scheuring, S. Software for drift compensation, particle tracking and particle analysis of high-speed atomic force microscopy image series. J. Mol. Recognit. 25, 292–298 (2012). g for drift compensation, particle tracking and particle analysis of high-speed atomic force microscopy image series. J. Mol. Recognit. 25, 292–298 (2012). ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms12789 was washed with buffer SB with 0.2% DM and 40 mM imidazole. The protein was eluted with buffer SB with 0.2% DM and 500 mM imidazole. Detergent-solubilized MloK1 was mixed with E. coli polar lipid extract (Avanti Polar Lipids) at a lipid-to- protein ratio of 0.8–1.0 and dialyzed against detergent-free buffer (20 mM KCl, 20 mM Tris-HCl pH 7.6, 1 mM BaCl2, 1 mM EDTA, 0.2 mM cAMP) for 2D-crystallization in dialysis buttons for 5–10 days (in buffer with cAMP ligand) at 37 C (the dialysis buffer was exchanged every other day). After crystallization and before HS-AFM analysis, the C-terminal hexahistidine-tagged was cleaved for 2–3 h at 20 C with 200 NIH units of thrombin (Sigma) per mg of protein. To stop the reaction, 0.1 mM Pefabloc (Sigma) was added and sample was washed three times with crystallization buffer. Supernatant was removed after the 2D crystals had sedimented and fresh crystallization buffer added. was washed with buffer SB with 0.2% DM and 40 mM imidazole. The protein was eluted with buffer SB with 0.2% DM and 500 mM imidazole. Detergent-solubilized MloK1 was mixed with E. coli polar lipid extract (Avanti Polar Lipids) at a lipid-to- protein ratio of 0.8–1.0 and dialyzed against detergent-free buffer (20 mM KCl, 20 mM Tris-HCl pH 7.6, 1 mM BaCl2, 1 mM EDTA, 0.2 mM cAMP) for 2D-crystallization in dialysis buttons for 5–10 days (in buffer with cAMP ligand) at 37 C (the dialysis buffer was exchanged every other day). After crystallization and before HS-AFM analysis, the C-terminal hexahistidine-tagged was cleaved for 2–3 h at 20 C with 200 NIH units of thrombin (Sigma) per mg of protein. To stop the reaction, 0.1 mM Pefabloc (Sigma) was added and sample was washed three times with crystallization buffer. Supernatant was removed after the 2D crystals had sedimented and fresh crystallization buffer added. 14. Clayton, G. M., Aller, S. G., Wang, J., Unger, V. & Morais-Cabral, J. H. Combining electron crystallography and X-ray crystallography to study the MlotiK1 cyclic nucleotide-regulated potassium channel. J. Struct. Biol. 167, 220–226 (2009). 15. Kowal, J. et al. Ligand-induced structural changes in the cyclic nucleotide- modulated potassium channel MloK1. Nat. Commun. 5, 3106 (2014). 16. Mari, S. A. et al. Gating of the MlotiK1 potassium channel involves large rearrangements of the cyclic nucleotide-binding domains. Proc. Natl Acad. Sc USA 108, 20802–20807 (2011). 17. Ando, T. et al. Acknowledgements This work was funded by the ANR grants A*MIDEX program (ANR-11-IDEX-0001-02), ANR-Nano (ANR-12-BS10-009-01) and ANR-BBMS (ANR-12-BSV8-0006-01), and a European Research Council (ERC) Consolidator Grant (#310080). Data availability. The data that support the ﬁndings of this study are available from the corresponding author upon request. Data availability. The data that support the ﬁndings of this study are available from the corresponding author upon request. References S.S., C.M.N. and M.R. conceived and designed the research and wrote the manuscript and prepared the ﬁgures. C.M.N. provided the construct and bacterial strain. J.K. and H.S. expressed and puriﬁed protein, and performed 2D crystallization. M.R. performed HS-AFM experiments. M.R. and S.S. analysed the data. A.M. supported HS-AFM experiments and programmed data processing scripts. 1. Ashcroft, F. M. From molecule to malady. Nature 440, 440–447 (2006). 2. Hille, B. Ion Channels of Excitable Membranes 507 (Sinauer 3. Craven, K. B. & Zagotta, W. N. CNG and HCN channels: two peas, one pod Annu. Rev. Physiol. 68, 375–401 (2006). y 4. Kaupp, U. B. & Seifert, R. Cyclic nucleotide-gated ion channels. Physiol. Rev 82, 769–824 (2002). 5. Kaupp, U. B. & Seifert, R. Molecular diversity of pacemaker ion channels. Annu. Rev. Physiol. 63, 235–257 (2001). Additional information 6. Robinson, R. B. & Siegelbaum, S. A. Hyperpolarization-activated cation currents: from molecules to physiological function. Annu. Rev. Physiol. 65, 453–480 (2003). Supplementary Information accompanies this paper at http://www.nature.com/ naturecommunications Supplementary Information accompanies this paper at http://www.nature.com/ naturecommunications Competing ﬁnancial interests: The authors declare no competing ﬁnancial interests. Competing ﬁnancial interests: The authors declare no competing ﬁnancial interests. Methods Ml K1 MloK1 expression and puriﬁcation. Full-length MloK1 channels were expressed and puriﬁed as previously described8,13,15. Brieﬂy, a C-terminal hexahistidine- tagged MloK1 construct in a pASK90 expression vector was transformed in Escherichia coli BL21(DE3) cells (New England Biolabs) and protein expression was induced by addition of 0.2 mg l 1 anhydrotetracycline for 2 h. The cells were harvested, sonicated and the membranes solubilized with 1.2% n-decyl-maltoside (DM, Anatrace) for 2.5 h at 4 C. The solubilization buffer (SB) had 295 mM NaCl, 5 mM KCl, 20 mM Tris-HCl pH 8.0, 0.2 mM cAMP. During sonication 10% glycerol, 1 mM phenylmethylsulphonyl ﬂuoride were added. The extract was spun down at 37,000g and the supernatant was applied to a Co2 þ afﬁnity column, which MloK1 expression and puriﬁcation. Full-length MloK1 channels were expressed and puriﬁed as previously described8,13,15. Brieﬂy, a C-terminal hexahistidine- tagged MloK1 construct in a pASK90 expression vector was transformed in Escherichia coli BL21(DE3) cells (New England Biolabs) and protein expression was induced by addition of 0.2 mg l 1 anhydrotetracycline for 2 h. The cells were harvested, sonicated and the membranes solubilized with 1.2% n-decyl-maltoside (DM, Anatrace) for 2.5 h at 4 C. The solubilization buffer (SB) had 295 mM NaCl, 5 mM KCl, 20 mM Tris-HCl pH 8.0, 0.2 mM cAMP. During sonication 10% glycerol, 1 mM phenylmethylsulphonyl ﬂuoride were added. The extract was spun down at 37,000g and the supernatant was applied to a Co2 þ afﬁnity column, which Why is the ligand released in low cAMP conditions upon tapping and not in high cAMP? In the presence of ligand, the cAMP released from the binding pocket upon AFM tip tapping is immediately replaced and the probability of going into a cAMP-free state is low. At 10nM cAMP, the binding pocket remains empty and the subunit starts blinking. This predicts that reintroduction of ligand should ﬁll the empty CNBDs and recover 7 Competing ﬁnancial interests: The authors declare no competing ﬁnancial interests. 7. Matulef, K. & Zagotta, W. N. Cyclic nucleotide-gated ion channels. Annu. Rev Cell Dev. Biol. 19, 23–44 (2003). Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ Nimigean, C. M., Shane, T. & Miller, C. A cyclic nucleotide mo 8. Nimigean, C. M., Shane, T. & Miller, C. A cyclic nucleotide modulated prokaryotic K þ channel. J. Gen. Physiol. 124, 203–210 (2004). 9. Clayton, G. M., Altieri, S., Heginbotham, L., Unger, V. M. & Morais-Cabral, J. H. Structure of the transmembrane regions of a bacterial cyclic nucleotide-regulated channel. Proc. Natl Acad. Sci. USA 105, 1511–1515 (2008). How to cite this article: Rangl, M. et al. Real-time visualization of conformational changes within single MloK1 cyclic nucleotide-modulated channels. Nat. Commun. 7:12789 doi: 10.1038/ncomms12789 (2016). How to cite this article: Rangl, M. et al. Real-time visualization of conformational changes within single MloK1 cyclic nucleotide-modulated channels. Nat. Commun. 7:12789 doi: 10.1038/ncomms12789 (2016). 10. Altieri, S. L. et al. Structural and energetic analysis of activation by a cyclic nucleotide binding domain. J. Mol. Biol. 381, 655–669 (2008). This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ 11. Clayton, G. M., Silverman, W. R., Heginbotham, L. & Morais-Cabral, J. H. Structural basis of ligand activation in a cyclic nucleotide regulated potassium channel. Cell 119, 615–627 (2004). 12. Schu¨nke, S., Stoldt, M., Lecher, J., Kaupp, U. B. & Willbold, D. Structural insights into conformational changes of a cyclic nucleotide-binding domain in solution from Mesorhizobium loti K1 channel. Proc. Natl Acad. Sci. USA 108, 6121–6126 (2011). 13. Chiu, P.-L. et al. The structure of the prokaryotic cyclic nucleotide-modulated potassium channel MloK1 at 16 Å resolution. Structure 15, 1053–1064 (2007). r The Author(s) 2016 NATURE COMMUNICATIONS \| 7:12789 \| DOI: 10.1038/ncomms12789 \| www.nature.com/naturecommunications 8
https://openalex.org/W2537578685	https://avehjournal.org/index.php/aveh/article/download/146/115	English	null	Evaluating the cytotoxicity of contact lens multi-purpose solutions in an in vitro lens system	African vision and eye health	2,009	cc-by	6,245	S Afr Optom 2009 68(1) 4-11 S Afr Optom 2009 68(1) 4-11 Abstract Purpose: To investigate the relative cytotoxic ef fects of contact lens multipurpose solutions on cul tured crystalline lenses.l Conclusions: The results show that OPTI-FREE Express and ReNu MultiPlus solutions exhibited more cytotoxic effect compared to COMPLETE MoisturePlus solution. The findings support reports from previous clinical and laboratory studies. These results suggest that the in vitro approach herein pre sented would be a valuable system for relatively in expensive and repeatable laboratory investigations of the possible ocular surface reactions of ophthal mic solutions, cosmetics and pharmaceuticals at pre- and during commercial phases. Methods: A comparison of the fluorescence emis sion levels of cultured bovine lenses as affected by three hour experimental exposure to three contact lens multipurpose solutions (COMPLETE Mois turePlus, AMO; OPTI-FREE Express, Alcon; and ReNu MultiPlus, Bausch & Lomb) was carried out. The pre- and post-exposure fluorescence levels of the lenses were obtained and values were compared to baseline and control measurements. Results: The solutions yielded varying degrees of cytotoxicity, demonstrating significant (p < 0.01) reversible reduction of cellular viability levels of the cultured crystalline lenses as revealed by the degree of fluorescence emissions in the following Keywords: Contact lens multipurpose solutions; ocular lens; cell viability; cytotoxicity; Alamar Blue; biochemical assay; fluorescence. <matoriowo@yahoo.com> Received 2 November 2008; revised version accepted 5 March 2009 order (OPTI-FREE Express > ReNu MultiPlus > COMPLETE MoisturePlus multi-purpose solu tions). Evaluating the cytotoxicity of contact lens multi purpose solutions in an in vitro lens system O Matthew Oriowo O Matthew Oriowo Department of Optometry, College of Applied Medical Sciences, King Saud University, PO Box 10219, Riyadh, 11433 Saudi Arabia <matoriowo@yahoo.com> Department of Optometry, College of Applied Medical Sciences, King Saud University, PO Box 10219, Riyadh, 11433 Saudi Arabia Introduction will arm the practitioner with useful knowledge with respect to what to tell patients regarding the transient ocular irritation that is possible from the use of mul tipurpose contact lens solutions. Currently, the most common products for disinfecting contact lenses are The need for contact lens practitioners to have a basic understanding of the components and the tem porary ocular surface reactions from using contact lens care solutions cannot be overemphasised. This The South African Optometrist 4 BScOptom MSc PhD FAAO BScOptom MSc PhD FAAO The South African Optometrist The South African Optometrist O Matthew Oriowo - Evaluating the cytotoxicity of contact lens multipurpose solutions in an in vitro lens system S Afr Optom 2009 68(1) 4-11 multi-purpose solutions (MPS) that can be used to clean, disinfect and wet contact lenses. Such a solu tion must be potent enough to kill microbial patho gens that may be harboured on the contact lenses yet must also be particularly gentle to the eye. A recent study found that contact lens wear failure was related to the product or practitioner factor rather than pa tient-specific problems1, implying that practitioners should possess adequate knowledge and be able to advise patients on the relative ocular surface reactions to different contact lens solutions. and laboratory studies have observed that the COM PLETE® multi-purpose solution has minimal toxic ity compared to OPTI-FREE and ReNu solutions3, 4. The fact that a solution is more comfortable, that is, has minimal cytotoxic or sensitivity effects does not guarantee full efficacy against opportunistic patho gens. For instance, recent results from two inde pendent epidemiologic studies5, 6, found that ~50% - 55% of Acanthamoeba keratitis (AK) cases used COMPLETE MoisturePlus multi-purpose solution, resulting in a more than 15-fold increase in the risk of AK with COMPLETE MoisturePlus solution use. This led the manufacturer, Advanced Medical Optics (AMO) to voluntarily recall the COMPLETE Mois turePlus multi-purpose solution7. It is a common view among eye care providers that efficacy against pathogenic microbes cannot be compromised in an attempt to produce an irritancy- free contact lens care solution since contact lens wear and contaminated contact lens solutions are the main causes of microbial keratitis2 . For example clinical Contact lens multipurpose solutions (MPS) have different compositions as indicated in Table 1. Table 1. Composition of the various multipurpose solutions. Solutions Preservatives Surfactant/cleaner Buffer Other components (e.g. Multipurpose contact lens solutions Fourteen bottles of each of the three multipurpose solutions: ReNu MultiPlus (Bausch & Lomb, Ro chester, NY, USA); OPTI-FREE Express (Alcon Lab oratories, Fort Worth, TX, USA); and COMPLETE moistureplus (Advanced Medical Optics, Dublin, Ireland) were randomly purchased from commercial retail pharmacy stores as encountered by the public. The composition of the solutions is as shown in Table 1. Since contact lens MPS(s) have varying levels of antimicrobial, cleaning and lubricating activities26-29, they will inevitably present some variations in their level of ocular surface sensitivity. The OPTI-FREE Express MPS has been found to show the highest an tibacterial activity against Pseudomonas aeruginosa compared to ReNu and COMPLETE solutions29, which should suggest more ocular surface cytotoxic or cytosensitive effect. However, a recent study re ported that ReNu MultiPlus showed a more significant adverse ocular surface sensitivity effect compared to OPTI-FREE and COMPLETE solutions3. Hence, the objective of the present study was to investigate the relative cytotoxicity of three commonly used contact lens multipurpose solutions (COMPLETE moisture plus, OPTI-FREE Express and ReNu MultiPlus) to bovine lenses, using an in vitro approach. This in vitro approach employing the Alamar Blue™ biochemical assay with cultured ocular lens was recently intro duced30-32. A repeatable in vitro approach to perform comparative sensitivity evaluations among contact lens solutions would be essential, particularly when considering cost effectiveness, the need for rapid screening information and to avoid the traditional large variation that occurs with in vivo studies such as the Draize test using rabbits16, 17, 19, 33-36. The Alamar Blue™ bioassay method utilizes the fluorescence emission levels of cultured whole crystalline lens tissue, as measured with a fluorescence multi-plate reader. The Alamar Blue™ assay model has shown consistent repeatability in its ability to detect subtle cytotoxic changes in ocular lens culture and human conjunctival cell lines4, 30-32. The use of bovine crys talline lens is relevant practically and experimentally. The crystalline lens was chosen as an ocular tissue model for studying ocular tissue irritancy because; (1) The epithelium of the cornea and the lens have the same embryologic origin and are physiologically similar. (2) The main function of both structures is to focus an image on the retina. (3) The structural adap Crystalline Lens culture All culture ingredients were purchased from the Sigma Chemical Co. (St. Louis, MO, USA) unless otherwise stated. Whole crystalline lenses were ex cised under aseptic conditions from abattoir-obtained bovine eyes and placed in a custom 25-ml two-cham bered container. The bovine eyes, from two year old cattle stock were obtained within 1-2 hour post-mor tem, and held at room temperature until the dissection of the lenses, which occurred within 2 to 5 hour post- mortem. To isolate the lens, the posterior portion of the eyeball was aseptically dissected, the suspensory ligaments of the lens were cut, and the adhering vit reous removed. The lenses were immersed in M199 culture medium with 3% fetal bovine serum and 1% antibiotics (penicillin/streptomycin 100 units per ml) with sodium bicarbonate and HEPES as buffers for pH stability. The cultured lenses were incubated at 37°C in an air, 5% CO2 atmosphere when not under going experimental measurements. Introduction Electrolyte) COMPLETE Moisture Plus™ 0.0001% polyhexam ethylene biguanide (PHMB) Poloxamer 237 Sodium phos phate Potasium chloride, NaCl, EDTA (0.01%), Taurine, Propylene Glycol. ReNu MultiPlus 0.0001% polyaminopro pyl biguanide (PAPB) Poloxamine, hy droxyalkyl phospho nate Sodium bo rate/boric acid EDTA (0.1%), NaCl OPTI-FREE Express 0.001% polidronium chloride, 0.0005% myr istamidopropyl dimeth ylamine (MAPD) AMP-95, Tetronic 1304 Sodium citrate/Boric acid Sodium chloride (NaCl), sorbital, Ede tate disodium (EDTA, 0.5%) The COMPLETE® Moisture Plus™ multi-purpose solution has lubricant/conditioner called Hydroxypropyl methyl cellulose (HPMC) in its formulation. Table 1. Composition of the various multipurpose solutions. functions13-15. The reason for non-compliance among contact lens wearers is multifactorial and well docu mented in the literature16-20. The notion that ocular surface sensitivity to MPS may contribute to non- compliance and contact lens dropout is still controver sial3, 21. The manufacturers’ efforts to provide simpli fied care systems may lead practitioners and patients into a false sense of security and the use of disposable or frequent replacement lenses may cause them to place less emphasis on the cleaning of lenses22. The incidence and morbidity of contact lens-related mi Manufacturers indicate that the preservatives (that is, antimicrobial agents) and other additives in the so lutions have high molecular weight materials which should not normally penetrate the contact lens matrix. Hence preventing any build up to toxic levels8, 10. It has been a concern that MPS preserved with even low percentages of antimicrobial agents could cause ocular surface irritation. Within the last twenty years, a number of no-rub MPS have been introduced4, 10- 13. MPSs are convenient and simple to use, but may present a compromise of the cleaning and disinfecting 5 The South African Optometrist O Matthew Oriowo - Evaluating the cytotoxicity of contact lens multipurpose solutions in an in vitro lens system S Afr Optom 2009 68(1) 4-11 crobial keratitis have shown little change compared with reports in the late 1980’s6, 23, 24. Despite changes or claimed improvements to contact lens care solu tions, microbial keratitis is still a concern in contact lens wear today, particularly in extended contact lens wear24, 25. Thus, contact lens care solutions must be efficacious against any pathogenic expressions by the microbial flora in the ocular surface. tations of both tissues are designed to minimize light scatter, and (4) both the lens and cornea are avascu lar32, 36. Exposure of crystalline lenses to contact lens multi- purpose (MPS) solutions The dissected lenses were kept in culture medium for 48 hours to allow for adaptation of the lenses to the medium. After the 48 hours of placing the lenses in culture medium, 40 out of 55 excised lenses were randomly allotted to treatment (utilizing 10 lenses per each experimental solution) group, and 10 lenses for the control group. The same set of 10 lenses was utilized as control for the three experimental solution groups. Fifteen lenses were not included in the study due to physical damage during dissection. The 30 The South African Optometrist 6 The South African Optometrist O Matthew Oriowo - Evaluating the cytotoxicity of contact lens multipurpose solutions in an in vitro lens system S Afr Optom 2009 68(1) 4-11 lenses selected (10 for each MPS solution treatment) for exposure were completely submerged in the three experimental solutions, respectively, for three hours at room temperature under the biological flow hood. The experimental exposure solutions comprised undiluted contact lens MPS, poured directly from the dispens ing bottles into the culture chamber, each containing one lens. Untreated control lenses remained in the culture medium for three hours at room temperature under the flow hood. All procedures were done un der sterile conditions utilizing a biohazard approved biological flow hood. After the three hour exposure period to MPS, the treated lenses were rinsed and the MPS replaced with fresh culture medium, and all lenses were returned to the incubator. lenses selected (10 for each MPS solution treatment) for exposure were completely submerged in the three experimental solutions, respectively, for three hours at room temperature under the biological flow hood. The experimental exposure solutions comprised undiluted contact lens MPS, poured directly from the dispens ing bottles into the culture chamber, each containing one lens. Untreated control lenses remained in the culture medium for three hours at room temperature under the flow hood. All procedures were done un der sterile conditions utilizing a biohazard approved biological flow hood. After the three hour exposure period to MPS, the treated lenses were rinsed and the MPS replaced with fresh culture medium, and all lenses were returned to the incubator. Freshly prepared assay solution before each use at every measurement time point was to avoid possible precipitation of the assay dye. Biochemical assay and fluorescence measurementsl y fl Baseline fluorescence measurements were obtained for control and treated lenses prior to exposure, and at the 6, 12, 24, 48, and 96 hour time intervals post the 3-hour experimental exposure to contact lens MPS. Following each measurement session, all lenses were rinsed and returned to fresh culture medium. The biochemical assay system consisted of the Alamar Blue™ assay, 12 multiwell plates, and a fluorescence plate reader. The system quantifies and records the fluorescence intensity levels of cultured ocular lenses. The fluorescence measurement is based on the princi ple that as radiant light wavelength is absorbed by a substance (in this case, the crystalline lens immersed in Alamar Blue™ assay) with the excitation wave length of 530 nm, a certain amount of fluorescence emission will result at a longer wavelength (in this case at 590 nm). The Alamar Blue™ dye (obtained from MEDICORP Inc., Montreal, Canada) is used to quantify the viability level of living cells in vitro37. It incorporates resazurin and resorufin as a fluoromet ric-colorimetric oxidation-reduction (Redox) indica tor that fluoresces and changes colour in response to reduction resulting from cell metabolism37, 38. It has been reported that serum interferes with Alamar Blue™ fluorescence readings by inducing reduction of the dye39, therefore it was ensured that the experi mental assay medium was serum free. Exposure of crystalline lenses to contact lens multi- purpose (MPS) solutions Both the treated and control lenses were transferred into sterile 12-well flat bottom tissue culture plates (Costar, Cambridge, MA, USA) with one lens per well. The culture me dium containing serum was carefully aspirated, and the lenses rinsed twice with 6-ml of experimental me dium with no serum. Then 3.8-ml of the assay solu tion was added to each well containing a lens, using a sterile 250- µl adjustable pipette tip and an Eppendorf repeater pipette. A prior pilot study by the present in vestigator showed a two hour incubation period to be optimum for the assay to diffuse into the lenses for fluorescence measurement. Therefore at each measurement session, both control and experimental lenses were incubated for two hours in the assay solu tion, after which the fluorescence measurements were performed with a CytoFluor™ II fluorescence multi- well plate reader (PerSeptive Biosystems Inc., Fram ingham, MA, USA). Thirty minutes prior to perform ing the fluorescence measurements, the excitation / emission wavelength settings on the CytoFluor™ plate reader were adjusted to 530/590 nm with the sensitiv ity gain set at 50, and temperature at 37ºC. The plate reader probe was set to scan 10 different positions in each lens. Thus, an average of 10 readings was ob tained for every lens. Therefore, each fluorescence level data represent an average of 10 readings per scan for each lens at least six times through the study duration, amounting to a total of 2400 quantitative lens fluorescence intensity measures. The South African Optometrist Statistical analysis Results were expressed as the mean ± the standard deviation (S.D) of the mean. The data were analysed using the paired t-test and repeated-measures analy sis of variance (ANOVA). Repeated measures ANO VA was used to compare the control group with the treated groups of lenses across all six measurement time periods. A repeated measures ANOVA found a significantly interaction between time and group (p < 0.001). For within group analysis, the baseline (that is, pre-exposure) and follow up post-exposure fluo rescence readings of lenses in the same group at the predetermined intervals were compared. The data were also compared between groups at each time point. The Dunnett multiple statistical test was used for within group comparisons (comparing baseline to The Alamar Blue™ dye was diluted into the culture medium (modified M199) without serum, to 8% (v/v). The assay solution was prepared immediately be fore use at each measurement session using a 100 l Eppendorf® “Tip-Ejector” microlitre pipette. µ 7 The South African Optometrist O Matthew Oriowo - Evaluating the cytotoxicity of contact lens multipurpose solutions in an in vitro lens system S Afr Optom 2009 68(1) 4-11 and lenses treated with COMPLETE solution (n = 10) did not show significant changes in fluorescence profiles throughout the duration of the experiment (Table 4). The OPTI-FREE treated lenses exhibited a significant recovery at 12 hr, but with a rebounce at 24 hr (p = 0.00001) and eventually back to recov ery by 96 hr. The fluorescence levels of ReNu treated lenses did not show a recovery at 12 hr (p = 0.012), but exhibited gradual recovery beginning from 24 hr to baseline and control levels by the end of the 96 hr study (Table 3). subsequent measurements in the same group), and the Tukey-Kramer multiple comparisons test for between group comparison at baseline and the respective fol low-up time points. Any probability p value less than 0.01 was considered significant. The extent of cyto toxicity is judged by each p value which indicates the degree of significant effects as follows: p values equal or less than 0.01(significant); ≤ 0.001 > 0.0001 (high ly significant); and ≤ 0.0001 > 0.00001 (extremely significant). The total number of times each solution exhibited minimal to extremely significant effect will be used to determine and discuss the relative rank or der of cytotoxicity among the solutions. Results The results for the different relative cytotoxic ef fects of the solutions are shown in Tables 2 to 4 as the mean (±S.D). At 6 hours, lenses treated with OPTI- FREE and ReNu (Tables 2 and 3) demonstrated sig nificant cytosensitive effects with p values of 0.001 and 0.01, respectively. The control lenses (n = 10), Table 2. Descriptive statistics of the fluorescence data for lenses exposed to OPTI-FREE multipurpose contact lens solution (n = 10 for exposed lenses, and n = 10 for control lenses). Measurement time (hr) Fluorescence level (mean ± S.D.) p values (within group) Contrast to baseline p values (Between groups) Exposed versus Control Exposed Control Exposed Control Baseline 34656± 2855 34835± 3811 0.907 6 28007± 3866 37042± 3379 0.001 0.10 0.00003 12 33820± 3287 35692± 4192 0.574 0.62 0.281 24 27751± 3132 35431± 2360 0.00001 0.71 0.00002 48 27007 ± 2819 33647± 2834 0.00001 0.13 0.00005 96 32952± 3651 35725± 4585 0.245 0.60 0.153 Note: Baseline readings were taken before exposing lenses to MPS. The same set of control lenses were utilised for OPTI-FREE, Complete, and ReNu solution exposures. Values are presented as arbitrary fluorescent units. Grading the level of cytotoxicity at different time points: p = 0.01 (minimal), 0.001 (very adverse), < 0.0001 (extremely adverse). Table 3. Descriptive statistics of the fluorescence data for lenses exposed to ReNu multipurpose contact lens solution (n = 10 for exposed lenses, and n = 10 for control lenses). Measurement time (hr) Fluorescence level (mean ± S.D.) p values (within group) Contrast to baseline p values (Between groups) Exposed versus Control Exposed Control Exposed Control Baseline 34720± 3313 34835± 3811 0.944 6 28392± 5295 37042± 3379 0.005 0.10 0.001 12 28141 ± 5745 35692± 4192 0.012 0.62 0.002 24 31161 ± 2887 35431± 2360 0.016 0.71 0.002 48 35088± 2973 33647± 2834 0.820 0.13 0.282 96 35157± 4166 35725± 4585 0.585 0.60 0.775 The South African Optometrist 8 Table 2. Descriptive statistics of the fluorescence data for lenses exposed to OPTI-FREE multipurpose contact lens solution (n = 10 for exposed lenses, and n = 10 for control lenses). Statistical analysis In order to rank the order of cytotoxicity among the three solutions, it was considered that the level of significance at each time point would indicate the degree of cytotoxic effect of each solution. Therefore, judging from the p values for each solution at differ ent intervals as shown in Tables 2 - 4, the descending rank order of the cytotoxic effect is as follows: OPTI- FREE > ReNu > COMPLETE solutions, with OPTI- FREE showing the most cytotoxicity effect. Discussion The fact that the last decade has witnessed con tact lens wearers changing to disposable and frequent replacement soft lenses with multipurpose solutions requires the contact lens practitioner to have informed knowledge on the efficacy and relative irritancy of multipurpose contact lens care regimens3, 10, 25. Con tact lens multipurpose solutions contain quaternary ammoniums or polymers of hexamethylene biguanide (PHMB) as active preservative agents. Preservatives are widely used in agricultural and food chemistry to keep food fresh, and in the cosmetic industry to avoid spoilage or chemical changes by microbes such as bacteria and fungi. Preservatives are also commonly used as sanitizers for baby wipes, pool and spa, and as disinfection products in medical preparations such as eye drops or contact lens solutions4. i p From the findings in the present study it might be assumed that the COMPLETE moistureplus™ MPS would have relatively little sensitivity effect com pared to OPTI-FREE or ReNu. However, there is a caveat in the assumption in that the present study is an exploratory in vitro investigation as opposed to in vivo experiment in which actual ocular surface irritancy ef fects can be directly obtained. A future investigation is required to conduct a parallel study of in vitro and in vivo evaluation of sensitivity effects of contact lens MPSs, and study the relationship between ocular lens cytotoxicity findings and actual ocular surface sensi tivity effects. One possible explanation for the differ ence in the MPS cytotoxic effects is that OPTI-FREE solution has a completely different antimicrobial agent (0.0005% myristamidopropyl dimethylamine (MAPD), while ReNu and COMPLETE moisturep lus MPSs are both 0.0001% of polyhexamethylene biguanide (PHMB)-preservative based solution sys tems. The antimicrobial action of MAPD is not fully known but has been proposed as similar to the action of PHMB and chlorhexidine which causes cytoplas mic membrane damage leading to loss of essential cel lular components following binding to the cell wall40. Also, as shown in Table 1, the COMPLETE solution has hydroxypropyl methylcellulose (HPMC) as lubri cant in its formulations compared to ReNu and OPTI- FREE solutions. The findings in the present study indicate that the chemical variations which exist be tween the solutions would yield differential cytotoxic reactions. However, the results show that there will be Multipurpose solutions are classified as medical devices (class 2b) and can impregnate the contact lens during the soaking time and insertion on ocular surface. Results Descriptive statistics of the fluorescence data for lenses exposed to COMPLETE multipurpose contact lens solution (n = 10 for exposed lenses, and n = 10 for control lenses). Table 4. Descriptive statistics of the fluorescence data for lenses exposed to COMPLETE multipurpose contact lens solution (n = 10 for exposed lenses, and n = 10 for control lenses). Measurement time (hr) Fluorescence level (mean ± S.D.) p values (within group) Contrast to baseline p values (Between groups) Exposed versus Control Exposed Control Exposed Control Baseline 35599± 3881 34835± 3811 0.66 6 36381± 2389 37042± 3379 0.55 0.10 0.62 12 35537± 2763 35692± 4192 0.96 0.62 0.92 24 34470± 1903 35431± 2360 0.42 0.71 0.33 48 35205± 1727 33647± 2834 0.70 0.13 0.16 96 35744± 3658 35725± 4585 0.91 0.60 0.99 produced the most adverse ocular surface effect com pared to OPTI-FREE and COMPLETE solutions3. i Results Measurement time (hr) Fluorescence level (mean ± S.D.) p values (within group) Contrast to baseline p values (Between groups) Exposed versus Control Exposed Control Exposed Control Baseline 34656± 2855 34835± 3811 0.907 6 28007± 3866 37042± 3379 0.001 0.10 0.00003 12 33820± 3287 35692± 4192 0.574 0.62 0.281 24 27751± 3132 35431± 2360 0.00001 0.71 0.00002 48 27007 ± 2819 33647± 2834 0.00001 0.13 0.00005 96 32952± 3651 35725± 4585 0.245 0.60 0.153 N t B li di t k b f i l t MPS Th t f t l l tili d f OPTI FREE tistics of the fluorescence data for lenses exposed to OPTI-FREE multipurpose contact lens solution (n = nd n = 10 for control lenses). Table 2. Descriptive statistics of the fluorescence data for lenses exposed to OPTI-FREE multipurpose con 10 for exposed lenses, and n = 10 for control lenses). Note: Baseline readings were taken before exposing lenses to MPS. The same set of control lenses were utilised for OPTI-FREE, Complete, and ReNu solution exposures. Values are presented as arbitrary fluorescent units. Grading the level of cytotoxicity at different time points: p = 0.01 (minimal), 0.001 (very adverse), < 0.0001 (extremely adverse). Table 3. Descriptive statistics of the fluorescence data for lenses exposed to ReNu multipurpose contact lens solution (n = 10 for exposed lenses, and n = 10 for control lenses). Table 3. Descriptive statistics of the fluorescence data for lenses exposed to ReNu multipurpose contact lens solution (n = 10 for exposed lenses, and n = 10 for control lenses). Measurement time (hr) Fluorescence level (mean ± S.D.) p values (within group) Contrast to baseline p values (Between groups) Exposed versus Control Exposed Control Exposed Control Baseline 34720± 3313 34835± 3811 0.944 6 28392± 5295 37042± 3379 0.005 0.10 0.001 12 28141 ± 5745 35692± 4192 0.012 0.62 0.002 24 31161 ± 2887 35431± 2360 0.016 0.71 0.002 48 35088± 2973 33647± 2834 0.820 0.13 0.282 96 35157± 4166 35725± 4585 0.585 0.60 0.775 tistics of the fluorescence data for lenses exposed to ReNu multipurpose contact lens solution (n = 10 for 10 for control lenses). 5 8 8 O Matthew Oriowo - Evaluating the cytotoxicity of contact lens multipurpose solutions in an in vitro lens system S Afr Optom 2009 68(1) 4-11 Table 4. References 1. Young G, Veys J, Pritchard N, Coleman S. A multi-centre study of lapsed contact lens wearers. Ophthal Physiol Opt 2002 22 516-527. According to the findings in the present study, there is no indication that any of these solutions would result in permanent adverse cytotoxic dam age to the ocular surface tissue with clinical or pa tient care use. Concerning the trade-off between relative efficacy and comfort, the findings of relative cytotoxic effect between the three solutions as dem onstrated in the present study appear to agree with the findings of Leung et al.29, who found that OPTI- FREE Express showed the highest antimicrobial ac tivity against Pseudomonas aeruginosa compared to ReNu, COMPLETE and Solo-care solutions at 4°C, 25°C and 30°C, however this is not the focus of the present study. An ideal contact lens MPS would have both low cytotoxic effect and high antimicrobial ef ficacy. Efficacy will always be of paramount impor tance in contact lens multipurpose solutions because it has been found that even a standard contact lens care hygiene regime does not seem to be sufficient in preventing the development of corneal infection and ulcers in contact lens (particularly in convention al and frequent replacement daily wear soft contact lenses) wearers25, 43. In terms of in vitro methodology, 2. Dart JK, Stapleton F, Minassian D. Contact lenses and other risk factors in microbial keratitis. Lancet 1991 338 650-653. 2. Dart JK, Stapleton F, Minassian D. Contact lenses and other risk factors in microbial keratitis. Lancet 1991 338 650-653. 3. Lievens, C.W. Hakim, N., Chinn, A. The effect of multi purpose solutions on the ocular surface. Eye Contact Lens 2006 32 8-11. 3. Lievens, C.W. Hakim, N., Chinn, A. The effect of multi purpose solutions on the ocular surface. Eye Contact Lens 2006 32 8-11. 4. Dutot M, Warnet JM, Baudouin C, Rat P. Cytotoxicity of contact lens multipurpose solutions: Role of oxidative stress, mitochondrial activity and P2X7 cell death receptor activation. Euro J Pharmaceutical Sciences 2008 33 138- 145. 4. Dutot M, Warnet JM, Baudouin C, Rat P. Cytotoxicity of contact lens multipurpose solutions: Role of oxidative stress, mitochondrial activity and P2X7 cell death receptor activation. Euro J Pharmaceutical Sciences 2008 33 138- 145. 5. Centers for Disease Control and Prevention. Acanthamoeba keratitis multiple states, 2005-2007. MMWR Morb Mortal Wkly Rep 2007 56 532-534. 6. Discussion Chemicals including preservatives contained in contact lens solutions could initiate ocular surface cytotoxic reactions or contact lens intolerance. The findings of the present study show that the three con tact lens MPSs induced varying levels of reversible lens cytotoxic reactions, with OPTI-FREE Express No Rub® solution showing the most effect. Simi lar observations on cultured human conjunctival cell lines showed that OPTI-FREE Express solution was significantly more toxic than ReNu MultiPlus No Rub® and COMPLETE moistureplus™ solution, re spectively4. In contrast, an in vivo observation from a recent clinical study found that ReNu MultiPlus The South African Optometrist 9 The South African Optometrist O Matthew Oriowo - Evaluating the cytotoxicity of contact lens multipurpose solutions in an in vitro lens system S Afr Optom 2009 68(1) 4-11 recovery from such irritancy or cytotoxic effect from the solutions. i the Alamar Blue™ biochemical assay has been used in many fields, especially in pharmacology to screen for agents toxic to mammalian cells44. The results in the present study agree with the previous clinical/lab oratory findings3, 45, and those of Pharm and Huff 46 who used the Alamar Blue™ assay to study cytotoxic effect of contact lens solutions on bovine corneal epi thelial cultures.i In the field of toxicology, the Draize41 test has been the standard in vivo measure of ocular toxicity for over fifty years. It is based on observations of ir ritation and injury to the cornea, conjunctiva, and iris after the application of test chemicals to the eyes of live rabbits33, 34. However, because of poor sensitiv ity and repeatability and ethical concerns about the suffering of live animals, there is an increasing need for more in vitro alternatives33, 34, 36, and researchers have continued to develop more in vitro approaches for determining ocular toxicity30, 36, 42. Crystalline lens culture has increasingly been used for in vitro alter native methods in ocular toxicology30, 32, 36, 42. Unlike the cornea, the ocular lens can be cultured as an intact organ for long periods as it can retain its physiologi cal integrity during the culture period, particularly with its repair mechanisms preserved. As earlier men tioned the crystalline lens has a number of similarities to the cornea to support its use as a model for corneal irritancy testing. It is an avascular tissue and its prin cipal function is to transmit light to the retina. Discussion Both the lens and cornea have structural and physiological adaptations to refract light. i In conclusion, these results confirm that OPTI-FREE is more cytotoxic compared to ReNu and COMPLETE contact lens multipurpose solu tions. The in vitro system herein presented offers a quick and quantitative in vitro assessment of the effi cacy and potential irritability of contact lens solutions. 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https://openalex.org/W4296995391	https://www.frontiersin.org/articles/10.3389/fgene.2022.886487/pdf	English	null	Epigenetic factors in breast cancer therapy	Frontiers in genetics	2,022	cc-by	11,302	OPEN ACCESS EDITED BY César López-Camarillo, Universidad Autónoma de la Ciudad de México, Mexico REVIEWED BY Dilek Cansu Gurer, Izmir Institute of Technology, Turkey Amir Yunus, Universiti Sains Malaysia (USM), Malaysia EDITED BY César López-Camarillo, Universidad Autónoma de la Ciudad de México, Mexico Runjhun Mathur 1,2, Niraj Kumar Jha1,3,4, Gaurav Saini5, Saurabh Kumar Jha1,4, Sheo Prasad Shukla6, Zita Filipejová7, Kavindra Kumar Kesari8, Danish Iqbal9,10, Parma Nand1, Vijay Jagdish Upadhye11, Abhimanyu Kumar Jha1, Shubhadeep Roychoudhury12 and Petr Slama13 REVIEWED BY Dilek Cansu Gurer, Izmir Institute of Technology, Turkey Amir Yunus, Universiti Sains Malaysia (USM), Malaysia 1Department of Biotechnology, School of Engineering and Technology, Sharda University, Greater Noida, India, 2Dr. A.P.J Abdul Kalam Technical University, Lucknow, India, 3Department of Biotechnology, School of Applied and Life Sciences (SALS), Uttaranchal University, Dehradun, India, 4Department of Biotechnology Engineering and Food Technology, Chandigarh University, Mohali, India, 5Department of Civil Engineering, Netaji Subhas University of Technology, Delhi, India, 6Department of Civil Engineering, Rajkiya Engineering College, Banda, India, 7Small Animal Clinic, University of Veterinary Sciences Brno, Brno, Czechia, 8Department of Applied Physics, School of Science, Aalto University, Espoo, Finland, 9Department of Medical Laboratory Sciences, College of Applied Medical Sciences, Majmaah University, Al Majma’ah, Saudi Arabia, 10Health and Basic Sciences Research Center, Majmaah University, Al Majma’ah, Saudi Arabia, 11Center of Research for Development (CR4D), Parul Institute of Applied Sciences (PIAS), Parul University, Vadodara, Gujarat, 12Department of Life Science and Bioinformatics, Assam University, Silchar, India, 13Department of Animal Morphology, Physiology, and Genetics, Faculty of AgriSciences, Mendel University in Brno, Brno, Czechia CITATION Mathur R, Jha NK, Saini G, Jha SK, Shukla SP, Filipejová Z, Kesari KK, Iqbal D, Nand P, Upadhye VJ, Jha AK, Roychoudhury S and Slama P (2022), Epigenetic factors in breast cancer therapy. Front. Genet. 13:886487. doi: 10.3389/fgene.2022.886487 Epigenetic modiﬁcations are inherited differences in cellular phenotypes, such as cell gene expression alterations, that occur during somatic cell divisions (also, in rare circumstances, in germ line transmission), but no alterations to the DNA sequence are involved. Histone alterations, polycomb/trithorax associated proteins, short non-coding or short RNAs, long non—coding RNAs (lncRNAs), & DNA methylation are just a few biological processes involved in epigenetic events. These various modiﬁcations are intricately linked. The transcriptional potential of genes is closely conditioned by epigenetic control, which is crucial in normal growth and development. Epigenetic mechanisms transmit genomic adaptation to an environment, resulting in a speciﬁc phenotype. TYPE Mini Review PUBLISHED 23 September 2022 DOI 10.3389/fgene.2022.886487 TYPE Mini Review PUBLISHED 23 September 2022 DOI 10.3389/fgene.2022.886487 TYPE Mini Review PUBLISHED 23 September 2022 DOI 10.3389/fgene.2022.886487 Epigenetic factors in breast cancer therapy OPEN ACCESS EDITED BY César López-Camarillo, Universidad Autónoma de la Ciudad de México, Mexico REVIEWED BY Dilek Cansu Gurer, Izmir Institute of Technology, Turkey Amir Yunus, Universiti Sains Malaysia (USM), Malaysia *CORRESPONDENCE Abhimanyu Kumar Jha, abhimanyujha630@gmail.com Shubhadeep Roychoudhury, shubhadeep1@gmail.com SPECIALTY SECTION This article was submitted to Cancer Genetics and Oncogenomics, a section of the journal Frontiers in Genetics RECEIVED 28 February 2022 ACCEPTED 20 July 2022 PUBLISHED 23 September 2022 CITATION Mathur R, Jha NK, Saini G, Jha SK, Shukla SP, Filipejová Z, Kesari KK, Iqbal D, Nand P, Upadhye VJ, Jha AK, Roychoudhury S and Slama P (2022), Epigenetic factors in breast cancer therapy. Front. Genet. 13:886487. doi: 10.3389/fgene.2022.886487 OPEN ACCESS The purpose of this systematic review is to glance at the roles of Estrogen signalling, polycomb/trithorax associated proteins, DNA methylation in breast cancer progression, as well as epigenetic mechanisms in breast cancer therapy, with an emphasis on functionality, regulatory factors, therapeutic value, and future challenges. Front. Genet. 13:886487. doi: 10.3389/fgene.2022.886487 cancer, breast, epigenetics, estrogen, therapy COPYRIGHT COPYRIGHT © 2022 Mathur, Jha, Saini, Jha, Shukla, Filipejová, Kesari, Iqbal, Nand, Upadhye, Jha, Roychoudhury and Slama. 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. KEYWORDS Frontiers in Genetics frontiersin.org 01 Mathur et al. 10.3389/fgene.2022.886487 Genes Function BRCA1 Wu et al. (2010) APC Saelee and Pongtheerat, (2020) GSTP1 Kanwal et al. (2014) Cyclin D2 Evron et al. (2001) PTEN Ramadan and Hashmin, (2021) p16INK4α Hui et al. (2000) RASSF1A Li et al. (2019) RARβ Wang et al. (2020) ZMYND10 Wang et al. (2019) DNA damage repair Catenin, cell proliferation, migration, and adhesion inhibitor Prevention of oxidative DNA damage by conjugation to glutathione Regulators of CDK kinases Regulating the AKT/PBK signalling pathway negatively Cyclin-dependent kinase inhibitor Ras effector homologue, cell cycle arrest Retinoic acid receptor Inhibitor of cancer cell colony formation patterns, locus speciﬁc hypermethylation, as well as nucleosomal remodelling. DNA methylation is deﬁned by Hinshelwood and Clark (Hinshelwood and Clark, 2008) (2008) as an enzyme-driven chemical modiﬁcation to DNA sequence that happens most frequently at CpG dinucleotides among mammals. tissue types. Repeating elements and transposons, which constitute roughly one-third of the human genome, include more than 90% of all methylation cytosines. Owing to the inherent carcinogenic potential of methylated cytosine residues, the proportion of Nucleotide bases in the genomes has been lowered over time, resulting in reduced number of CpGs than the quantitatively expected. DNA hypomethylation has been linked to gene reactivation and chromosomal instability, which can result in proto-oncogene overexpression, Imprinting loss, skewed or missing X-chromosomal inactivation, and increased recombination and mutation rates (De Smet et al., 2004). Gene suppression and genomic instability are connected to DNA hypermethylation as well as the suppression of tumour suppressor genes. In humans, PCDHB15 is a member of the cadherin superfamily of calcium- dependent cell-cell adhesion molecules that encodes for the PCDHB15 protein. CDH1 (also known as E-cadherin) and other cell adhesion molecules operate as epithelial-mesenchymal transition suppressors. In this CDH1 epigenetic silencing has been reported often in human cancer cases, including breast cancer (de Ruijter et al., 2020). Cytosines that are methylated are more vulnerable to endogenous or exogenous mutagenesis mechanisms than other DNA bases, with CpG site mutation rates projected to be higher than other transitional mutations (Jones et al., 2008). Transitions from C to T at CpG dinucleotides account for almost a 1/3 of all known germ line and somatic and mutations, albeit the distribution varies depending on the tumour type (Lo and Sukumar, 2008). CpG islands are tiny DNA fragments (ranging in size from 200 base pairs to several kilo base pairs) found in 60% of all genes. Introduction the world and more than 270,000 instances projected in the United States by 2020. The greatest serious hazard to women’s health in developed countries is breast cancer. Breast cancer is the most frequent cancer in women around the world (Bray et al., 2004; Chen et al., 2016) and distant metastasis is the major cause of poor survival (Gupta and Massague, 2006; Hanahan and Weinberg, 2011). The most common cause of death among breast cancer patients is metastasis. The molecular pathways that drive tumour cells to become metastatic have been thoroughly investigated, leading to major advances in prediction and treatment techniques. However, the signiﬁcant high percentage of breast cancer related fatalities continues to be a major source of concern. As a result, elucidating novel metastasis-related molecular processes is critical for improving breast cancer therapy outcomes. It is vital to completely understand the molecular pathways that enable breast cancer cell metastasis in order to create strategies to improve breast cancer patient survival and prognosis. Long non-coding RNAs (lncRNAs) have subsequently been revealed to play an important role promoting breast cancer metastasis through a variety of molecular pathways, albeit their exact functional characteristics have yet to be deﬁned. Long noncoding RNAs (lncRNAs) have recently been linked to breast cancer metastasis in a number of studies (Bin et al., 2018; Klinge, 2018; Zhou et al., 2018; Arun and Spector, 2019; Tomar et al., 2020). Long noncoding RNAs are noncoding RNAs with a size of more than 200 nucleotides that have a role in a range of biological processes, particularly cancer cell invasion. In transformed cells, epigenetic modiﬁcations include changes in DNA methylation, such as global hypomethylation or altered histone tail modiﬁcation Since 2004, invasive breast cancer has been on the rise, in 2018, more than two million instances were reported around FIGURE 1 Epigenetic Deregulation in Cancer. A vast number of epigenetic modiﬁers are mutated or activated inappropriately during cancer genesis. Simultaneously, epigenetic alterations such as DNA methylation, histone modiﬁcations, and microRNAs cause aberrant gene expression, resulting in genomic instability. FIGURE 1 Epigenetic Deregulation in Cancer. A vast number of epigenetic modiﬁers are mutated or activated inappropriately during cancer genesis. Simultaneously, epigenetic alterations such as DNA methylation, histone modiﬁcations, and microRNAs cause aberrant gene expression, resulting in genomic instability. 02 Frontiers in Genetics frontiersin.org 10.3389/fgene.2022.886487 Mathur et al. TABLE 1 Breast cancer genes that are hypermethylated. TABLE 1 Breast cancer genes that are hypermethylated. Introduction Genes Function BRCA1 Wu et al. (2010) DNA damage repair APC Saelee and Pongtheerat, (2020) Catenin, cell proliferation, migration, and adhesion inhibitor GSTP1 Kanwal et al. (2014) Prevention of oxidative DNA damage by conjugation to glutathione Cyclin D2 Evron et al. (2001) Regulators of CDK kinases PTEN Ramadan and Hashmin, (2021) Regulating the AKT/PBK signalling pathway negatively p16INK4α Hui et al. (2000) Cyclin-dependent kinase inhibitor RASSF1A Li et al. (2019) Ras effector homologue, cell cycle arrest RARβ Wang et al. (2020) Retinoic acid receptor ZMYND10 Wang et al. (2019) Inhibitor of cancer cell colony formation Function Epigenetically regulated genes in breast cancer For a long time, it was considered that methylation cytosines on DNA and deacetylated histones were two separate processes that could regulate chromatin structure and gene expression independently (Turner, 2000; Roth et al., 2001). HDACs have been linked to Epigenetic modiﬁcations by methyl group associated protein like MeCP2, which may read methylated sites on DNA and attract HDACs to them, or by HDACs directly interacting with DNA methyltransferases (Lo and Sukumar, 2008) (DNMTs). (Robertson et al., 2000; Bachman et al., 2001). Histone H3 lysine nine gets acetylated in functional chromatin regions, but when a genes is silenced, it becomes methylated, creating a binding domain for hetero-chromatin protein1 (HP1) (Litt et al., 2001; Peters et al., 2002). When serine 10 is phosphorylated, another epigenetic change, phosphorylation, prevents lysine nine from becoming methylated (Rea et al., 2000). PolyADP ribosylation is another alteration. PolyADP ribosylation has really been reported to affect chromatin structure through two methods, either covalently, by establishing short chains of Adenosine Diphosphate ribose polymers to histone proteins, or non- covalently, thereby attracting histones to the extended and branching polymers. Several research have sought to investigate the role of hypermethylation of TSG genes’ promoters in breast cancer, as well as the relationship between methylation of certain CGIs in TSGs and a variety of breast cancer clinical states. Table 1 lists the most important hypermethylated genes implicated in breast cancer functions so far. Methylation of these TSG promoters is linked to cancer cells losing all TSG protein products and developing a malignant phenotype. This DNA hypermethylation is a reversible signal, possibly due to the activity of Demethylase, which reverses the reaction of DNA methyltransferase and is a strong contender to be one of its key partners in shaping genome methylation patterns (Strahl and Allis, 2000; Ito et al., 2002). As a result, many recent research has focused on a novel strategy to cancer treatment that aims to block DNA hypermethylation and/ or re-expression of silenced TSGs. To create the transcriptional regulatory platform, DNA is packed into chromatin, a highly structured and dynamic protein–DNA complex. Histone modiﬁcations and composition interact with the binding of a variety of nonhistone proteins to control open (euchromatin) and closed (heterochromatin) chromatin states. Genes CpG islands that are ordinarily unmethylated in cancer cells may become methylated, potentially silencing critical genes such as tumour suppressor genes. At the same time, due to insufﬁcient transcriptional regulation of typically silent genes like oncogenes or retrotransposons, CpG dinucleotides in other places can become unmethylated. DNA methylation silences tumour suppressor genes (TSGs) that govern tumor development, DNA repair genes, oestrogen receptor genes, or genes that regulate angiogenesis. Because transcription factors which interface with methylated DNA differ from those that interact with unmethylated DNA, DNA methylation has an impact on gene expression (Figure 1) Hypermethylation of promoter regions silences the gene, which is a critical step in carcinogenesis with substantial implications for cancer prevention. Another epigenetic process that can regulate gene expression by altering chromatin shape is post- translational histone tail modiﬁcations, which are linked to DNA methylation (Martin and Zhang, 2005; Baylin and Ohm, 2006). Furthermore, it has been demonstrated that several nucleosomal remodelling regulators are also engaged in DNA methylation and histone modiﬁcation regulation (Esteller et al., 2000; Bird, 2002; Martin and Zhang, 2005; Baylin and Ohm, 2006). Understanding all of these epigenetic modiﬁcations and their role in breast carcinogenesis is critical for further advancements in breast cancer detection, prognosis, and treatment. In the presence of the dinucleotide sequence CpG, DNA hypermethylation is a post-replication alteration that nearly exclusively affects cytosines’ pyrimidine ring (Pfeifer and Besaratinia, 2009). In mammalian genomes, the bulk of CpG dinucleotides (75%) are methylated. The quantity of 5- methylcytosine in 1% of all bases varied somewhat between In human malignancies and primary tumours, some tumour suppressor genes and other malignant genes have been discovered to be hypermethylated (Frigola et al., 2006). Cell -cycle control, DNA repair, cell death, cellular maintenance, and invasion are among their physiologic Frontiers in Genetics 03 frontiersin.org 10.3389/fgene.2022.886487 Mathur et al. 10.3389/fgene.2022.886487 to DNA binding proteins during gene transcription initiation (Ito et al., 2002). Each histone modiﬁcation acts as a chromatin organisation signal. Histone acetylation (hyperacetylation) is associated with increased transcriptional activity, whereas hypoacetylation (hypoacetylation) is associated with gene repression (Forsberg and Bresnick, 2001; Wade, 2001). Transcription related Protein (, p53, p73, E2F1, STAT1, GATA1, HMGB1, YY1, and NFkB etc), hormone response (GR, ER, and AR), nuclear transporter (Importin7), WNT signalling (catenin), DNA repair (Ku70) and heat shock/ chaperone reaction (HSP90) are examples of HDAC substrates (Bolden et al., 2006; Kim et al., 2006). functions. Epigenetically regulated genes in breast cancer The nucleosome is chromatin’s most basic component, wrapping 146 bp of DNA around an octamer made up of four core histones, an H3/ H4 tetramer, and two H2A/H2B dimers (Strahl and Allis, 2000; Ito et al., 2002). The importance of local chromatin architecture in the regulation of gene expression is now widely acknowledged. Posttranslational changes to the N terminal tails of histones have a big impact on chromatin architecture. Genes Table 1 shows the genes which are most commonly methylation in breast cancer. Epigenetic cancer research has taken on a new dimension with the ﬁnding of long-range gene silence induced by epigenetic alterations (Stransky et al., 2006). Long-range epigenetic silencing appears to be ubiquitous during carcinogenesis, according to a recent study that revealed transcriptional dysregulation that can be regulated by epigenetic processes (Stransky et al., 2006). Epigenetic mechanism in breast cancer therapy Diagnostic and prognostic methods based on epigenetics play an important role in precision medicine. Precision oncology beneﬁts substantially from epigenetics-based diagnostic and prognostic techniques. Numerous DNA methylation diagnostic tests, in particular, are now being tested in clinics or are already in use. Precision oncology efforts to address dysregulated epigenetic pathways resulted in the development of epidrugs, or drugs that target epigenetic modulators. The FDA has approved only nine epidrugs, many more are in clinical studies for solid and hematological tumours (NCT01928576, NCT03179943), including antagonists of DNA methyltransferases (DNMTs) (NCT03164057, NCT02717884), EZH2, IDH and HDAC.The phase II trials (NCT00676663 and Methylation, acetylation, phosphorylation, ubiquitination, sumoylation, ADP ribosylation, deamination, and proline isomerization are all covalent alterations to core histones (Schubeler et al., 2004; Shilatifard, 2006). The discovery of multiple histone modiﬁcations with varied functions in gene regulation aided the identiﬁcation of a regulatory histone code, that deﬁnes at least partially overall transcriptional possibilities of a gene or genomic region (Xu et al., 2009). Altering the N terminus histone tail, which affects nucleosome density and positioning, enables this packed, inaccessible DNA accessible Frontiers in Genetics 04 frontiersin.org 10.3389/fgene.2022.886487 Mathur et al. et al., 2016). The maternal allele encodes H19, a 2.3-kb lncRNA that is regarded as an oncogene in several malignancies. At the H19/ IGF2 locus, a novel lncRNA called 91H is being produced in the H19 antisense direction. In breast cancer, the 91H lncRNA is in charge of preserving the genomic imprinting of the H19/IGF2 locus by preventing histone and DNA methylation on the maternal allele (Hu et al., 2018; Wang et al., 2018). E2F1 stimulates H19, which aids the G1-S transition in breast cancer cells (Vennin et al., 2017). Through the activation of Akt, the miR-675 produced by H19 downregulates the c-Cb1 and Cb1-b proteins and activates EGFR and c-Met to encourage cell growth (Berteaux et al., 2005; Zhang et al., 2017) discovered that overexpression of the lncRNA MEG3 inhibits cancer growth in a mouse model of breast cancer by inhibiting Akt signalling, in addition to causing cell cycle arrest in the G0/G1 phase. (Chen et al., 2017). demonstrated that lncRNA PTENP1 restricts the growth of breast cancer cells by downregulating the MAPK and AKT signalling pathways. NCT00828854) exploring the epidrugs’ effectiveness when used in conjunction with normal treatment in oestrogen receptor positive (ER+) breast cancer are signiﬁcant, reﬂecting recent developments in the knowledge of the epigenetic process. MALAT lncRNA Multiple BC types have abnormal expression of the lncRNA MALAT (MALAT), and this abnormal expression is associated with metastasis and a poor prognosis (Jadaliha et al., 2016; Wu et al., 2019). Further evidence suggests that high concentrations of 17- oestradiol can impede this lncRNA activity (Zhao et al., 2014). In a fascinating study, individuals with early post-BC-resection fever had higher MALAT levels (Li et al., 2018), which was associated with a worse prognosis. Additionally, MALAT deletion in mouse 4T1 xenografts markedly reduced inﬂammation and the lung metastases that are frequently observed in BC (Li et al., 2018). Following is a description of the lncRNAs H19, TINCR, MALAT, and NEAT1 DANCR, whose aberrant expression is linked to the growth and metastasis of BC. TINCR lncRNA In 2018 (Liu et al., 2018), it was found that TINCR lncRNA (TINCR) inﬂuences how primary BC tumours develop and how they spread later. In a certain study of 24 patients, the qPCR technique identiﬁed greater TINCR BC expression compared to non-BC participants. Additionally, SP1-zinc ﬁnger transcriptional factor, which normally identiﬁes the Guanine Cytosine -rich sequences in promoter regions, causes greater TINCR activity (Liu et al., 2018). H19 LncRNA It has been established that BC development and dysregulated long non-coding RNA H19 (H19) expression are related (Yang et al., 2016; Hu et al., 2018). Over 70% of BC tumours, including ER+ and ER-, HER2+ and HER2-positive tumours, have highly expressed H19 (Yang et al., 2016; Wang et al., 2018). This lncRNA has greater expression in BC for a number of usual mutational polymorphisms as well (Vennin et al., 2017; Wang et al., 2018). Apoptosis suppression and cell proliferation promotion are two biological reactions in which the Akt signalling pathway is involved (Yang Long non coding RNAs LncRNAs are RNA molecules having a length of more than 200 nucleotides but no apparent protein-coding function. Over 10,000 lncRNAs have been identiﬁed in the human transcriptome, with their genes located inter- or intra-genes in the genome. However, only a few have been thoroughly described. RNA polymerase II transcribes LncRNA genes, which then go through 5′ capping, splicing, 3′ cleavage, and polyadenylation. LncRNA loci are comparable to those of protein-coding genes at the chromatin level, although they frequently lack introns or have one or two. Splicing matures lncRNAs in the same way that it matures pre- mRNAs. n general, lncRNAs are found in the nucleus, although they have also been found in the cytoplasm and exosomes, and their expression levels are often lower than those of coding genes. Many investigations have found that their expression differs depending on the cell type24. In compared to protein-coding genes, lncRNAs are under low selected pressure, but their selective pressure is stronger than genomic repeat sequences. When comparing the sequences of lnRNAs from different species, brief highly conserved sequences can be found, demonstrating that they have preserved information about their cellular location and structure during evolution (Shang et al., 2000; Métivier et al., 2003; Moore and Proudfoot, 2009; Derrien et al., 2012; Rinn and Chang, 2012). Epigenetic mechanism in breast cancer therapy Due to ER expression, over 80% of all affected individuals are classiﬁed as ER+, and over 90 percent of all these patients have a 5 year cumulative rate of survival. Endocrine-based therapies such as ER-blockade, oestrogen synthesis inhibition, and selective ER degradation are used to treat most ER + cancers since ER is the primary oncogenic driver (Zardo et al., 2003; Thomas and Potter, 2013). Epigenetic mechanism underlying Erα signalling Epigenetic mechanisms are involved in ER signalling. In response to E2 stimulation, multitudes of ER co-regulators are transported to chromosomes in a synchronised way to ensure appropriate transcriptional and repressive activity at ER target sites. Regardless of the fact that every ER molecule usually stays on the chromatin for few moments at most, ER has been observed cycling on and off the chromatin for minutes and hours after E2 stimulation (Djebali et al., 2012; Johnson and O’Malley, 2012; Paakinaho et al., 2017; Wils and Bijlsma, 2018; Zhang et al., 2020). PRMTs, the SWI/SNF complex, P300/CBP & the Mediator complex, as well as the p160 family of proteins, are all signiﬁcant epigenetic ER coactivators. Members of the p160 family the co—activators i.e., SRC-1, SRC-2, and SRC-3, bind to ER directly and act as a recruiting platform for other activating enzymes and proteins to be recruited by ER to change chromatin, including chromatin remodelling complexes Breast cancer messes up epigenetic mechanisms that are essential for mammary gland development. The mammary gland’s balance self-renewal, and tissue integrity is regulated by a variety of signalling cascades and chromatin moderators, and also hormonal factors. Embryonic, pubertal, & reproductive stages are all three stages in the development of the mammary gland. WNT and Hedgehog (HH) signalling pathways coordinate embryonic mammary gland development, whereas hormones control pubertal and reproductive stages (Suzuki et al., 2008). The link between both the WNT & ER signalling pathways, particularly via Polycomb protein EZH231, it has been suggested that DKK’s involvement of WNT signalling activity can relate forward into the ER dependent pathway (and vice versa) to strengthen survival and growth with DKK3 promoter methylation being associated with positive ER status.5-azacytidine and trichostatin A, for example, have been shown to restore DKK3 expression in vitro (Figure 2). In the clinic, however, attempts to re-establish ER expression with hypomethylating medications have failed, EMT inﬂuences the polarisation of mammary cells, milk ﬂow patterns, particularly during pregnancy and during wound healing, cell movements are important. Mediated by ZEB1, SNAIL, and TWIST, among other transcription factors (TFs). SNAIL, for example, activates the Methyltransferase DNMT1 and inhibits CDH1 through DNA methylation. Furthermore, TGF-induced EMT modulates SNAIL transcription reactivation via the H3K27me3 demethylase KDM6B. WNT signalling epigenetic modiﬁcation in ER + breast cancer WNT signalling abnormalities have been associated to the onset and progression of a variety of cancers, including breast cancer. Breast cancer is aided by epigenetic suppression of WNT antagonist genes such as SFRP and DKK37. Chronic WNT signalling in breast cancer, which is linked to a poor prognosis, is caused by the methylation of these genes, which silences them (Bell et al., 2019). As a result of these changes, catenin stays constitutively active, resulting in enhanced stem cell replacement and division, which has been linked to disease resurgence (Serrano-Gomez et al., 2016). One of the constituent of the DKK family, i.e DKK3 had signiﬁcantly more promoter methylation in tumours from individuals with lymph node metastases, advanced stage disease, or breast cancer samples with positive ER status. status of mammary cancer samples. Estrogen subtypes and ER signalling pathways malignancies, is connected to the longevity of a mammary gland stem population of cells in cancer patients (Xiang et al., 2013). Derailment of important epigenetic mechanisms during breast development currently plays an essential role in the pathogenesis of breast cancer, according to research conducted in the last few years with the introduction of technical breakthroughs like as next-generation sequencing. This article discusses how the functional relationship between epigenetic alterations and developmental signalling cascades contributes to breast cancer. Estrogen promotes a variety of developmental processes in the body, involving reproductive maturity and bone growth, as well as energy balance via glycaemic control, intake rate, and thermoregulation. Estrogen also regulates mammary gland development through coordinating mitogenic and epigenetic processes. Several chemicals, as well as naturally occurring substances like polyphenols, which serve as a hypermethylation agent, can reverse the epigenetic silencing of tumour suppressor genes (Mathur and Jha, 2020). .Estrone, 17- Estradiol,Estriol, Estetrol (i.e, E1,E2,E3,E4) & Estrone-sulfate are the ﬁve major oestrogen subtypes (E1s). E1 and E2, the body’s two major estrogens, are reversibly transformed to E2, the physiologically active variety. Only E3 and E4 are identiﬁed throughout pregnancy, with E3 being the most prevalent. Because steroid sulfatases convert it to its active metabolite, E1 and E2, in situ, E1s is largely employed as an oestrogen reservoir (Mukherjee et al., 2017). NEAT1 lncRNA NEAT1 is a crucial oncogene in cancer and has a big impact on BC’s ability to induce EMT (Lu et al., 2016). In a sample of 179 BC patients, abnormal NEAT1 activity inﬂuenced chemoresistance and cancer cell stemness, and it has been expressed 6.86 times greater in BC patients than that in 192 controls (Shin et al., 2019). Frontiers in Genetics 05 frontiersin.org 10.3389/fgene.2022.886487 Mathur et al. Epigenetic mechanism underlying Erα signalling Increased levels of SNAIL and KDM6B have been associated to cancer recurrence, metastases, and poor ﬂatline survival in invasive breast carcinomas (Beatson, 1896; Liu et al., 2006; Saez-Ayala et al., 2013; Hanker et al., 2020). As a result, one can expect that targeting H3K27me3 demethylases The reactivation of various developmental pathways, which would be a common characteristic of many Frontiers in Genetics 06 frontiersin.org Mathur et al. 10.3389/fgene.2022.886487 FIGURE 2 Wnt Signalling Pathway: DKK3 binds to LRP, a WNT pathway coactivator of Frizzled, in normal mammary epithelial cells, preventing the pathway from being activated in the presence of the WNT ligand. In the absence of WNT activation, E-Cadherin binds to cytoplasmic -catenin, which is destroyed by GSK3. The DKK3 promoter, on the other hand, is hypermethylated in breast cancer, resulting in its downregulation. LRP can coactivate Frizzled in the presence of the WNT ligand in the absence of DKK3, resulting in phosphorylation of DSH, which prevents GSK3 from degrading -catenin. FIGURE 2 FIGURE 2 Wnt Signalling Pathway: DKK3 binds to LRP, a WNT pathway coactivator of Frizzled, in normal mammary epithelial cells, preventing the pathway from being activated in the presence of the WNT ligand. In the absence of WNT activation, E-Cadherin binds to cytoplasmic -catenin, which is destroyed by GSK3. The DKK3 promoter, on the other hand, is hypermethylated in breast cancer, resulting in its downregulation. LRP can coactivate Frizzled in the presence of the WNT ligand in the absence of DKK3, resulting in phosphorylation of DSH, which prevents GSK3 from degrading -catenin. (BMI1), a constituent of the PRC1 complex (Jeselsohn et al., 2015). Breast cancer stem cells have already been related to hormonal therapy resistance; however, it is unclear whether the appearance of stem cell like features in resistant cells is attributable to the multiplication of pre- existing highly specialised tumour cells or to epigenetic modiﬁcations that promote dynamic reprogramming (Jeselsohn et al., 2015). in combination with DNA hypomethylating medicines, which are prospective treatment targets in other solid tumours such as castration-resistant prostate cancer, could reduce recurrence (Liu et al., 2001). Polycomb complexes and HH signalling Epigenetic changes in breast cancer impair HH signalling, which is an important developmental pathway. Cancer progression is driven by increased ligand-dependent pathway activation and unregulated cell division (Early Breast Cancer Trialists’ Collaborative Group, 2005). Whenever the promoters of the SHH, HH Ligand, or its subsequent receptors, PTCH, is hypomethylated, the pathway is activated more ligand-dependently, resulting in uncontrolled cell division and cancer progression (Early Breast Cancer Trialists’ Collaborative Group, 2005). HH signalling also helps to promote normal and tumorigenic breast stem cells by boosting the production of PCGF4 According to the ﬁndings, combining medicines that directly block HH signalling with epigenetic modiﬁers like DNMTs to recover HH antagonistic control could alter cancer stem cell viability and differentiation. It has previously been reported on the utilisation of two-step approaches that combine several classes of medicines to trigger a process known as targeted phenotypic ﬂipping to treat resilient melanoma cells to lineage-speciﬁc therapy (Razavi et al., 2018). WNT and Hedgehog signalling pathways are required for the development of embryonic mammary glands, and their activation must be carefully coordinated spatially and temporally (SHH). In healthy mammary Frontiers in Genetics 07 frontiersin.org Mathur et al. 10.3389/fgene.2022.886487 epithelial cells, DKK3 binds to LRP, a Frizzled WNT pathway coactivator, preventing the route from becoming activated in the vicinity of the WNT ligand. E-Cadherin attaches to cytoplasmic -catenin in the lack of WNT activation, which is eliminated by GSK3. The role of tumorigenic cell pathways in modulating these microenvironmental and extracellular stimulus has previously been characterised (Munzone et al., 2006; Xia et al., 2006), inferring potential signalling components (e.g., SRC Kinase/ integrin/FAK) that might be targeted to overcome endocrine resistance (Osborne et al., 2003; Shi et al., 2009). In addition, the list of other host genome–associated variables inﬂuencing endocrine sensitivity is rising as a consequence of new pharmacogenomic and high-throughput research. The oncogenic E2-ER axis is the focus of endocrine treatment. The very ﬁrst-time steroid hormonal signalling being linked to breast tumor progression when both ovaries were surgically removed from patients with breast cancer, resulting in tumour regression and pave the way for endocrine therapy (Hanker et al., 2020). Treatments that decrease estrogen synthesis as well as techniques that speciﬁcally target the estrogen receptor are used in hormonal therapy, which is really the benchmark for ER + breast cancer (ER). Resistance mechanisms associated with tumors However, as previously indicated, the most of pathways that may play a role in endocrine resistance originates in tumour cells. These pathways can be split into three categories, each of which has components and mechanisms that overlap. p p The two different types of ER regulator are ER and ER coregulators. The ﬁrst group includes the ER, with coregulators, as well as other factors that alter the normal ER activity and modify receptor functions in relation to endocrine therapy. In refractory endocrine cancers, reduction of ER synthesis (i.e., the ER isoform) culminates in an endocrine-insensitive phenotype, that is rare (Lavinsky et al., 1998; Shou et al., 2004; Musgrove and Sutherland, 2009). Treatments that block growth factor receptors pathways that are known for down regulation, ER can enhance ER expression and endocrine sensitivities both in experimental and clinical situations (Schiff et al., 2004; Levin and Pietras, 2008; Santen et al., 2009) Reduced endocrine responsiveness has also been associated to the expression of various ER splicing variation, speciﬁcally the recently found minor variance ER36 (Musgrove and Sutherland, 2009) and oestrogen-related receptors. Furthermore, statistics suggest that ER coregulators, whether negative (corepressors) or positive (coactivators), have a role in deﬁning endocrine sensitivity and resistance by inﬂuencing the balance of agonistic vs. antagonistic SERM activity. Both in clinical and experimental contexts, dysregulation of a ER co- activator AIB1 (also abbreviated as SRC3 or NCoA3) has been associated to tamoxifen resilience (Span et al., 2003; Butt et al., 2005), while reduced expression of the co-repressor NCoR has been detected in tamoxifen-resistance experimental malignancies (Arpino et al., 2008). The ER as well as its coregulators are heavily inﬂuenced by posttranslational modiﬁcations. Growth factor receptor [e.g., FGFR (ﬁbro-blast growth factor receptor, EGFR/HER2, and IGF1-R ) and other cellular and stress-related kinases [e.g.p42/44, JNK , AKT, and PKA (protein kinase A and p38 MAPKs ), PAK1] regulate several posttranslational modiﬁcations (p21-activated kinase). Ubiquitination, Methylation, Phosphorylation, and other posttranslational modiﬁcations of ER and its co-regulators have been identiﬁed to alter ER activity and susceptibility to various endocrine therapies (Chakraborty et al., 2010) Outside of the nucleus, ER interfaces with cytoplasmic and Mechanisms Of endocrine therapy resistance, as well as possible alternatives Considering the fact that the endocrine therapy is effective in the treatment of ER + breast cancer patients, resistance develops in around 25% of early-stage patients and virtually all metastatic patients, results in a poor clinical prognosis (Liu et al., 2006; Serrano-Gomez et al., 2016; Bell et al., 2019); (Jansen et al., 2005; Generali et al., 2006; Pontiggia et al., 2009). Resistance to endocrine therapy has been classiﬁed as either intrinsic or acquired. Patients with breast cancer frequently experience clonally distinct progression as a result of the selection of genetic changes under treatment (Folgiero et al., 2008). Polycomb complexes and HH signalling Selective oestrogen receptor degraders (SERDs), Selected Oestrogen Receptor Modulators (SERMs), and Aromatase Inhibitors are the three categories (AIs) (Fanning et al., 2016). In addition, next-generation ER targeting medications are presently being investigated in ER+/ HER2 metastatic melanoma as adjuvant therapy or in combination with other therapy (Helleman et al., 2008). Growth factor receptor pathways In the case that the ER system is effectively inhibited, the third set of regulatory mechanisms in endocrine resistance would comprise those that can provide alternate proliferation and migration inputs to tumours. Importantly, through bi-directional interactions and control of the ER, these mechanisms—such as growth factors and other cellular-kinase pathways—might be able to offset the inhibitory activity of endocrine therapy. Many of these pathways, on the otherhand, it might develop into ER-independent drivers of tumour development and survival, making patients susceptible to all kinds of endocrine therapy, either early or later on. It has been suggested that ﬁbroblast growth factor (FGF), insulin/IGF1 receptors HER, tyrosine kinase receptors, and vascular endothelial growth- factor (VEGF) receptors are all involved (Harrod et al., 2017; Jeselsohn et al., 2018; Morel et al., 2020). Resistance mechanisms associated with the tumor microenvironment and the host The tumour microenvironment’s role as a regulator of these pathways and contribution to endocrine responsiveness has recently been established. This theory has been supported by studies including gene expression studies and biomarkers linked to hormonal therapy outcomes (Encarnacion et al., 1993; Brinkman et al., 2010) as well as the more complex in vitro or in vivo existing experimental systems (Gutierrez et al., 2005). Endocrine resistance is linked to stromal cells (endothelial, ﬁbroblasts and immune cells), structural features of the microenvironment and soluble substances (e.g., interleukins and growth factors) as well as other micro-environmental variables including hypoxia and acidity (Lopez-Tarruella and Schiff, 2007). Frontiers in Genetics 08 frontiersin.org 10.3389/fgene.2022.886487 Mathur et al. to glycine (ERD538G). From a structural standpoint, these changes remain stable ER in an agonists conﬁguration, resulting in constitutively active state (Oronsky et al., 2014). ESR1 mutations are detected in less than 1% of original tumours, however they are reported in 20–40% of tumours after endocrine therapy and have been associated to poor AI & tamoxifen efﬁcacy (O’Neil et al., 2017; Zucchetti et al., 2019; Xu et al., 2020). The nearly complete detection of ESR1 alterations in hematologic malignancies after the AI therapy shows that under the constraints of endocrine treatment, uncommon, resistant clones can be selected. membrane signalling complexes to activate and regulate a variety of growth factor receptors as well as other cell - signalling cascades (Kern et al., 1994; Morgan et al., 2009; Shi et al., 2009; Spoerke et al., 2016). Cell cycle signalling molecules Molecules associated in cellular and biological responsiveness to endocrine therapy, such as cell growth inhibition and apoptosis induction, are included in endocrine resistance–related pathways. The majority of information on the participation of these pathways comes from preclinical investigations. Positive cell-cycle regulators, notably those directing G1 phase progression, and also negative cell cycle regulator, have both been demonstrated to disrupt and decrease endocrine therapy’s antiproliferative action, tends to result in resistance (Fribbens et al., 2016). Endocrine resistance is caused by overexpression of positive cell-cycle regulators MYC & cyclins E1 and D1, which activate cyclin-dependent kinases (CDKs) for G1 phase or reduce the inhibitory effects of negative cell-cycle regulators (p21 & p27) (Gates et al., 2018). Several studies have focused on establishing new therapeutic strategies for tumor tissues with ESR1 mutations in recent years. Continuous ER signalling encourages hormone independent development and thus is linked to a distinct transcription network involved in signaling pathways and metastasis as a result of this process (Fukumoto et al., 2018). Activating kinases, epigenetic modifying enzymes and ER co-regulators, are required for the development of ESR1 mutants (Fukumoto et al., 2018). .As a consequence, they could be employed to treat ESR1 mutant malignancies in the preclinical stage. Another type of genetic mutation discovered in metastatic ER + breast cancer is ESR1 gene fusion events, which are likely to represent novel resistance drivers. As a result of ESR1 chromosomal rearrangement occurrences, the ER’s LBD is replaced by another protein. Endocrine-resistant breast cancer is caused by epigenetic factors According to a whole-genome sequencing study, epigenetic factors are among the most commonly changed factors in human malignancies. The most frequent genetic modiﬁcations in many types of cancer are inactivating mutations as well as the loss of SNF/SWI subunits. In breast cancer, ARID1A impacts breast luminal lineage adherence and sensitivity to endocrine treatment. Patients’ poor response to SERDs suggests that ARID1A loss- of-function mutations are more frequent in endocrine-resistant metastatic situations, implying that they can also cause endocrine resistance. Alteration Of ESR1 And Genes Involved In Estrogen- Mediated Signalling. ARID1A deﬁciency affects chromatin accessibility and transcription factor binding, as well as the binding of ER and FOXA1 to chromatin, all of which inﬂuence luminal cell destiny. The tumour cell’s reliance on ER for growth and survival is targeted by endocrine treatment. As a result, bypassing pharmacological inhibition relies on the accumulation of changes in the ER and its downstream targets. The main mechanism of resistance in most cases is ligand independent ER reactivation (Oronsky et al., 2014). Constitutive ER activation can be mediated by mutations in the ESR1 gene (which codes for ER) and is a major driver of acquired resistance. The majority of ER mutations occur in the LBD at two neighbouring amino acids: tyrosine at position 537 transformed to asparagine, cysteine, or serine (ERY537 N/C/S) and aspartic acid at position 538 altered According to Xu et al., long-term ER suppression could result in the generation of individuals with ARID1A inactivating mutation, promoting a luminal-to-basal phenotypic transition (Bitler et al., 2015). In the clinic, ER + tumours cured with endocrine therapy, reduce ER expression, by becoming resistant to hormone therapy. The increasing prevalence of ARID1A mutation in endocrine resistant breast cancer, including its prevalence in other cancers, highlights the need of treating ARID1A mutant tumours with targeted therapeutic strategies (Morel et al., 2020). Frontiers in Genetics 09 frontiersin.org Mathur et al. 10.3389/fgene.2022.886487 10.3389/fgene.2022.886487 One of the therapy paradigms examined in ARID1A mutant cancers is synthesised lethality, which relates to the lethal consequence of simultaneous alteration of two genes which, when separately disrupted, do not impact cell viability (Morel et al., 2020). For example EZH2 suppression and ARID1A mutations are synthetically fatal in ovarian cancer, and HDAC2 inhibition ampliﬁes this effect. Conclusion New ﬁndings, as with all parts of science, produce new questions, and some of the most important unanswered questions like. Is it possible to use the dynamic nature of epigenetic modiﬁcations to develop short-term treatment techniques to avoid selection toward a resistant phenotype, or are epidrugs’ underlying processes contributing to resistance formation is possible to follow disease progression and therapy response using epigenetic markers. Endocrine therapy has been proven to be an important treatment option for hormone-responsive breast cancers. However, there is still an urgent need to develop strategies to combat the phenotype of resistance that appears to be unavoidable. Recent epidrug breakthroughs attest to the developing new era of epigenetic-based treatments for screening and treating a variety of disorders, including breast cancer. Acknowledgments The authors would like to acknowledge the support from their respective institutes throughout the review writing process. Author contributions All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication. Tumorigenesis and medication resistance are both inﬂuenced by epigenetic instability. Epidrugs have primarily been used to treat haematological malignancies, with limited efﬁcacy in solid tumors. The failure to treat solid tumours, on the other side, can be traced back to a one size ﬁts all approach. Epigenetic reprogramming’s plasticity enhances cancerous cells’ overall ﬁtness, making individualised cancer treatment much more challenging. According to multiple preclinical and clinical studies, epidrugs have synergistic beneﬁts with a number of therapeutic methods, including immunotherapy, radiation, and endocrine therapy. One of the most notable areas of current drug discovery operations is the development of small compounds that target chromatin regulators (Shiino et al., 2016). 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. The majority of the studies focused on epigenetic changes that occur when cancer progresses and resistance develops. Small molecule HDACi blockers (vorinostat and entinostat ) and also DNA hypomethylating drugs (decitabine & 5-azacytidine) have been explored as re-sensitizing strategies to endocrine therapy in ER + preclinical models. The modes of action of DNMT inhibitors (DNMTs) have been proposed as de-methylation of tumour suppressor genes and an unique viral mimicking mechanism. Furthermore, in endocrine-resistant breast cancer, Epigenetic avenues in the endocrine therapy In the realm of endocrine therapy, there are a variety of epigenetic options. Despite the fact that endocrine and molecularly targeted have been shown to cure the great majority of breast cancers, they have failed to target a tiny percentage of the population, leading to recurrence and therapeutic resistance. Compounding variables like tumour genetic instability enables tumours to adapt to a range of stresses, including the selective pressure produced by therapeutic drugs, which we addressed previously. As a result, precise patient classiﬁcation and personalised treatment approaches would be required to reduce the signiﬁcant morbidity and mortality of individuals with ER + metastatic breast cancer (Shiino et al., 2016). Endocrine-resistant breast cancer is caused by epigenetic factors In ARID1A defective cells, HDAC2 is attracted to EZH2/ARID1A co-target genes including such PIK3IP1, a PI3K/AKT signalling inhibitor, leading in incorrect stimulation of this mitogenic system (Shiino et al., 2016). These two processes, ARID1A loss of function and enhanced PI3K/AKT signalling, are typically detected in endocrine-resistant breast cancer cells (Shiino et al., 2016). As a consequence, one can believe that targets EZH2 in breast cancer patients with ARID1A mutations might be a promising treatment option. epigenetic dysregulation is a prevalent occurrence. 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https://openalex.org/W3006217404	https://zenodo.org/record/3687452/files/141.pdf	English	null	BROILER POULTRY FARMING OPEN HOUSE SYSTEM PATTERNS STOCHASTIC FRONTIER OF ECONOMIC, TECHNICAL AND ALLOCATIVE EFFICIENCY OF IN LAMONGAN REGENCY EAST JAVA	International journal of advanced research	2,020	cc-by	4,934	ISSN: 2320-5407 ISSN: 2320-5407 Int. J. Adv. Res. 8(01), 1053-1060 Journal Homepage: - www.journalijar.com Article DOI: 10.21474/IJAR01/10402 DOI URL: http://dx.doi.org/10.21474/IJAR01/10402 BROILER POULTRY FARMING OPEN HOUSE SYSTEM PATTERNS STOCHASTIC FRONTIER OF ECONOMIC, TECHNICAL AND ALLOCATIVE EFFICIENCY OF IN LAMONGAN REGENCY EAST JAVA 1053 Niswatin Hasanah1, Zaenal Fanani2, Suyadi2 and Bambang Ali Nugroho2 1. Animal Husbandry Departmentof Polythecnic State Jember. 2. Postgraduate Animal Husbandry of Brawijaya University. …………………………………………………………………………………………………….... Manuscript Info Abstract ……………………. ……………………………………………………………… Manuscript History Received: 30 November 2019 Final Accepted: 31 December 2019 Published: January 2020 The purpose of this study was to examine and analyze the level of economic, technical and allocative efficiency of the use of production factors for the type of open house system cages per breeder individual in Lamongan Regency and study and analyze what factors affect economic efficiency, allocative efficiency and technical efficiency in the closed house system cages.The sampling technique used in this study was a multistage sampling method which consisted of 14 breeders who partner with PT. X Lamongan Regency, East Java. The result of thestudy was the R/C ratio of broiler poultry farming with Closed House System (CHS) of 1.13. Input variables that affected the production function of broiler poultry farming with a Open House System (OHS) patterns are DOC, feed, vaccines, and electricity & water. Input variables that affect the cost function of poultry farming with a Open House System (OHS) pattern were DOC, feed, vaccines, and electricity & water. The Open House System (OHS) broiler poultry farming business in Lamongan Regency has not been technically efficient even though a high level of technical efficiency was obtained in each of the business patterns of 0.958 in the Open House System (OHS) pattern. The diversity of technical efficiency level in the broiler poultry farming business with aOpen House System (OHS) pattern of 0.031 was influenced by inefficiency sources, namely age and education because the age of Open House System breeders in the average productive age and the average education of scholars who supported the operation of broiler poultry cages. The ability of breeders in minimizing costs to achieve average broiler production with a Open House System (OHS) pattern of 15,142.8 live chickens was at satisfaction level but did not meet economic efficiency with an average economic efficiency of 0.9999750 with Open House System (OHS) pattern. The average level of allocative efficiency in broiler poultry farming business with the pattern of Open House System (OHS) the average level of allocative efficiency obtained was 1.045. This showed that the Open House System (OHS) broiler poultry farming business has been allocatively efficient. BROILER POULTRY FARMING OPEN HOUSE SYSTEM PATTERNS STOCHASTIC FRONTIER OF ECONOMIC, TECHNICAL AND ALLOCATIVE EFFICIENCY OF IN LAMONGAN REGENCY EAST JAVA The suggestions of this studyare the real time monitoring from the core parties namely reporting sapronak data, mortality and daily events on plasma parties by using the application of Corresponding Author:- Niswatin Hasanah Address: Animal Husbandry Department of Polythecnic State Jember. . Materials And Methods:- Research Time and Place: The study was carried out from November 2018 to August 2019. The location was in Lamongan Regencywith a closed house system cages in partnership with PT. X in Lamogan Regency. Introduction:- The Indonesian's local poultry market faces uncertain market conditions because Indonesia lost the 2019 World Trade Organization (WTO) session. Indonesia's defeat triggered the opening of opportunities for imported Brazilian broilers to enter the Indonesian market which is predicted to have lower prices because it has a stock of used vaccine thigh meat overflow. The cost of poultry industry feed is high because the price of corn is expensive. The government must protect traditional markets so that the majority of products sold are dominated by local breeds rather than imports. The Indonesian government has made several efforts, namely limiting the production of DOC in breeding with scheduling programs, parent stock rejects early and infertile hatching eggs sold in the market as consumption eggs. The Indonesian government needs to increase the efficiency of national chicken production, one of them is to improve an efficient cage management system, provide stable and inexpensive chicken feed, increase national chicken production efficiency and regulation of poultry partnership management. By looking at these conditions, the availability of cheap chickens is needed from the efficient management of broiler poultry farming thereby increasing the competitiveness of local poultry products. The purpose of this study was to study and analyze the level of economic, technical and allocative efficiency of the use of production factors for the type of closed house system cages per breeder individual in Lamongan Regency and study and analyze any factors that affectedeconomic efficiency, allocative efficiency and technical efficiency in the Open House Systemcages. Sampling Method:- p g The Multistage Sampling Method was used to select PT'. X to determine the sample size of 14 respondents from a total population of 104 respondents from the open house and close house breedersby using the Slovin formula: N n = ——— 1 + N(e)² Int. J. Adv. Res. 8(01), 1053-1060 the FMDC tool for monitoring and evaluating the implementation of partnership cooperation, strict criminal and civil law sanctions for plasma parties which are proven to commit fraud in implementing partnership cooperation, an increase in the price of guarantee certificates for each new plasma that wants to join as a breeder. Copy Right, IJAR, 2020,. All rights reserved. ……………………………….... Copy Right, IJAR, 2020,. All rights reserved. Abstract 1053 ISSN: 2320-5407 ISSN: 2320-5407 Int. J. Adv. Res. 8(01), 1053-1060 Int. J. Adv. Res. 8(01), 1053-1060 Abstract ……………………………………………………………… The purpose of this study was to examine and analyze the level of economic, technical and allocative efficiency of the use of production factors for the type of open house system cages per breeder individual in Lamongan Regency and study and analyze what factors affect economic efficiency, allocative efficiency and technical efficiency in the closed house system cages.The sampling technique used in this study was a multistage sampling method which consisted of 14 breeders who partner with PT. X Lamongan Regency, East Java. The result of thestudy was the R/C ratio of broiler poultry farming with Closed House System (CHS) of 1.13. Input variables that affected the production function of broiler poultry farming with a Open House System (OHS) patterns are DOC, feed, vaccines, and electricity & water. Input variables that affect the cost function of poultry farming with a Open House System (OHS) pattern were DOC, feed, vaccines, and electricity & water. The Open House System (OHS) broiler poultry farming business in Lamongan Regency has not been technically efficient even though a high level of technical efficiency was obtained in each of the business patterns of 0.958 in the Open House System (OHS) pattern. The diversity of technical efficiency level in the broiler poultry farming business with aOpen House System (OHS) pattern of 0.031 was influenced by inefficiency sources, namely age and education because the age of Open House System breeders in the average productive age and the average education of scholars who supported the operation of broiler poultry cages. The ability of breeders in minimizing costs to achieve average broiler production with a Open House System (OHS) pattern of 15,142.8 live chickens was at satisfaction level but did not meet economic efficiency with an average economic efficiency of 0.9999750 with Open House System (OHS) pattern. The average level of allocative efficiency in broiler poultry farming business with the pattern of Open House System (OHS) the average level of allocative efficiency obtained was 1.045. This showed that the Open House System (OHS) broiler poultry farming business has been allocatively efficient. The suggestions of this studyare the real time monitoring from the core parties namely reporting sapronak data, mortality and daily events on plasma parties by using the application of Corresponding Author:- Niswatin Hasanah Address: Animal Husbandry Department of Polythecnic State Jember. Corresponding Author:- Niswatin Hasanah Address: Animal Husbandry Department of Polythecnic State Jember. q Data collection in this research was carried out as follows: q Data collection in this research was carried out as follows: Interview, namely in-depth interview with breedersby using a questionnaire, in the form of semi-closed and open questions. Data collection in this research was carried out as follows: y in-depth interview with breedersby using a questionnaire, in the form of semi-closed and open Interview, namely in-depth interview with breedersby using a questionnaire, in the form of se questions. Description; p n = the total of samples desired n = the total of samples desired N t t l l ti p N = total population ion d 10% which is the degree of deviation from the characteristics of the sample to the population. e = precision used 10% which is the degree of deviation from the characteristics of the sample to the population. The research data used were 6 times the last production process from each breeder. The total of data used was the multiplication between the total of broiler poultry breeders and the total of production processes. The Illustration matrix of Open House System contract partnership research data is presented in the following table. Table 1:- Open House System (OHS) Research Data Matrix. Periode Produksi 1 2 3 4 5 6 7 8 9 10 dst.. 16 I 1.I 2.I 3.I 4.I 5.I 6.I 7.I 8.I 9.I 10.I 16.I II 1.II 2.II 3.II 4.II 5.II 6.II 7.II 8.II 9.II 10.II 16.II III 1.III 2.III 3.III 4.III 5.III 6.III 7.III 8.III 9.III 10.III 16.III IV 1.IV 2.IV 3.IV 4.IV 5.IV 6.IV 7.IV 8.IV 9.IV 10.IV 16.IV V 1.V 2.V 3.V 4.V 5.V 6.V 7.V 8.V 9.V 10.V 16.V Table 1:- Open House System (OHS) Research Data Matrix. 1054 Int. J. Adv. Res. 8(01), 1053-1060 ISSN: 2320-5407 Description: Y = Production of Open house broiler poultry systems or open per production period (kg live weight/production period) p ) XI = Total of broiler poultry seeds (DOC) per production period (chick in tail/production period) X2 = Total of feed used per production period (kg feed intake/production period) X3 = Total of medicine used per production period (liters of orange vitamin medicine/production period) X4 = Total of electricity used per production period (kwh/pp) X5 = Total of fuel used per production period (liters of gasolek/production period) X6 = Total of workers used per production period (JKSP/production period) β0 = constant βi chs or ohs - β6 chs or ohs = Predicted variable fixed input parameter Ln = Natural Logarithm e = 2,718 Vi = An error has been made because of a random retrieval Ui = Effect of technical efficiency that appears p XI = Total of broiler poultry seeds (DOC) per production period (chick in tail/production period) X2 = Total of feed used per production period (kg feed intake/production period) = Total of fuel used per production period (liters of gasolek/production period) βi chs or ohs - β6 chs or ohs = Predicted variable fixed input parameter Ln Nat ral Logarithm e 2 718 Analysis of Production Function of Broiler Business: Analysis of Production Function of Broiler Business: Factors that directly affect the production of broiler poultry in the Open House System of the contract partnership pattern are: chicken seedlings (DOC), feed, medicine, electricity, fuel and labor. The functional form of Cobb- Douglas is mathematically formulated as follows: LnYchs or ohs = β0 + βILnXl +β2 LnX2+β3 LnX3+β4 LnX4 + β5 LnX5 + β6 LnX6 + β7 LnX7+ β8 LnX8 Vi – rUi Data Analysis Methods: The stages of data analysis in this study were as follows: 1) the analytical method used to determine the level of income of closed house system breeders was income analysis. 2) To find out the data used in this study was the Best Linear Unlimited Estimator (BLUE) classic assumption test was performed. 3) To find out the factors that affected the production function and the cost function of broiler poultry farming business with closed house system pattern by using Ordinary Least Squares (OLS) and Maximum Likelihood Estimation (MLE) methods. 4) To analyze the level of technical and economic efficiency per individual broiler poultry breeder with the Closed House System pattern, an efficiency analysis with the Technical Efficiency Effect Model option and on the level of technical and economic efficiency obtained was determined by allocative efficiency value per individual broiler poultry breeder. 5) Based on the level of income, technical efficiency, economic efficiency and allocative efficiency achieved per individual broiler poultry breeder with the Open House System pattern the average difference test was performed. All data in this study were analyzed by using Minitab 16 and Frontier software version 4.1c. to obtain in-depth information and perceptions from respondents. y y y p p y 5) Based on the level of income, technical efficiency, economic efficiency and allocative efficiency achieved per individual broiler poultry breeder with the Open House System pattern the average difference test was performed. All data in this study were analyzed by using Minitab 16 and Frontier software version 4.1c. to obtain in-depth information and perceptions from respondents. Function cost analysis of Broiler Poultry Farming Business: Factors that directly affect the cost function in the broiler poultry farming business with the open house system (OHS) contract partnership patterns are: chicken seed costs (DOC), feed costs, medical costs, electricity costs, fuel costs, labor and production costs. The mathematical formula is as follows: LnCi chs or ohs = α0 + α1LnW1 + α 2LnW2 + α3LnW3 + α4LnW4 + α5 Ln W5 + α 6LnW6 + α 7LnY + α 8LnY Vi + Ui costs, labor and production costs. The mathematical formula is as follows: LnCi chs or ohs = α0 + α1LnW1 + α 2LnW2 + α3LnW3 + α4LnW4 + α5 Ln W5 + α 6LnW6 + α 7LnY + α 8LnY Vi + Ui Description: LnCi = Production costs of broilers poultry closed house system or open house system per production period (IDR/head/production period) W1 = Cost of broiler seedlings (DOC) per tail (IDR/ tail/production period) W2 = Feed cost per kilogram in one production period (IDR/tail/production period) W3 = Medical cost per unit in one production period (IDR/tail/production period) W4 = Cost of electricity used per production period (IDR/kwh/tail/production period) Sources of Technical Inefficiency y Table 3:- Estimating the Technical Inefficiency Parameters of Production Function of Broiler Poultry Business with the Open House System (OHS) pattern in Lamongan Regency. Variable Coefficient Std Error z count Sig. Age -0.669 42.431 -0.020 0.987 Education 263.940 141.697 1.860 0.063* Experience -72.818 104.666 -0.700 0.487 Family 489.779 245.258 2.000 0.046** Job status 965.283 652.514 1.480 0.139 Constant 1484.436 10048.760 0.150 0.883 Source: Processed Primary Data (2019) y ting the Technical Inefficiency Parameters of Production Function of Broiler Poultry Business with System (OHS) pattern in Lamongan Regency Table 3:- Estimating the Technical Inefficiency Parameters of Production Function of Broiler Poult h O H S t (OHS) tt i L R The estimation of the effect of the technical inefficiency of production in the broiler poultry farming business with the Open House System (OHS) pattern shows that the Age and Education variable has a significant effect on the production of the broiler poultry farming business, while the other variables do not have a significant effect. Allocative Efficiency Analysis: Every entrepreneur in production always uses several inputs in an optimal total to get the maximum number of production results. The use of several production factors will be different between one entrepreneur and another entrepreneur so that the production results and the profits obtained will also be different. The difference is also experienced by broiler poultry breeders with an open house system pattern. The difference is thought to be caused by differences in the ability of both knowledges about livestock business and financial capabilities. The difference in ability between broiler poultry breeders causes differences in the proportion of the use of production factors and the price of production factors. The use of production factors and the different price of production factors between breeders in the broiler poultry farming business with a closed house system pattern and broiler poultry breeders with a Open House System pattern will have an impact on the profits obtained by breeders. Therefore, with different abilities owned by breeders, it will be different in maximizing profits. The efforts of broiler poultry breeders with a closed house system pattern in maximizing profits can be seen from the achievement of allocative efficiency values. The estimation of the allocative efficiency level in most previous studies uses the ratio between the Marginal Product Value (MPV) and the price of production factors. The estimation aims to determine the level of use of production factors. ISSN: 2320-5407 Int. J. Adv. Res. 8(01), 1053-1060 Figure 1 shows the technical efficiency per individual in a broiler poultry farming business with a Open House System (CHS) pattern. From these results, it is obtained the highest value of 0.971 in breeder no. 6 and the lowest value of 0.923 in breeder no. 5. nical Inefficiency ting the Technical Inefficiency Parameters of Production Function of Broiler Poultry Business with Description: 1055 Int. J. Adv. Res. 8(01), 1053-1060 ISSN: 2320-5407 W5 = Fuel cost per liter in one production period (IDR/liter/tail/production period) W6 = Labor costs used per production period (IDR JKSP/day/production period) Y = Total of broiler poultry production closed house system or open house system per production period (IDR/tail/production period) α0 = Constant α1 - α6 = Parameter for expected input variable Ln = Natural Logarithm e = 2,718 Vi = An error has been made because of a random retrieval Ui = Effect of technical efficiency that appears W5 = Fuel cost per liter in one production period (IDR/liter/tail/production period) p p p p p W6 = Labor costs used per production period (IDR JKSP/day/production period) Result And Discussion:- Result And Discussion:- Technical Efficiency Analysis: Table 2:- Technical Efficiency Description of the Broiler Business in Lamongan District. Technical Efficiency Closed House System (OHS) Average 0.958 Standard Deviation 0.031 Minimum 0.704 Maximum 0.993 Source: Processed Primary Data (2019) Table 2is found that the Open House System (OHS) has been technically efficient. The achievement of a high technical efficiency index value reflects the achievements of broiler poultry breeders in both business groups that are very satisfying in managing livestock business, especially in the mastery of information and the decision making the process for managing factors production. The Open House System (OHS) is a measure that breeders still have the opportunity to add several input variables to increase broiler poultry production, but the possibility to increase productivity is very small because of the narrow interval between the level of productivity that has been achieved with the maximum level of productivity. The decision on the addition of several variables became the choice of the second breeder of the broiler poultry farming business group. The input variables that are recommended to be added by broiler poultry breeders with a Closed House System (CHS) pattern are chicken feed and seedlings (DOC). Figure 1:- Distribution of Technical Efficiency per Individual in Broiler Poultry Business with Closed House System (CHS) Pattern Technical Efficiency Figure 1:- Distribution of Technical Efficiency per Individual in Broiler Poultry Business with Closed House System (CHS) Pattern 1056 Int. J. Adv. Res. 8(01), 1053-1060 ISSN: 2320-5407 Int. J. Adv. Res. 8(01), 1053-1060 ISSN: 2320-5407 Int. J. Adv. Res. 8(01), 1053-1060 ISSN: 2320-5407 These results indicate that the overall broiler poultry farming business with closed house system pattern has maximized the level of profit or in other words that the overall broiler poultry farming business has used several inputs optimally to obtain the maximum total of production. The achievement of the average allocative efficiency values between the two groups of the same broiler poultry farming (rounding numbers) is thought to be caused by several factors of production used originating from the same source and the similarity in the price of these factors of production. Allocative Efficiency per Individual in Broiler Business with Open House System (OHS) Pattern Figure 13:- Distribution of Allocative Efficiency per Individual in Broiler Poultry Farming Business with Open House System (OHS) Pattern. 0.99 1.01 1.03 1.05 1.07 1.09 1.11 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Allocative Efficiency cative Efficiency per Individual in Broiler Business with Open House System (OHS) Pattern Allocative Efficiency Figure 13:- Distribution of Allocative Efficiency per Individual in Broiler Poultry Farming Business with Open House System (OHS) Pattern. Figure 13 shows the technical efficiency per individual in a broiler poultry farming business with a Open House System (OHS) pattern. From these results obtained the highest value of 1,029 in breeders No.5 and the lowest value of 1.099 in breeders No. 6. Sources of Technical Inefficiency In this study, the allocative efficiency level is assumed to use the ratio between the results of the level of economic efficiency derived from the frontier cost function and the result of the level of technical efficiency derived from the frontier production function. The purpose of using this method is so that the level of allocative ability per individual breeder both broiler poultry breeders with a closed house system pattern can be known. The distribution of allocative efficiency values achieved by broiler poultry breeders with the closed house system pattern is presented in the following table. Table 4:- Description of the Allocative Efficiency of Broiler Poultry Farming in Lamongan Regency. Technical Efficiency Open House System (OHS) Average 1.045 StandardDeviation 0.043 Minimum 1.007 Maximum 1.421 Source: Processed Primary Data (2019) Table 4 shows that the allocative efficiency values obtained by broiler poultry breeders with a open house system pattern range from 1,000 to 1,211. Based on the highest and lowest allocative efficiency values achieved by the two broiler poultry farming businesses, there is diversity in the ability of breeders to maximize profits, but this is not the case when seen from the average allocative efficiency values obtained. The average value of the allocative efficiency of a broiler poultry farming with the closed house system pattern obtained is 1.0 (rounding number). 1057 Int. J. Adv. Res. 8(01), 1053-1060 Int. J. Adv. Res. 8(01), 1053-1060 ISSN: 2320-5407 Suggestion:- 1. Real time supervision from the core parties namely reporting sapronak data, mortality and daily events on plasma parties by using the application of the FMDC tool for monitoring and evaluating the implementation of partnership cooperation. 1. Real time supervision from the core parties namely reporting sapronak data, mortality and daily events on plasma parties by using the application of the FMDC tool for monitoring and evaluating the implementation of partnership cooperation. 2. Strict criminal and civil law sanctions for plasma parties which are proven to commit fraud in implementing partnership cooperation. 2. Strict criminal and civil law sanctions for plasma parties which are proven to commit fraud in implementing partnership cooperation. p p n increase in the price of guarantee certificates for each new plasma that wants to join as a breeder. 4. The government needs to involve the Association of Indonesian Poultry Slaughterhouse (ARPHUIN) as an element of control of import volumes because it has data on the supply capacity of demand for chicken, the amount of stock in cold storage so that over supply in the market can be avoided. 4. The government needs to involve the Association of Indonesian Poultry Slaughterhouse (ARPHUIN) as an element of control of import volumes because it has data on the supply capacity of demand for chicken, the amount of stock in cold storage so that over supply in the market can be avoided. Conclusion:- Based on the results and discussion it can be concluded that: Based on the results and discussion it can be concluded that: 1. R/C ratio of broiler poultry farming business with Open House System (OHS) is 1.12. 2. Input variables that affect the production function of broiler poultry farming business with a Open House System (OHS) pattern are DOC, feed, vaccines, electricity & water, and labor. 2. Input variables that affect the production function of broiler poultry farming business with a Open House System (OHS) pattern are DOC, feed, vaccines, electricity & water, and labor. y ( ) p y 3. Input variables that affect the cost function of a poultry farming business with the Open House System (OHS) pattern are DOC, feed, vaccines, and electricity & water. y p y 3. Input variables that affect the cost function of a poultry farming business with the Open House System (OHS) pattern are DOC, feed, vaccines, and electricity & water. 4. Broiler poultry farming with Open House System (OHS) in Lamongan Regency has not been technically efficient even though a high level of technical efficiency is obtained in each of the business patterns of 0.958 in the Open House System (OHS) pattern. 4. Broiler poultry farming with Open House System (OHS) in Lamongan Regency has not been technically efficient even though a high level of technical efficiency is obtained in each of the business patterns of 0.958 in the Open House System (OHS) pattern. p y ( ) p 5. The diversity of technical efficiency level in the broiler poultry farming business with Open House System (OHS) pattern of 0.031 is influenced by inefficiency sources, namely age and education. p y ( ) p 5. The diversity of technical efficiency level in the broiler poultry farming business with Open House System (OHS) pattern of 0.031 is influenced by inefficiency sources, namely age and education. 6. The ability of breeders in minimizing the cost to achieve average broiler poultry production with a Open House System (OHS) pattern of 5687,5 live chickens is at a satisfactory level but does not meet economic efficiency with an average economic efficiency of 0, 9999740 with the Open House System (OHS) pattern. Economic Efficiency Analysis: The success rate of the performance of broiler poultry breeders with a open house system pattern can be known through the use of production factors in producing high production with minimum costs to the production factors. The success rate of broiler poultry farming can be said to be economically efficient if technically the breeders are efficient using production factors by streamlining the prices of these production factors. The level of success achieved between breeders will differ from one another due to differences in the level of knowledge and ability to the broiler poultry farming business. Achieving economic efficiency from broiler poultry breeders with a closed house system pattern is presented in the following table. Table 5:- Description of the Economic Efficiency of the Broiler Poultry Farming Business in Lamongan Regency. Economic Efficiency Closed House System (CHS) Average 0.9999740 StandardDeviation 0.0000001 Minimum 0.9999737 Maximum 0.9999746 Source: Processed Primary Data (2019) Based on table 5,it shows the average value of economic efficiency achieved by the broiler poultry farming business with a closed house system pattern is 0.9999740 (rounding number). This shows that on average all breeders in the two broiler poultry farming business groups have almost the same ability in minimizing production costs for the use of several production factors. The ability which is almost the same between the two broiler poultry farming businesses is thought to be caused by all the production factors used originating from one source which causes the uniformity of the price of the production factor. 1058 Int. J. Adv. Res. 8(01), 1053-1060 ISSN: 2320-5407 Distribution of Economic Efficiency per Individual in the Broiler PoultryFarming Business with a Open House System (OHS) Pattern Figure 15:- Distribution of Economic Efficiency per Individual in Broiler Poultry Farming Business with Open House System (OHS) Pattern Economic Efficiency Economic Efficiency per Individual in the Broiler PoultryFarming Business with a Open OHS) Pattern Figure 15:- Distribution of Economic Efficiency per Individual in Broiler Poultry Farming Business with Open House System (OHS) Pattern Figure 15 shows the economic efficiency per individual in a broiler poultry farming business with a Open House System (OHS) pattern. From these results obtained the highest value of 1,0000261 in breeders 9 and the lowest value of 1,0000259 in breeders no 5. 1. Abayie Eric FosuOteng, Kofi Amanor and Joseph Magnus Frimpong. 2011. 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https://openalex.org/W4323364276	https://bmcpublichealth.biomedcentral.com/counter/pdf/10.1186/s12889-023-15227-4	English	null	Community-based approaches to infant safe sleep and breastfeeding promotion: a qualitative study	BMC public health	2,023	cc-by	10,145	© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract Background In the U.S., sudden unexpected infant deaths (SUID) due to accidental suffocation and strangulation in bed (ASSB) are increasing, with disparities by race/ethnicity. While breastfeeding is a protective factor against infant mortality, racial/ethnic disparities are present in its uptake, and motivations to breastfeed are also often coupled with non-recommended infant sleep practices that are associated with infant sleep deaths. Combining infant safe sleep (ISS) and breastfeeding promotion on the community level presents opportunities to address racial/ethnic disparities and associated socioeconomic, cultural, and psychosocial influences. Methods We completed a descriptive qualitative hermeneutical phenomenology using thematic analysis of focus group data. We examined the phenomenon of community-level providers promoting ISS and breastfeeding in com- munities vulnerable to ISS and breastfeeding disparities. We asked eighteen informants participating in a national quality improvement collaborative about i.) areas requiring additional support to meet community needs around ISS and breastfeeding, and ii.) recommendations on tools to improve their work promoting ISS and breastfeeding. Results We identified four themes: i.) education and dissemination, ii.) relationship building and social support, iii.) working with clients’ personal circumstances and considerations, and iv.) tools and systems. Conclusions Our findings support embedding risk-mitigation approaches in ISS education; relationship building between providers, clients, and peers; and the provision of ISS and breastfeeding supportive material resources with educational opportunities. These findings may be used to inform community-level provider approaches to ISS and breastfeeding promotion. Keywords Infant safe sleep, Breastfeeding, Community health promotion, Perinatal education Community‑based approaches to infant safe sleep and breastfeeding promotion: a qualitative study Meera Menon1, Rebecca Huber1* , Dana D. West1, Stacy Scott1, Rebecca B. Russell1 and Sc Background Since the late 1990s, there has been significant progress in reducing the number of sudden unexpected infant deaths (SUID) in the U.S., yet troubling trends and dis- parities remain [1]. SUID encompasses deaths from sud- den infant death syndrome (SIDS), accidental suffocation and strangulation in bed (ASSB), and other ill-defined and unspecified causes of infant deaths [2, 3]. Despite declining SUID, ASSB and deaths due to unknown causes are increasing (see Fig. 1), and there are substantial dis- parities between racial/ethnic and geographic groups [1, Correspondence: Rebecca Huber rhuber@nichq.org 1 The National Institute for Children’s Health Quality (NICHQ), 308 Congress Street, 5th Floor, Boston, MA 02210, USA 2 Department of Pediatrics, Warren Alpert Medical School of Brown University, Providence, RI, USA 3 Department of Health Services, Policy and Practice, Brown University School of Public Health, Providence, RI, USA Correspondence: Rebecca Huber rhuber@nichq.org 1 The National Institute for Children’s Health Quality (NICHQ), 308 Congress Street, 5th Floor, Boston, MA 02210, USA 2 Department of Pediatrics, Warren Alpert Medical School of Brown University, Providence, RI, USA 3 Department of Health Services, Policy and Practice, Brown University School of Public Health, Providence, RI, USA BMC Public Health BMC Public Health Menon et al. BMC Public Health (2023) 23:437 https://doi.org/10.1186/s12889-023-15227-4 Open Access © 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. Menon et al. BMC Public Health (2023) 23:437 Page 2 of 12 Fig. 1 Trends in sudden unexpected infant deaths by cause, 1990-2020. Source: Data from Centers for Disease Control and Prevention, National Center for Health Statistics. National Vital Statistics System, Mortality Files, analyzed by the National Institute for Children’s Health Quality Fig. 1 Trends in sudden unexpected infant deaths by cause, 1990-2020. Source: Data from Centers for Disease Control and Prevention, National Center for Health Statistics. National Vital Statistics System, Mortality Files, analyzed by the National Institute for Children’s Health Quality 3–7]. Recent rates of SUID in the U.S. are shown to be higher among non-Hispanic Black and American Indian/ Alaskan Native infants compared to Hispanic, non-His- panic White, and Asian/Pacific Islander infants [3, 4, 6– 8]. Furthermore, within the U.S., there are higher rates of infant deaths in rural compared to urban communities [4, 6]. together, these findings stress the need to promote ISS, particularly to prevent infant sleep related deaths among historically marginalized populations. Despite recommendations from the World Health Organization (WHO) and the AAP encouraging infants to be exclusively breastfed for at least 6 months and up to 2 years, the U.S. ranks among the lowest in breastfeed- ing rates compared with other industrialized countries, and displays racial/ethnic and socioeconomic disparities in breastfeeding practices that mirror those present with SUID [13–17]. The reasons for not initiating or maintain- ing breastfeeding are complex and involve cultural, psy- chosocial, and policy-level factors, all generally leading to a lack of support for a breastfeeding individual [18–21]. In addition, factors such as preconceptions of breast- feeding in one’s social circle influence breastfeeding out- comes [16, 18, 22]. Furthermore, while breastfeeding is an important protective factor against SIDS, strong moti- vations to breastfeed are often coupled with non-recom- mended infant sleep practices such as bedsharing, and are often most pronounced among groups most vulner- able to SUID, making it challenging to rectify disparities among both ISS and breastfeeding uptake [23–28]. Thus, ] As SUID often occurs during infant sleep or in an infant’s sleeping space, there is a strong need to educate on strategies to prevent infant sleep related deaths using the American Academy of Pediatrics (AAP)‘s infant safe sleep (ISS) guidelines [9]. AAP ISS guidelines include having infants room-share with parents without bedshar- ing, keeping soft objects such as pillows and comforters out of the crib, and placing infants to sleep supine [10]. Despite the widespread nature of these recommenda- tions and successful public health messaging around ISS practices to prevent SIDS and SUID, such as the Safe-to- Sleep campaign (formerly the Back-to-Sleep campaign), racial/ethnic and geographic variation in the adoption of ISS practices persist, with non-Hispanic Black parents demonstrating higher rates of non-recommended infant sleep practices compared to other groups [11, 12]. Taken Menon et al. BMC Public Health (2023) 23:437 Menon et al. BMC Public Health (2023) 23:437 Page 3 of 12 Further, there is a dearth of research on the experi- ences of community-level organizations and providers in promoting both ISS and breastfeeding. Given the impor- tance of community settings in reaching historically mar- ginalized populations as well as coordinated messages to support ISS and breastfeeding, it is critical to understand the needs of community-level direct service providers to build systems to promote ISS and breastfeeding. Accord- ingly, our study examined the phenomenon of commu- nity-level providers and organizations promoting ISS and breastfeeding for their communities vulnerable to ISS and breastfeeding disparities (e.g., rural communities or those with high concentrations of Black or Indigenous populations). Methods Data collected for this analysis came from a larger evaluation of the National Action Partnership to Pro- mote Safe Sleep Improvement and Innovation Network (NAPPSS-IIN), a multi-year project running from 2017- 2022. NAPPSS-IIN aimed to make ISS and breastfeed- ing a national norm. Specifically, the project focused on increasing infant caregivers’ adoption of ISS and breast- feeding, as recommended by the AAP, by empower- ing champions within systems that serve historically marginalized families. The goal of NAPPSS-IIN was to reduce rates of SUID, increase rates of breastfeeding, and decrease disparities in these outcomes among Black and Indigenous infants. The initiative was funded by the Maternal Child Health Bureau (MCHB) of the Health Resources and Services Administration (HRSA) and led by the National Institute for Children’s Health Quality (NICHQ). Providing material resources for ISS and breastfeed- ing (e.g., bassinets, breast pumps), parent education, and connecting parents to supports are all successful strategies to promoting uptake of ISS and breastfeeding [31–33]. Community baby showers combine the above opportunities and demonstrate increased uptake of AAP- supported ISS practices among participants while more successfully reaching vulnerable populations compared to similar events in healthcare settings [31–33]. Further, community-driven models such as peer counseling are shown to be effective in providing breastfeeding support and enhancing breastfeeding outcomes, often more than in-hospital breastfeeding support alone [36–38]. Finally, in a community-based intervention targeting African American parents that combined ISS and breastfeeding messaging together, researchers found that participants were able to successfully maintain exclusive breastfeed- ing rates without bedsharing [24]. As part of the NAPPSS-IIN evaluation, in Spring 2021 NICHQ hosted a series of listening sessions held in focus groups with community-level partners working in ISS and breastfeeding promotion and participating in the NAPPSS-IIN initiative. All NAPPSS-IIN participants served communities vulnerable to ISS and breastfeeding disparities (e.g., rural communities or those with high concentrations of Black or Indigenous populations). We employed descriptive hermeneutical phenomenology as the research design [46–48]. The phenomenon studied was the lived experiences and perceptions of community- level providers promoting ISS and breastfeeding. Focus groups were selected over key informant interviews While ISS and breastfeeding interventions delivered in community settings have been successful, interventions and services may not be available in all communities [39]. Specifically, our aims were: i.) to identify areas requiring additional support to meet communities’ needs around ISS and breastfeeding, and ii.) to capture recommendations and opinions on tools and resources that would improve their abilities to promote ISS and breastfeeding within their communities. it is important to consider education that supports the combination of ISS and breastfeeding recommendations while accounting for parents’ contexts, preferences, and culture. While hospital-based interventions are successful in bundling ISS and breastfeeding [29, 30], promotion in the community setting may better reach populations vul- nerable to disparities [21, 24, 31–33]. For the purposes of this study, we define community as encompassing the following elements: contained by specific place-based and geographic boundaries and comprised of individu- als that share social ties and connections including, but not limited to culture, socio-economic status, and race/ ethnicity [34]. In a systematic review of breastfeeding interventions, Segura-Pérez and colleagues identified that policy- and community-level interventions (e.g., delivered through community agencies) were more likely to improve uptake of optimal infant care practices [21]. In addition, research depicts parents are more likely to change their behaviors and practices when they hear messages from multiple sources within their communi- ties, underscoring the importance of delivering ISS and breastfeeding education across multiple venues [35]. Methods Moreover, community-led work to promote ISS and breastfeeding is not immune to barriers such as the influ- ence of parents’ social networks and cultural beliefs that can hinder uptake of recommended practices [16, 35, 40– 43]. In addition, community settings are often limited by resources, such as those to train and provide continuing support for educators [44, 45]. Menon et al. BMC Public Health (2023) 23:437 Page 4 of 12 for the opportunity to foster shared discussion on the phenomenon. the data. NVivo was used for data analysis and manage- ment [50]. To maximize trustworthiness of the analysis, investi- gator triangulation was used. All coders have expertise in community-based ISS and breastfeeding promotion. The researchers followed the six phases outlined by Braun and Clarke [49] which embedded reflexivity into all stages of the analysis. A combination of inductive and deductive coding was applied with initial codes based on predetermined areas of challenge and opportunity in ISS and breastfeeding promotion identified within the litera- ture. Deductive coding was coupled with inductive cod- ing to allow for the experience of informants to guide the domains of analysis. Initial review of two transcripts and coding were completed by MM and RH independently. Upon initial review, MM and RH met to discuss coding structure and application. This process continued in an iterative fashion until an initial codebook was established and appropriate reliability was met (pooled K > 0.80). RH coded the remaining two cases. A third analyst, DW, reviewed all coding applications and the codebook holis- tically. Disagreements were mediated with the other analysts until thematic saturation was achieved and reli- ability was achieved (pooled K > 0.80). Theme generation proceeded iteratively with the coding team discussing and refining themes independently and collaboratively. Themes were mapped onto codes to verify accuracy and exemplar quotes were identified. Memo generation and collaborative discussions around codes and themes occurred with the entire research team to maintain rigor and reflexivity. We recruited a convenience sample of 18 informants participating in the NAPPSS-IIN project who consented to participate in interviews. Focus groups followed a semi-structured format and aimed to identify barriers and opportunities for promoting ISS and breastfeeding as well as to inform NAPPSS-IIN activities for the final years of the initiative. The interview guide was developed specifically for the focus groups and is available as a Sup- plementary Material, Additional file 1. Methods Overall, a total of four focus groups were conducted, with the number of informants per session ranging from two to six. Table 1 provides demographics of informants’ organizations along with their roles. Focus groups were conducted vir- tually on the Zoom platform and included informants as well as up to four members of the research team. Tran- scripts from each call were obtained and transcribed using the Rev.com service. All identifying information was redacted prior to transcript review. The study proto- col was reviewed and approved by Solutions Institutional Review Board. Some informants had more than one role/certification, service area, and organization/sector type Analysish The research team utilized thematic analysis as the pri- mary methodology and employed inductive coding to develop the code structure [49]. This method was used for the flexibility of approach and because limited research was available to develop a predetermined cod- ing structure. Instead, the team developed coding and themes based on the explicit meanings emerging from Table 1 Informant roles, organizations, and service areas in focus groups Some informants had more than one role/certification, service area, and organization/sector type Case Informant roles Organization/Sector types Service areas Case 1 • Lactation Consultant (2) • Program Director/Manager (1) • Program Coordinator (1) • Registered Nurse (1) • Non-profit (2) • Health system (1) • Head Start/Early Head Start (1) • Statewide (1) • Urban (1) • Rural (1) • Unspecified service area (1) Case 2 • Program Director/Manager (2) • Program Coordinator (1) • Clinical Consultant (1) • Data Analyst (1) • Lactation Consultant (1) • Quality Assurance Manager (1) • Registered Nurse (1) • Department of Health (2) • Non-profit (2) • Health insurance (1) • Healthy Start (1) • Urban (3) • Statewide (2) • Rural (1) • National (1) Case 3 • Program Director/Manager (1) • Case Manager (1) • Non-profit (1) • Health insurance (1) • Statewide (1) • Urban (1) • Rural (1) Case 4 • Program Director/Manager (2) • Program Coordinator (2) • Registered Nurse (1) • Doula (1) • Mental Health Clinician (1) • Department of Health (3) • Non-profit (2) • Independent healthcare professional (1) • Urban (4) • Statewide (2) Table 1 Informant roles, organizations, and service areas in focus groups Page 5 of 12 Menon et al. BMC Public Health (2023) 23:437 Table 2 Theme and sub-theme titles, references, and exemplar quotes Exemplar quotes not provided for themes in which there were exemplar quotes from sub-themes Theme/Sub-theme title N references % references Exemplar quote Theme 1: Education and dissemination 271 37% Sub-theme 1a: Education and dissemination challenges 213 29% “Not all nurses teach the same thing…some of them [were educated] in nursing school [which could be] 35 years ago. [At that time,] some [nurses] didn’t even [learn] information on safe sleep or, let alone, breastfeeding and safe sleep.” Sub-theme 1b: Education opportunities 49 7% “I think what is needed in our state is tools for open, candid conversations to talk with families about this intersection between breastfeeding and safe sleep. Analysish We see that they’re often [taught] separate but parents experience them together...And so I would like more tools that would help…to have conversations that are less prescriptive, less... preachy.” Sub-theme 1c: Dissemination opportunities 9 1% “[Nighttime parenting plans are] a chance to [establish], ‘Let’s walk through what’s going to happen at 2 AM and you’re exhausted and all the best intentions in the world [around ISS and breastfeeding] are gone out the window.’” Theme 2: Relationship building and social support 252 34% Sub-theme 2a: Patient-provider relationship building 171 23% “Having the Maternal Nurse Navigators that...reach out, even if it’s a small population, [to] give them information, make sure they have resources... I think that’s a really great start. I know we’re catching some of the moms [who we would normally miss].” Sub-theme 2b: Peer-to-peer connections 81 11% “I’ve also learned to maintain connections with mothers and families that I have served in the past because they can tell their [breastfeeding and ISS] stories to clients that I’m serving now and I think that stories are so powerful.” Theme 3: Working with clients’ personal circumstances and considerations 144 20% Sub-theme 3a: Capacity 88 12% “The biggest resource I think moms need is someone to help them, but I don’t know how you...take the load off them, because we do see...they’re just at their wits end sometimes. They’ve got so many things going on in their life, especially if they’ve got socio-economic factors affecting them.” Sub-theme 3b: Social determinants 38 5% “[ISS promotion makes] assumptions that [the client] has a crib or room for a crib. [But,] we don’t always know their living circumstances and how that impacts what they’re able to do”. Sub-theme 3c: Generational barriers 18 2% “Things have changed a lot since our clients’ mothers and grandmothers were having babies. [Extended family members will say]...‘you’ve got to give both [formula and breastmilk] because the baby is not [eating] enough,’ or, ‘you put the baby on their stomach to sleep because that’s what we did.’ We educate our moms, but then there’s that missing piece – how does it get from the mom, to the grandma, and the auntie and the older generation who did things differently?” Theme 4: Tools and systems 71 10% “All of our clients are Black mothers...Our initiation rate is excel- lent, but we run into issues [when they] go back to work. TOTAL Exemplar quotes not provided for themes in which there were exemplar quotes from sub-themes Analysish A lot of them don’t have [comprehensive] maternity leave...or they work in jobs that don’t allow them time to pump. That’s where we see the breastfeeding [rates] fall off.” TOTAL 738 100% Table 2 Theme and sub-theme titles, references, and exemplar quotes N references % references Exemplar quote “[Nighttime parenting plans are] a chance to [establish], ‘Let’s walk through what’s going to happen at 2 AM and you’re exhausted and all the best intentions in the world [around ISS and breastfeeding] are gone out the window.’” “Having the Maternal Nurse Navigators that...reach out, even if it’s a small population, [to] give them information, make sure they have resources... I think that’s a really great start. I know we’re catching some of the moms [who we would normally miss].” “I’ve also learned to maintain connections with mothers and families that I have served in the past because they can tell their [breastfeeding and ISS] stories to clients that I’m serving now and I think that stories are so powerful.” “The biggest resource I think moms need is someone to help them, but I don’t know how you...take the load off them, because we do see...they’re just at their wits end sometimes. They’ve got so many things going on in their life, especially if they’ve got socio-economic factors affecting them.” “[ISS promotion makes] assumptions that [the client] has a crib or room for a crib. [But,] we don’t always know their living circumstances and how that impacts what they’re able to do”. “Things have changed a lot since our clients’ mothers and grandmothers were having babies. [Extended family members will say]...‘you’ve got to give both [formula and breastmilk] because the baby is not [eating] enough,’ or, ‘you put the baby on their stomach to sleep because that’s what we did.’ We educate our moms, but then there’s that missing piece – how does it get from the mom, to the grandma, and the auntie and the older generation who did things differently?” “All of our clients are Black mothers...Our initiation rate is excel- lent, but we run into issues [when they] go back to work. A lot of them don’t have [comprehensive] maternity leave...or they work in jobs that don’t allow them time to pump. That’s where we see the breastfeeding [rates] fall off.” Sub‑theme 2a: Patient‑provider relationship building Activities and meaningful connections with providers were noted as both resources and challenges to ISS and breastfeeding promotion. Informants discussed indi- vidualized attention as a key area to support uptake of breastfeeding and ISS. However, six informants discussed these techniques as subject to individual discretion. One informant shared, “[What’s lacking is] the support after- wards... [parents are] not getting lactation nurses that are supportive in the hospital. I know they have lacta- tion consultants that … [are] there for five minutes … and then they walk out.” “I think [we could use] guidance on phrasing and word- ing [for] when we are providing education to [moms] postpartum or [prenatally]. How do we come about it in a non-combative or...a non-aggressive manner? Because I feel like as soon as they step into that hospital … [ISS is] being pushed down their throat.” Sub‑theme 1b: Education opportunities Several informants noted that educational and dissemi- nation challenges were often intertwined. Specifically, informants referenced ISS and breastfeeding messages as disconnected from clients’ everyday realities. An example included abstinence-based approaches to ISS, of which eleven informants were critical, noting a preference for promoting risk-mitigation. Abstinence-based ISS educa- tion was often discussed alongside the difficulty of com- bining ISS and breastfeeding messaging, as evidenced in the excerpt below: Five informants successfully employed client-provider relationship building to promote breastfeeding and ISS which resulted in positive outcomes, such as reaching underserved groups, as was described by one informant: “Having the Maternal Nurse Navigators that...reach out, even if it’s a small population, [to] give them infor- mation, make sure they have resources... I think that’s a really great start. I know we’re catching some of the moms [who we would normally miss].” Five informants successfully employed client-provider relationship building to promote breastfeeding and ISS which resulted in positive outcomes, such as reaching underserved groups, as was described by one informant: “Having the Maternal Nurse Navigators that...reach out, even if it’s a small population, [to] give them infor- mation, make sure they have resources... I think that’s a really great start. I know we’re catching some of the moms [who we would normally miss].” “I think what is needed in our state is tools for open, candid conversations to talk with families about this intersection between breastfeeding and safe sleep. We see that they’re often [taught] separate but parents expe- rience them together...And so I would like more tools that would help … to have conversations that are less pre- scriptive, less... preachy.” Theme 2: Relationship building and social supporth The second most discussed theme was activities that build meaningful and intentional connections for ISS and breastfeeding promotion. This theme entailed two topics: client-provider relationship building and peer-to- peer connections. While all informants agreed that these methods were positive resources to promote ISS and breastfeeding, informants also shared barriers in building relationships. Sub‑theme 1a: Education and dissemination challenges Sub‑theme 1c: Dissemination opportunities Dissemination opportunities referred to spreading infor- mation about ISS and breastfeeding. Four informants shared examples such as helping parents with nighttime decision-making. Nighttime decision-making was dis- cussed as a pragmatic method to prepare clients for the realities of adhering to ISS and breastfeeding practices as an overwhelmed new parent. One informant elaborated that their organization developed nighttime parenting plans in partnership with each client, which were well- received: “It’s a chance to [establish], ‘Let’s walk through what’s going to happen at 2AM and you’re exhausted and all the best intentions in the world [around ISS and breastfeeding] are gone out the window.’” Challenges to education and dissemination included an absence of effective teaching guidance (related to messaging and tone, education standards), an absence of effective messaging guidance for clients and providers (related to promoting abstinence of unsafe sleep practices, discussing ISS and breastfeed- ing jointly), and ignoring AAP guidelines. Thirteen informants indicated that clinician education can be dependent on when and where they received their initial training and whether they engaged in continued educational opportunities. One informant elaborated, “Not all nurses teach the same thing … some of them [were educated] in nursing school [which could be] 35 years ago. [At that time,] some [nurses] didn’t even [learn] information on safe sleep or, let alone, breast- feeding and safe sleep.” While inconsistent education standards and a lack of realistic messaging guidance were challenges to education and dissemination, nine informants shared progress and success in these areas. Informants referenced needing guidance on messaging and tone to support ISS and breastfeed- ing, and several shared a desire to learn best com- munication practices, noting how guidance on conversational approaches could result in more trust- ing relationships with clients. One informant elaborated on how this could enhance their ISS and breastfeeding promotion: Theme 2: Relationship building and social supporth Theme 1: Education and disseminationh The education and dissemination theme included chal- lenges and opportunities around teaching, learning, and spreading information about ISS and breastfeeding promotion. Of the four themes identified, this theme was the most discussed in focus groups. We identified four themes discussed by community-level providers in focus groups as areas where they needed support, tools, or resources in promoting ISS and breast- feeding (Table 2). Menon et al. BMC Public Health (2023) 23:437 Page 6 of 12 Menon et al. BMC Public Health (2023) 23:437 Page 6 of 12 Sub‑theme 3b: Social determinants Nine informants discussed broader conditions that hin- dered ISS and breastfeeding adoption such as opioid use, poverty, mental health, and language barriers. As with capacity, informants expressed sensitivity on these topics and took efforts to address SDOH in their work. Yet, informants reported continued structural barriers to sufficiently address SDOH while promoting ISS and breastfeeding. One informant expressed how their abil- ity to promote ISS was limited by SDOH: “[ISS promo- tion makes] assumptions that [the client] has a crib or room for a crib. [But,] we don’t always know their living circumstances and how that impacts what they’re able to do.” However, the four informants who were able to broker successful peer-to-peer connections among their clients reported positive outcomes as described by the following informant: “I’ve also learned to maintain connections with moth- ers and families that I have served in the past because they can tell their [breastfeeding and ISS] stories to cli- ents that I’m serving now and I think that stories are so powerful.” Sub‑theme 3c: Generational barriers ISS d b f di i ISS and breastfeeding experiences differing from current recommendations were discussed by four informants as hindering ISS and breastfeeding adoption. Inform- ants noted the influence of cultural/family practices on ISS and breastfeeding adherence. While some sought to engage all extended family members who interact with a newborn to remedy this barrier, they reflected on their limited capacity to do so, as evidenced in the quote below: This theme referred to clients’ experiences and/or context hindering ISS and breastfeeding guideline adoption. All informants were sympathetic to clients’ circumstances and many reported efforts to address them in service delivery. Yet, population needs continued to be a barrier to ISS and breastfeeding adoption, and fell under three topics: capacity, social determinants of health (SDOH), and generational barriers. “Things have changed a lot since our clients’ mothers and grandmothers were having babies. [Extended family members will say]...‘you’ve got to give both [formula and breastmilk] because the baby is not [eating] enough,’ or, ‘you put the baby on their stomach to sleep because that’s what we did.’ We educate our moms, but then there’s that missing piece – how does it get from the mom, to the grandma, and the auntie and the older generation who did things differently?” Sub‑theme 2b: Peer‑to‑peer connections Informants discussed building peer-to-peer connections in support of ISS and breastfeeding as both a resource and challenge. Examples shared included support groups, virtual/in-person events, and connections to external specialists/organizations. Some informants noted that Menon et al. BMC Public Health (2023) 23:437 Page 7 of 12 they knew of peer-to-peer resources around ISS and breastfeeding, but that organizations may not be actively sharing this information with parents: “When it comes to … bed sharing, a lot of times par- ents know it’s a risk … but they do it anyway because...it’s easy for them. There’s this fine line that you walk some- times, because...We don’t want to make them feel bad or guilty, but...you also want to provide them with truth and research and information.” “It would be really nice if the hospital...[developed] a clear-cut list of community resources that mom could utilize, whether that were to be, where can they get assis- tance on finding a pump, [or] what support groups they have specifically for the breastfeeding community … where they have resources for in-home consultations...or for [ISS]...or to the health department to get a ‘Pack N’ Play’.” Sub‑theme 3a: Capacity Individual conditions that hampered ISS and breastfeed- ing adoption included client overwhelm which often led to clients following their intuition around ISS and breast- feeding. Thirteen informants expressed sensitivity to the overwhelming nature of new parenthood and impacts on breastfeeding and ISS. Few informants were able to offer practical solutions to address overwhelm beyond rela- tionship building, as discussed below:h “The biggest resource I think moms need is someone to help them, but I don’t know how you...take the load off them, because we do see...they’re just at their wits end sometimes. They’ve got so many things going on in their life, especially if they’ve got socio-economic factors affecting them.” Theme 4: Tools and systemsi Informants shared specific and actionable instruments and infrastructure as mechanisms for ISS and breast- feeding promotion. These mechanisms were framed by informants as components working together or failing to work together to promote ISS and breastfeeding. Seven informants found material resources such as “Pack N’ Plays”, bassinets, and baby gates helpful in promoting ISS and breastfeeding. One informant noted how the pres- ence of material resources in the home could “bring on f Six informants referenced how parents may rely on their own instincts during duress, which may not align with ISS and breastfeeding guidelines. The main mecha- nism shared by four informants to remedy this dynamic was social support, as evidenced by this quote: Menon et al. BMC Public Health (2023) 23:437 Page 8 of 12 Page 8 of 12 [important] conversations in the community” around ISS and breastfeeding when friends and family members visited.f conversations around bedsharing in their communities, especially as abstinence-based approaches to ISS educa- tion may deter parents from initiating breastfeeding or can serve to prematurely end breastfeeding [52].h Informants discussed different media portrayals of ISS and breastfeeding including videos, pamphlets, and liter- ature with positive and negative sentiments. While some reported their organization’s educational videos had potentially shaming messaging, others found their videos effective in promoting ISS and breastfeeding. Addition- ally, though some informants found providing pamphlets and literature on ISS and breastfeeding to be helpful, oth- ers were concerned about overwhelming clients with too much information. The Academy of Breastfeeding Medicine highlights the need to promote recommended ISS guidelines while incorporating messaging on risk-mitigation [52, 53]. These approaches holistically consider factors that may lead to risk of infant death (i.e., smoking, parent con- sumption of alcohol or drugs, and prematurity/low birth weight) and encourage providers to engage in conversa- tion around ISS while addressing strategies to reduce risk of adverse outcomes should unsafe sleep practices occur [52, 53]. Risk-mitigation approaches use flexible, stigma- free methods that allow for incremental changes to move towards ISS rather than parents feeling overwhelmed by fully overhauling practices [52]. Indeed, some study informants noted success in the provision of risk-miti- gation support and education, especially within the con- text of parents’ nighttime decision-making. Providing resources around risk-mitigation to a broader swath of providers, including those working on the community level, may rectify some tensions around ISS education. Discussion Community-based approaches demonstrate promise in promoting ISS and breastfeeding among underserved populations [21, 24, 31–33]; however, study informants noted substantial barriers to ISS and breastfeeding pro- motion within their communities, all of which were vul- nerable to ISS and breastfeeding disparities (e.g., rural communities or those with high concentrations of Black or Indigenous populations). Despite the challenges, informants also highlighted opportunities to promote ISS and breastfeeding practices in their communities. Our findings suggest that addressing the challenges inform- ants identified and bolstering work in areas deemed promising would help community-level organizations increase the reach of ISS and breastfeeding promotion. Another challenge related to education and dissemi- nation was variation in community-level providers’ edu- cation around ISS and breastfeeding and training on sensitive message delivery. While studies depict barri- ers for paraprofessional and community-focused educa- tion models on recommended ISS practices [44], efforts to train providers on education strategies improve client outcomes [45, 54]. Providing ISS and breastfeeding train- ing opportunities for paraprofessionals [45] as well as cultural sensitivity trainings targeted on message dissem- ination [54] enhance the quality of support clients receive and their ISS and breastfeeding outcomes [45, 54]. Taken together, the challenges noted by our study informants underscore the need for continued education and sup- port for community-level providers around educating and messaging current ISS and breastfeeding guidelines. Challenges and opportunities related to education and dissemination Education and dissemination around ISS and breastfeed- ing guidelines was discussed by most informants as a challenge; however, these concepts were also referenced as areas of opportunity. Providers shared difficulties in educating parents on effective ISS and breastfeeding strategies, mostly referring to tension around abstinence- based ISS education. Bedsharing is often the reality for many families as a sizeable number of parents will unintentionally bedshare [51], and many parents with strong breastfeeding intentions bedshare [23, 26, 28]. Study informants noted the struggle of not engaging in Theme 4: Tools and systemsi In this manner, providers may still support ISS practices as endorsed by the AAP while meeting parents where they are. Policies and work environments were referenced as mutually reinforcing mechanisms for ISS and breastfeed- ing promotion. Three informants reported that in the absence of comprehensive paid family leave in their state, parents returned to work earlier than they would have liked, and that work environments did not promote ISS and breastfeeding. One informant indicated that, “work- ing in jobs that don’t allow them time to pump [is] where we see breastfeeding [rates] fall off after those first couple of months” within their community. Challenges and opportunities related to relationship building and social support With informants highlighting relationship build- ing between providers and clients as an area of need, providing trainings and resources to community-level providers around shared decision-making could support efforts to promote ISS and breastfeeding. Study informants also noted that generational barriers influencing parents’ decision making as a challenge in their communities. Research shows that certain African American and American Indian/Alaskan Native groups may be aware of ISS guidelines but prefer to follow infant sleep practices that are deemed “unsafe” due to cul- tural preferences, making it challenging for providers to engage in conversations around shifting behavior [22, 43, 61, 62]. These challenges may be due to generational barriers, as American Indian/Alaskan Native parents and African American parents often look to older family members, who may be skeptical of recommendations on ISS and breastfeeding, as trusted sources around infant care [18, 22, 40, 41, 43, 63]. With research depicting that parents vulnerable to disparities in ISS and breastfeed- ing value grandparents and elders’ opinions on infant care, and that grandparents and elders tend to be wary of infant care recommendations that run counter to cultural beliefs [18, 22, 27, 63], it is critical that providers consider culturally congruent care in building relationships with families. f Similarly, peer-to-peer connections were discussed as a challenge to ISS and breastfeeding promotion among study informants who noted their limited ability to facili- tate connections within their programs. However, several informants shared preliminary efforts around supporting peer-to-peer connections, remarking that these relation- ships may support ISS and breastfeeding practice uptake. Parents utilize peer networks, including social media, as a source of information, support, and bonding regarding infant care [35, 40, 42]. Yet, peer-to-peer spaces have less formalized content moderation than those maintained by professionals, providing an area of opportunity for community-level organizations to train and engage peer counselors as moderators. Substantial research depicts that peer counselor support is associated with uptake of optimal infant care practices as counselors may provide individualized support for parents [36–38, 59]. Build- ing on opportunities identified by our study informants, training peer counselors and facilitating connections among peers may provide another avenue to support uptake of ISS and breastfeeding. Challenges and opportunities related to tools and systems The last barrier study informants shared was regarding tools and systems, such as media messaging, material resources, and societal factors supporting the uptake of ISS and breastfeeding in their communities. Challenges and opportunities related to relationship building and social support Informants noted their capacity to build relationships and provide client support both directly and through peers was a barrier to promoting ISS and breastfeeding in their communities. However, embedded in these chal- lenges were opportunities to strengthen community-led promotion. Study informants shared that clients found current interactions with providers lacking and preferred additional opportunities for connection. Parents look to Menon et al. BMC Public Health (2023) 23:437 Page 9 of 12 Menon et al. BMC Public Health (2023) 23:437 research demonstrates that parental life circumstances tend to dictate decisions around ISS and breastfeed- ing [18, 27, 40, 42, 43]. For instance, social and physical support at home is strongly associated with the uptake of breastfeeding and ISS [16, 18, 27, 35]. Further, mental health, poverty, and substance abuse may drive parental decisions around the uptake of ISS and breastfeeding practices. For example, conditions related to poverty and trauma can inform decisions around infant care [27, 60]; trauma histories are related to lower breastfeeding initia- tion [60] and in certain housing contexts, it may be safer for parents to sleep with their infants, rather than recom- mended ISS practices [27]. healthcare providers as trusted sources of information; yet, many parents are conflicted about querying provid- ers on infant care practices, noting they do not want to bother clinicians [40]. Moreover, research finds that minoritized parents feel they are overlooked or forgot- ten by providers and city services [43]. Providers, in turn, mention time limitations for client connections and edu- cation deter them from engaging in conversations around ISS practices [45].h healthcare providers as trusted sources of information; yet, many parents are conflicted about querying provid- ers on infant care practices, noting they do not want to bother clinicians [40]. Moreover, research finds that minoritized parents feel they are overlooked or forgot- ten by providers and city services [43]. Providers, in turn, mention time limitations for client connections and edu- cation deter them from engaging in conversations around ISS practices [45].h These challenges underscore that concerted strategies must be taken to build connections within parents’ rou- tine interactions with providers. Having providers engage in conversational, shared decision-making approaches to promoting ISS and breastfeeding could assist in fostering relationship building. Conversational approaches around infant care, particularly on ISS and breastfeeding, are positively associated with client uptake of these practices [55–58]. Challenges and opportunities related to relationship building and social support Media por- trayals were noted as an area of challenge for informants, who discussed that ISS and breastfeeding messaging could be shaming. Research depicts parents’ struggle to negotiate messaging related to infant care from edu- cational resources and health campaigns with informa- tion received from friends and family [22, 43, 63]. In addition, media portrayals of ISS and breastfeeding are not often culturally congruent in delivery and intended target population [22, 43]. As parents are more likely to change their behavior related to infant care when mes- saging received is additive and not contradictory [35], it is important to ensure that media resources and messag- ing convey aligned information. Challenges and opportunities related to working with clients’ personal circumstances and considerations Working with clients’ personal circumstances and con- siderations were barriers for informants to promote ISS and breastfeeding. These circumstances were primarily related to parents’ capacity, generational barriers, and SDOH. Informants shared that when parents’ capacities were limited, parents could revert to their own instincts regarding ISS and breastfeeding practices. Other Menon et al. BMC Public Health (2023) 23:437 Menon et al. BMC Public Health (2023) 23:437 Page 10 of 12 Page 10 of 12 Informants noted that the provision of material resources served as an opportunity to promote ISS and breastfeeding. Material resources such as portable cribs were discussed as an entry point to engage in conversa- tions about ISS and breastfeeding with clients. Prior studies find that events such as community baby show- ers effectively reach groups that are vulnerable to ISS and breastfeeding disparities [31–33]. In addition, the activi- ties conducted at community baby showers—providing physical and educational resources while connecting par- ents with providers in their communities—have demon- strated success in increasing the uptake of ISS [31–33]. Given the opportunities noted by our study informants, supporting activities that distribute material resources and promote social connections and education could ultimately increase uptake of ISS and breastfeeding. exploring whether organization needs differ based on their target populations and scope. Moreover, our analy- sis did not include the perspective of clients. To develop a holistic perspective of community needs around ISS and breastfeeding support, future studies may examine the con- cordance between provider and client responses. Addition- ally, researcher bias is a possibility in qualitative research; we took efforts to minimize bias by discussing discrepan- cies with a third coder and using investigator triangulation. Finally, qualitative research by nature cannot determine the prevalence of each opinion shared in focus groups. Conclusions Given the disparities that exist in ISS and breastfeed- ing practice uptake in the U.S. [11–13, 15], advocates look to the community setting to reach families vul- nerable to disparities. However, the unique considera- tions of providers and organizations that work within community-based settings have not been thoroughly explored. Accordingly, our study is among the first to qualitatively identify community-level provider and organizational needs to effectively promote both ISS and breastfeeding. Informants in our study under- scored challenges such as: navigating abstinence-based approaches in ISS education, while simultaneously pro- moting breastfeeding [51–53]; limited support to build meaningful connections between providers, clients, and peers [40, 45]; parents’ capacities, cultural contexts, and ability to access material resources [18, 27, 40, 43]; and the broader systems that serve as barriers to ISS and breastfeeding adherence [21, 22, 43]. Nonetheless, study informants also noted areas of innovation and opportunity including: embedding risk-mitigation and conversational approaches in ISS education [52, 53]; supporting relationship building and peer to peer sup- port [36–39]; and combining the provision of material resources to support ISS and breastfeeding with educa- tion [31–33]. By supporting community-level providers and organizations in addressing their unique challenges as well as building upon their successes and innova- tions, ISS and breastfeeding advocates may improve ISS and breastfeeding outcomes among historically margin- alized populations. Finally, informants shared societal factors such as cli- ents’ inability to take parental leave as hindering uptake of recommended ISS and breastfeeding practices. Research depicts policy-level, community-focused inter- ventions improve adherence to of ISS and breastfeed- ing compared to initiatives that address individual-level behavior [21]. In an analysis of policies protecting and supporting breastfeeding in the workplace, research- ers found that worker protection laws such as providing space to breastfeed and mandating breaks to facilitate breastfeeding and pumping benefited Hispanic and Afri- can American women more so than other groups [21]. As breastfeeding decision-making is strongly related to ISS behavior [23], policies providing supportive structures and environments related to breastfeeding may work to promote the uptake of both recommended ISS and breastfeeding practices. Limitations and future directionsi While our findings have implications for providing sup- port and promoting innovative approaches to com- munity-level promotion of ISS and breastfeeding, our results must be interpreted with the following consid- erations. Firstly, all focus groups were conducted vir- tually using the Zoom platform due to the COVID-19 pandemic, which may have hindered our ability to cap- ture non-verbal communication from informants. We were also unable to examine whether responses differed based on demographics of our study informants as well as the communities they serve. Future research may con- sider exploring similarities and differences shared by community-level providers in promoting ISS and breast- feeding based on demographic factors. In addition, we broadly categorized community-level organizations and providers for inclusion in our study, meaning inform- ants’ organizations could target either individual-level or policy-level issues. Additional research may consider Abbreviations AAP American Academy of Pediatrics ASSB Accidental suffocation and strangulation in bed HHS Department of Health and Human Services HRSA Health Resources and Services Administration ISS Infant safe sleep MCHB Maternal Child Health Bureau SDOH Social determinants of health SIDS Sudden infant death syndrome SUID Sudden unexpected infant death WHO World Health Organization Declarations 19. Mirkovic KR, Perrine CG, Scanlon KS. Paid maternity leave and breastfeed- ing outcomes. Birth. 2016;43(3):233–9. Funding Th This project was supported by the Health Resources and Services Adminis- tration (HRSA) of the U.S. Department of Health and Human Services (HHS) under grant number HRSA-17-094, National Action Partnership to Promote Safe Sleep Program, $4,988,565. This information or content and conclusions are those of the author and should not be construed as the official position or policy of, nor should any endorsements be inferred by HRSA, HHS or the U.S. Government. 14. Jones KM, Power ML, Queenan JT, Schulkin J. Racial and ethnic disparities in breastfeeding. Breastfeed Med. 2015;10(4):186–96. 15. McKinney CO, Hahn-Holbrook J, Chase-Lansdale PL, Ramey SL, Krohn J, Reed-Vance M, et al. Racial and ethnic differences in breastfeeding. Pediatrics. 2016;138(2):1–11. 16. Oniwon O, Tender JAF, He J, Voorhees E, Moon RY. Reasons for infant feeding decisions in low-income families in Washington. DC J Hum Lact. 2016;32(4):704–10. Availability of data and materials Audio transcriptions and recordings are not publicly available to protect informant privacy. Data for analysis were de-identified and are available upon reasonable request. 17. United Nations Children’s Fund (UNICEF). Breastfeeding: a mother’s gift for every child; 2018. p. 1–13. Available from: https://data.unicef.org/resou rces/breastfeeding-a-mothers-gift-for-every-child/. 18. Asiodu IV, Waters CM, Dailey DE, Lyndon A. Infant feeding decision-mak- ing and the influences of social support persons among first-time African American mothers. Matern Child Health J. 2017;21(4):863–72. Additional file 1. Additional file 1. 3. Parks SE, Erck Lambert AB, Shapiro-Mendoza CK. Racial and ethnic trends in sudden unexpected infant deaths: United States, 1995–2013. Pediat- rics. 2017;139(6):1–16. 3. Parks SE, Erck Lambert AB, Shapiro-Mendoza CK. Racial and ethnic trends in sudden unexpected infant deaths: United States, 1995–2013. Pediat- rics. 2017;139(6):1–16. Code availability Not applicable. 9. Centers for Disease Control and Prevention. About SIDS and SUID. 2022. Available from: https://www.cdc.gov/sids/about/index.htm. Cited 2022 Dec 7. 9. Centers for Disease Control and Prevention. About SIDS and SUID. 2022. Available from: https://www.cdc.gov/sids/about/index.htm. Cited 2022 Dec 7. Acknowledgements The authors would like to acknowledge all the individuals participating in the NAPPSS-IIN initiative as well as the study informants. The authors also thank Isabel Zuckoff, MPH, Colleen Bernard, and Karla Coleman, MBA for their assistance with data collection. In addition, the authors thank the HRSA team involved in this project including Sara Kinsman, MD, MSCE, PhD, Maureen Perkins, MPH, Bethany Miller, MSW, MEd, and Erin Reiney, MPH. 4. Drowos J, Fils A, Mejia de Grubb MC, Salemi JL, Zoorob RJ, Hennekens CH, et al. Accidental infant suffocation and strangulation in bed: disparities and opportunities. 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https://openalex.org/W3045438149	https://www.pure.ed.ac.uk/ws/files/161791594/CampbelEtalFS2020ParentalEthnicIdentity.pdf	English	null	Parental Ethnic Identity and Child Test Scores*	Fiscal studies	2,020	cc-by	15,508	Edinburgh Research Explorer General rights C i h f h General rights Copyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights. Parental ethnic identity and child test scores Citation for published version: Campbell, S, Nuevo‐chiquero, A, Popli, G & Ratcliffe, A 2020, 'Parental ethnic identity and child test scores', Fiscal Studies: The Journal of Applied Public Economics. https://doi.org/10.1111/1475-5890.12236 Citation for published version: Campbell, S, Nuevo‐chiquero, A, Popli, G & Ratcliffe, A 2020, 'Parental ethnic identity and child test scores', Fiscal Studies: The Journal of Applied Public Economics. https://doi.org/10.1111/1475-5890.12236 Citation for published version: Stuart Campbell,† Ana Nuevo-Chiquero,‡ Gurleen Popli§ and Anita Ratcliffe§ †University College London (s.campbell@ucl.ac.uk) ‡University of Edinburgh and IZA (ana.nuevo.chiquero@ed.ac.uk) §University of Shefﬁeld (g.popli@shefﬁeld.ac.uk, a.ratcliffe@shefﬁeld.ac.uk) †University College London (s.campbell@ucl.ac.uk) ‡University of Edinburgh and IZA (ana.nuevo.chiquero@ed.ac.uk) §University of Shefﬁeld (g.popli@shefﬁeld.ac.uk, a.ratcliffe@shefﬁeld.ac.uk) © 2020 The Authors. Fiscal Studies published by John Wiley & Sons Ltd. on behalf of Institute for Fiscal Studies. 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. Take down policy Take down policy The University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorer content complies with UK legislation. If you believe that the public display of this file breaches copyright please contact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately and investigate your claim. Download date: 24. Oct. 2024 FISCAL STUDIES, vol. 0, no. 0, pp. 1–31 (2020) 0143-5671 p We acknowledge ﬁnancial support from the University of Shefﬁeld Economics Departmental Research Investment Project fund. We are grateful to the Centre for Longitudinal Studies at the UCL Institute of Education for the use of the Millennium Cohort Study data, and to the UK Data Service for making them available. We thank the Editor and three referees for helpful comments. We would also like to thank Andy Dickerson, Emily McDool, Philip Powell, Jennifer Roberts and Sarah Salway for comments on an earlier draft. Finally, we would like to thank participants at the Racism, Ethnic Identity and Child Development Workshop and the Health Economics and Decision Science Seminar at the University of Shefﬁeld; the Economics Department Seminar at the University of Lancaster; the Simposio of the Asociación Española de Economía, and the European Society of Population Economics Annual Conference. All responsibility for the analysis and interpretation of these data lies with the authors. Declarations of interest: none. Keywords: Ethnic identity, national identity, test scores, child development. Parental Ethnic Identity and Child Test Scores* Stuart Campbell,† Ana Nuevo-Chiquero,‡ Gurleen Popli§ and Anita Ratcliffe§ Abstract We examine the relationship between parental ethnic identity and the test scores of ethnic minority children. We use standard survey measures of the strength of parental identity alongside validated cognitive test scores in a rich British cohort study. We show that children whose mothers report either an adoption or an active rejection of the majority identity tend to score lower in cognitive tests at age 7, compared with those children whose mothers report neutral feelings about the majority identity. We ﬁnd no consistent differences © 2020 The Authors. Fiscal Studies published by John Wiley & Sons Ltd. on behalf of Institute for Fiscal Studies. 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. Fiscal Studies 2 in test scores according to mothers’ minority identity. Our ﬁndings provide no support for education or citizenship policies that promote the adoption of the majority identity or discourage the maintenance of separate identities in ethnic minority communities. in test scores according to mothers’ minority identity. Our ﬁndings provide no support for education or citizenship policies that promote the adoption of the majority identity or discourage the maintenance of separate identities in ethnic minority communities. © 2020 The Authors. Fiscal Studies published by John Wiley & Sons Ltd. on behalf of Institute for Fiscal Studies , , 12The distinct experiences of minority and majority children may partly reﬂect language difﬁculties (Schnepf, 2007; Bleakley and Chin, 2008; Casey and Dustmann, 2008), average differences in parental human capital endowments more generally (Djaji´c, 2003; Nielsen et al., 2003; Van Ours and Veenman, 2003; Colding, 2006; Algan et al., 2010; Belzil and Poinas, 2010; Cobb-Clark and Nguyen, 2012), family or neighbourhood poverty levels (Brooks-Gunn, Klebanov and Duncan, 1996; Aber et al., 1997; Bradley et al., 2001), or ethnic discrimination (Ford et al., 2013; Bécares, Nazroo and Kelly, 2015). 11Carneiro, Heckman and Masterov, 2005. 1Bernhard, Fehr and Fischbacher, 2006; Chen and Li, 2009. 2Costa i Font and Cowell, 2015. 3Benjamin, Choi and Fisher, 2016. 4Chiswick, 2009. 5Constant, Roberts and Zimmermann, 2009. 6Depetris-Chauvin, Durante and Campante, 2020. 7Jurajda and Kovaˇc, 2020. 8Becker and Tomes, 1976; Becker, 1981. 9Cunha, Heckman and Lochner, 2006. 10Knudsen et al., 2006; Conti and Heckman, 2014. 11Carneiro, Heckman and Masterov, 2005. 12The distinct experiences of minority and majority children may partly reﬂect language difﬁculties (Schnepf, 2007; Bleakley and Chin, 2008; Casey and Dustmann, 2008), average differences in parental human capital endowments more generally (Djaji´c, 2003; Nielsen et al., 2003; Van Ours and Veenman, 2003; Colding, 2006; Algan et al., 2010; Belzil and Poinas, 2010; Cobb-Clark and Nguyen, 2012), family or neighbourhood poverty levels (Brooks-Gunn, Klebanov and Duncan, 1996; Aber et al., 1997; Bradley et al., 2001), or ethnic discrimination (Ford et al., 2013; Bécares, Nazroo and Kelly, 2015). 13Akerlof, 1997; Akerlof and Kranton, 2002, 2005; Battu, Mwale and Zenou, 2007; Bénabou and Tirole, 2011; Bisin et al., 2011b, 2016; Kranton, 2016. 14Earlier contributions in sociology and social psychology have also been inﬂuential (e.g. Merton and Merton, 1968; Tajfel and Turner, 1979; Wetherell, 1996; Berry, 1997). Akerlof and Kranton (2002) provide a review of the earlier social scientiﬁc literature with a focus on education. 15Berry, 1997; Phinney et al., 2001. 16Dustmann, 1996; Zimmermann, Zimmermann and Constant, 2007; Constant and Zimmermann, 2008; Zimmermann et al., 2008; Constant, Gataullina and Zimmermann, 2009; Casey and Dustmann, 2010; Manning and Roy, 2010; Georgiadis and Manning, 2013; Masella, 2013; Campbell, 2019; Chiang, Liu and Wen, 2019; Depetris-Chauvin et al., 2020. 17Mason, 2004; Pendakur and Pendakur, 2005; Battu and Zenou, 2010; Bisin et al., 2011a. 18Constant, Gataullina and Zimmermann, 2006; Constant and Zimmermann, 2009; Nekby and Rödin, 2010; Drydakis, 2013. 19Schüller, 2015. © 2020 The Authors. Fiscal Studies published by John Wiley & Sons Ltd. on behalf of Institute for Fiscal Studies I. Introduction In this paper, we examine the relationship between parental ethnic identity and the test scores of ethnic minority children. Existing work suggests that identity matters for a wide range of social and economic behaviours, including prosociality,1 redistributive preferences,2 and contributions to public goods.3 Existing work on the speciﬁc case of ethnic identity suggests that it may also matter for a diverse set of issues, from consumption patterns4 and housing tenure,5 to political violence6 and war.7 As we discuss further below, ethnic identity has also been widely implicated in education and labour market outcomes for ethnic minority individuals. We extend this strand of existing work by examining the role of parental ethnic identity in the cognitive performance of young children. This is important because early childhood circumstances and parental investment play a central role in the development of social and cognitive skills.8 Skills acquired in the early years not only persist into later periods, but also affect the productivity of later learning.9 Early experiences have therefore been linked to later outcomes in education, employment and health.10 Unexplained ethnic disparities in such outcomes are also often attributed to experiences in the early years.11 Factors that differentially affect the behaviour or social environment of ethnic minority parents are therefore of central interest for understanding ethnic disparities over life.12 Parental ethnic identity could be particularly inﬂuential in this respect. Parental ethnic identity and child test scores 3 Parental ethnic identity and child test scores A literature in economics now recognises identity as a key determinant of individual and group behaviour.13 Much of this work follows the framework provided by Akerlof and Kranton (2000), who deﬁne identity as a person’s ‘sense of self’, arising from their membership of different social categories.14 These categories can be based on characteristics such as gender, language, ethnicity, and nationality, and each is associated with different behavioural norms. Adherence to or deviation from these norms shapes the rewards associated with different actions. Ethnic identity is an area of particular interest, as it relates to the social and economic integration of minority groups, and therefore to the functioning of society as a whole. It is also a domain in which individuals may hold more than one identity: minority group members may identify with the majority group, the minority group, or some combination of the two.15 Several studies have examined the determinants of minority identity,16 but the central empirical question for economists working on this topic concerns how minority and majority identity shape labour market outcomes. There is evidence both from North America and from several European countries that a strong minority identity harms labour market prospects,17 while there are also some indications that a strong majority identity can be beneﬁcial in this regard, even when it is combined with a strong minority identity.18 However, strong labour market implications have not been detected in every setting. Islam and Raschky (2015) ﬁnd only small impacts of ethnic identity on labour market outcomes in Canada, while Casey and Dustmann (2010) ﬁnd only weak associations in Germany. Either rejection or acceptance of a minority or majority identity could be important for child development, and the expected sign of these relationships is uncertain. Parental investments and access to social resources could be improved by a strong parental majority identity, if majority afﬁliation is linked with stronger majority-group language skills, better knowledge of majority- group institutions, or a higher degree of cultural integration.19 However, a strong parental minority identity may also improve child outcomes, if 4 Fiscal Studies Fiscal Studies afﬁliation with the minority group improves self-esteem by afﬁrming heritage, or allows access to informal parenting support by signalling minority group commitment.20 For example, Nekby, Rödin and Özcan (2009) examine tertiary education among young adults with immigrant backgrounds in Sweden. 20A literature in developmental psychology suggests that personal minority identity may be beneﬁcial in education for adolescents (see Miller-Cotto and Byrnes, 2016, for a recent review). 21See, for example, Phinney (1992) and Phinney and Ong (2007). 22Exceptions include Bisin et al. (2011a), Constant, Gataullina and Zimmermann (2009) and Constant and Zimmermann (2008), who infer ethnic identity from other observed characteristics. 23Burton, Nandi and Platt, 2010; Nandi and Platt, 2012. © 2020 The Authors. Fiscal Studies published by John Wiley & Sons Ltd. on behalf of Institute for Fiscal Studies Parental ethnic identity and child test scores They ﬁnd that men in their early twenties who report both minority and majority identities are more likely to complete tertiary education than others, although they ﬁnd no such association for women. Schüller (2015) ﬁnds that immigrant children aged 10–14 years in Germany are more likely to be placed in the middle or upper tier of secondary education if their father reports a strong minority identity, or if their mother reports a strong majority identity. Alternatively, a strong minority identity could lead to over-investment in ethnicity-speciﬁc human and social capital, leaving parents and children isolated from the resources of the majority society, and therefore harming the cognitive outcomes of minority children. By the same logic, a strong majority identity could isolate parents from the resources of the minority community. In either scenario, minority parents are facing a trade-off between access to social and economic resources in the minority and majority society. The existence of such a trade-off is suggested by theoretical and empirical results elsewhere. For example, Austen-Smith and Fryer (2005) and Fryer and Torelli (2010) suggest that high-school students from some minority backgrounds face a trade-off between peer acceptance and academic attainment. Battu et al. (2007) and Battu and Zenou (2010) suggest that minority adults face a trade-off between identifying with their minority group and labour market success. The way we measure and model ethnic identity matters for how we understand these relationships. If a person’s sense of their identity is multidimensional, it may be difﬁcult to capture simply by asking them to answer a single survey question. While psychologists have traditionally used multiple-item inventories to capture ethnic identity in smaller samples,21 the convention in economics has been to use the single binary or Likert- scale based measures of identity, which are available in larger surveys.22 The simplicity of such one-dimensional measures has an intuitive appeal, and should yield easily interpretable results. However, this simplicity could come at a cost, if such measures are prone to error, or if the question is understood differently by people of different backgrounds.23 In keeping with the economics literature, we use a Likert-scale based measure to capture ethnic identity in this paper but, below, we discuss some of the limitations that may be associated with such measures. The relationship between the strength of ethnic © 2020 The Authors. Fiscal Studies published by John Wiley & Sons Ltd. 20A literature in developmental psychology suggests that personal minority identity may be beneﬁcial in education for adolescents (see Miller-Cotto and Byrnes, 2016, for a recent review). Parental ethnic identity and child test scores on behalf of Institute for Fiscal Studies Parental ethnic identity and child test scores 5 identity and economic outcomes is also often assumed to be linear, although Fryer and Torelli (2010) suggest non-linearities in the relationship between minority peer acceptance and academic attainment. Therefore, we allow for non-linearities in our empirical model below. The investigation of parental ethnic identity and child test scores is important for three reasons. First, as we note above, the early years are crucial for later development trajectories, and different experiences in the early years are often thought to be responsible for ethnic disparities later in life. It is therefore critical to understand the role of ethnic identity in outcomes at this age. Second, many developed countries have seen an increase in the size of ethnic minority populations in recent years, and the demographic proﬁle of these populations is often young compared with that of the ethnic majority. For example, although only around 15 per cent of the overall population of England is from an ethnic minority background, minority children make up nearly a third of the current primary school population.24 Close to half of the school-age population in the United States is from a minority background, compared to around 40 per cent of the country overall.25 The future economies of such diverse societies will therefore be substantially shaped by the current educational performance of minority as well as majority children. A ﬁnal reason for the importance of this topic relates directly to public policy. Several countries have responded to increased ethnic diversity by introducing education and citizenship policies that actively promote the majority identity,26 while generally discouraging the maintenance of separate identities in ethnic minority communities.27 The inﬂuence of parental ethnic identity on child development is therefore an active policy concern. Our main contribution is to show the relationship between parental ethnic identity and childhood outcomes using direct measures of cognitive development in young children. The two previous papers closest to our own, Schüller (2015) and Nekby et al. (2009), use broad measures of educational attainment, and do so in samples of older children and young adults, respectively. Our data allow us access to validated cognitive test scores at age 7, alongside measures of parental ethnic identity taken when the child is aged 5. 24These ﬁgures come from the 2016 Annual Population Survey (www.nomisweb.co.uk) and the 2016 School Census (www.gov.uk/government/statistics/schools-pupils-and-their-characteristics-january-2016, accessed on 20 January 2019). 25 © 2020 The Authors. Fiscal Studies published by John Wiley & Sons Ltd. on behalf of Institute for Fiscal Studies , 26For example, Manning and Roy (2010) cite language requirements, ‘citizenship’ classes in schools, citizenship ceremonies, and tests of cultural knowledge for those seeking citizenship, as measures intended to promote the majority identity. 27Berry, 1974; Uberoi and McLean, 2007. 28Ub i d M L 2007 M i d R 2010 25Musu-Gillette et al., 2017. 26For example, Manning and Roy (2010) cite language requirements, ‘citizenship’ classes in scho citizenship ceremonies, and tests of cultural knowledge for those seeking citizenship, as measures inten to promote the majority identity. 27 28Uberoi and McLean, 2007; Manning and Roy, 2010. 27Berry, 1974; Uberoi and McLean, 2007. 29The vast majority of cohort members are singletons, and, while the MCS does contain twins and triplets, we exclude these children from our sample. 30Oversampling of high poverty areas occurs throughout the UK, whereas oversampling of areas with large ethnic communities is conﬁned to England, where ethnic minorities are disproportionately concentrated. Hence, our sample is mostly drawn from England, and our results should be extrapolated to the rest of Great Britain with caution. Parental ethnic identity and child test scores We are also able to examine this topic in a new national context, in a country with a relatively large and heterogeneous ethnic minority population, and a recent history of relatively active policy promotion of the majority identity.28 6 6 Fiscal Studies Using an ethnic minority sample drawn from a rich UK cohort study, we examine the relationship between parental ethnic identity and children’s test scores. We employ separate measures of parental minority and majority identity, alongside tests of children’s cognitive functioning in three key domains. We show that children whose mothers report either an adoption or an active rejection of the majority identity tend to score lower in cognitive tests at age 7, compared with those children whose mothers report a neutral view of the majority identity. This result is driven by children from poor households, and by those from households who lack access to family support networks. We suggest two interpretations of our ﬁndings. The ﬁrst is that both maternal adoption and active rejection of the majority identity divert family resources away from investments more conducive to children’s cognitive development. The second is that mothers adopt a position on the majority identity in response to challenging circumstances, which are also reﬂected in children’s lower test scores. This second interpretation does not imply a direct relationship between parental ethnic identity and child test scores, but instead implies the presence of omitted variables in our empirical model. We also note that our estimates are sometimes imprecise and unstable across speciﬁcations, perhaps due to measurement error in the identity variables. The paper is organised as follows. In Section II, we describe the data. In Section III, we present our empirical model and our main results. In Section IV, we explore the characteristics of the families driving our main results. Finally, in Section V, we summarise our ﬁndings and discuss implications. © 2020 The Authors. Fiscal Studies published by John Wiley & Sons Ltd. on behalf of Institute for Fiscal Studies 29The vast majority of cohort members are singletons, and, while the MCS does contain twins and triplets, we exclude these children from our sample. 31Additional measures of cognitive ability are available in subsequent waves of the MCS, but we use only those at age 7, in order to minimise the gap in time between measures of parental identity and test scores. 32Connelly (2013) provides a useful discussion of tests available in the MCS. 33Jirout and Newcombe, 2015. 34Hofferth, 2009. Parental ethnic identity and child test scores 7 Data are collected through face-to-face interviews for generic information, and by a self-completion questionnaire for more sensitive topics. The main carer of each cohort member (the mother in 98 per cent of cases) provides information on the child and family setting, while both the main carer and father ﬁgure (if resident) provide more sensitive information via a self- completion questionnaire. Questions on ethnic identity are in the parents’ self-completion questionnaire in the age 5 survey. We take the outcome and control variables from the age 7 survey, in order to remove any inﬂuence of contemporaneous child cognitive outcomes on parental identity. We restrict the sample to families that are present in both waves. In our estimation sample, 20 per cent of cohort members do not live with their father, and among those that do, just 63 per cent have the necessary information supplied by fathers to carry out our analyses of paternal ethnic identity. Therefore, we use two samples: the ﬁrst comprises the children of all ethnic minority mothers, which we use to analyse maternal identity, and the second comprises the children of ethnic minority mothers in couples with complete partner information, which we use to analyse both paternal and maternal ethnic identity. After excluding those with missing information on the outcome and family variables, our main sample is composed of 1,249 children, of whom 629 have two parents with complete information on the father. © 2020 The Authors. Fiscal Studies published by John Wiley & Sons Ltd. on behalf of Institute for Fiscal Studies II. Data Our data come from the Millennium Cohort Study (MCS). The MCS follows around 19,000 children born in the UK between 2000 and 2001; of these, around 3,000 are from ethnic minority families.29 The initial survey design oversamples families living in high poverty areas and areas with large ethnic minority populations.30 Detailed information is collected on each cohort member, their families, and the home environment. We use only cohort members born in Great Britain (England, Scotland and Wales), as questions about ethnic identity are not asked in Northern Ireland. y Cohort members and their families are interviewed when the child is 9 months old, and then again when the child is aged 3, 5, 7, 11 and 14 years. © 2020 The Authors. Fiscal Studies published by John Wiley & Sons Ltd. on behalf of Institute for Fiscal Studies Parental ethnic identity and child test scores 31Additional measures of cognitive ability are available in subsequent waves of the MCS, but we use only those at age 7, in order to minimise the gap in time between measures of parental identity and test scores. 32 ll ( ) id f l di i f il bl i h 35Wai, Lubinski and Benbow, 2009; Uttal and Cohen, 2012. 36Wai et al., 2009. 1. Outcomes of interest At age 7, cohort members are tested in three key domains of cognitive functioning: maths, spatial problem solving, and reading skills.31 Proﬁciency in maths is determined using a shortened version of the ‘National Foundation for Educational Research Progress in Maths’ test, in which children perform calculations on a range of topics including numbers, shapes, measurement, and data handling. Spatial problem solving and reading skills are measured using the second edition of the ‘British Ability Scales’, through a pattern con- struction test (where children must replicate a design using patterned squares) and a word reading test (where children read words presented on a card).32 We consider these three test scores as separate outcomes. Parents may provide different inputs across the three areas. For example, some parents may prefer to play with puzzles, blocks and board games, which have been linked to higher levels of spatial ability,33 while others may prefer to read with their children.34 The three domains of cognitive functioning also have © 2020 The Authors. Fiscal Studies published by John Wiley & Sons Ltd. on behalf of Institute for Fiscal Studies 8 8 Fiscal Studies FIGURE 1 FIGURE 1 Outcome distribution by test. Note: Standardised score distributions by test type. The scores have been standardised with respect to the entire sample (ethnic minority and majority children) at age 7. Outcome distribution by test. Note: Standardised score distributions by test type. The scores have been standardised with respect to the entire sample (ethnic minority and majority children) at age 7. distinct implications for subsequent educational and occupational choices. For example, students with high ability in maths and spatial problem solving are more likely to take ‘STEM’ (Science, Technology, Engineering, or Mathematics) subjects at degree level and beyond, while students with higher verbal ability are concentrated within the arts and humanities.35 While maths and spatial problem solving abilities are highly correlated, spatial ability appears to play a role independently of maths in driving achievement in STEM subjects.36 We use the age-standardised versions of test scores available in the MCS, which take into account the extra time that older children within the cohort have had to develop their skills. For ease of interpretation and comparability, we standardise test scores in maths, spatial ability and reading by subtracting the mean and dividing by the standard deviation of test scores observed in the entire sample of MCS children. © 2020 The Authors. Fiscal Studies published by John Wiley & Sons Ltd. on behalf of Institute for Fiscal Studies 37Respondents have usually indicated their ethnic group in an earlier wave of the MCS. They may choose from 16 ethnic categories if they live in England, 17 categories if they live in Wales, and 14 categories if they live in Scotland. The categories are based on the 2001 census. For England, the categories are: ‘White - British’; ‘White - Irish’; ‘Any other White background’; ‘Mixed - White and Black Caribbean’; ‘Mixed - White and Black African’; ‘Mixed - White and Asian’; ‘Any other mixed background’; ‘Asian/Asian British - Indian’; ‘Asian/Asian British - Pakistani’; ‘Asian/Asian British - Bangladeshi’; ‘Any other Asian background’; ‘Black/Black British - Caribbean’; ‘Black/Black British - African’; ‘Any other Black background’; ‘Chinese’; ‘Any other background’. 38 d (2010) diff ( h h i l S f h i i i i ) h 1. Outcomes of interest Figure 1 shows the distribution of © 2020 The Authors. Fiscal Studies published by John Wiley & Sons Ltd. on behalf of Institute for Fiscal Studies Parental ethnic identity and child test scores 9 these standardised test scores for our sample of ethnic minority children. The distributions suggest that ethnic minority children perform slightly better than majority children in reading tests, and slightly worse in maths and spatial problem solving tests. © 2020 The Authors. Fiscal Studies published by John Wiley & Sons Ltd. on behalf of Institute for Fiscal Studies 39Burton et al., 2010. 38Battu and Zenou (2010) use a different survey (the Fourth National Survey of Ethnic Minorities) where the same question appears. 39Burton et al., 2010. 40 40Burton et al., 2010. © 2020 The Authors. Fiscal Studies published by John Wiley & Sons Ltd. on behalf of Institute for Fiscal Studies FIGURE 2 Minority and majority identity combinations, relative prevalence (Likert scale). 2. Measures of parental ethnic identity We capture parental ethnic identity using two questions from the self- completion module of the MCS. Parents who indicate that they belong to any non-White ethnic minority group are asked to what extent they agree with the following statements: In many ways I think of myself as British and In many ways I think of myself as [name of ethnic group].37 Respondents may choose any one of six responses: (1) Strongly agree, (2) Agree, (3) Neither agree nor disagree, (4) Disagree, (5) Strongly disagree, or (6) Can’t say. Battu and Zenou (2010) interpret these questions as addressing identiﬁcation with a country, with a place, and its way of living.38 Similar questions, which focus on the degree to which an individual values their ethnic origin and their sense of belonging to the adopted country, are used to measure ethnic identity in Islam and Raschky (2015), Nekby et al. (2009) and Schüller (2015). As we have noted above, such questions have the advantage of being widely available in large-scale data sets such as the MCS, and Likert response categories allow respondents to attach a weight to these two different aspects of their identity.39 The main ethnic identity categories in the survey are also linked to geographical ancestry, which is thought to reduce the ambiguity of ethnic identity questions.40 However, given the multidimensional nature of identity, answers may still be prone to measurement error. We return to this issue in our discussion of the results below. Figure 2 presents the distribution of responses to the minority and majority identity questions in the MCS, where the size of each circle represents the frequency of responses in each combination of identities. ‘Can’t say’ has been combined with ‘Neither agree nor disagree’. Although some of the other response categories contain low frequencies, we keep them separate in 10 Fiscal Studies Minority and majority identity combinations, relative prevalence (Likert scale). Note: Circle sizes are proportional to the number of mothers reporting both categories for minority and majority identity. Frequencies range from 298 mothers who ‘Agree’ to both the minority and majority statements, and 2 mothers who ‘Disagree’ to the majority statement and ‘Strongly disagree’ to the minority statement. The horizontal dashed line separates those in the ‘Agree’ and ‘Strongly agree’ categories for both minority and majority identities. Note: Circle sizes are proportional to the number of mothers reporting both categories for minority and majority identity. Frequencies range from 298 mothers who ‘Agree’ to both the minority and majority statements, and 2 mothers who ‘Disagree’ to the majority statement and ‘Strongly disagree’ to the minority statement. The horizontal dashed line separates those in the ‘Agree’ and ‘Strongly agree’ categories for both minority and majority identities. our analysis. We ﬁnd evidence of non-linearities in the relationship between these parental identity responses and children’s cognitive outcomes, and if we aggregate responses into larger categories these non-linearities are sometimes disguised. We therefore accept the limitations associated with smaller cell sizes. Around 40 per cent of ethnic minority mothers ‘Agree’ with the majority identity statement, and a quarter ‘Strongly agree’. 42 per cent ‘Agree’ with the minority identity statement and nearly a third (29 per cent) ‘Strongly agree’. Responses are relatively concentrated in the top- right quadrant, where respondents express some level of agreement with both the minority and majority identity statements. Nearly a quarter (24 per cent) ‘Agree’ with both statements and 12 per cent ‘Strongly agree’ with both. Summary statistics for all parental identity categories in both samples are presented in Table A.1 in the online Appendix. Levels of minority and majority Parental ethnic identity and child test scores 11 identity are similar for mothers in the two samples, and fathers’ levels of identity are similar to mothers, except that fathers are less likely to ‘Neither agree nor disagree’ with the majority identity statement (18 per cent versus 26 per cent of mothers), and more likely to be in the ‘Strongly disagree’ category (30 per cent versus 22 per cent of mothers). 41Ruhm, 2004; Todd and Wolpin, 2007; Bettinger, Hægeland and Rege, 2014. 42Mensah and Kiernan, 2010; Wilson, Burgess and Briggs, 2011. 43Dunifon and Kowaleski-Jones, 2002; Hawkes and Joshi, 2012. 44Black, Devereux and Salvanes, 2005. 45de La Rochebrochard and Joshi, 2013. © 2020 The Authors. Fiscal Studies published by John Wiley & Sons Ltd. on behalf of Institute for Fiscal Studies 1Ruhm, 2004; Todd and Wolpin, 2007; Bettinger, Hægeland and Rege, 2014. 3. Other controls A key concern when introducing control variables in this type of analysis is that parental ethnic identity could determine many of the household characteristics observed. For example, although parental employment status and educational attainment may be important in shaping parental inputs and the quality of the learning environment at home,41 several studies cited above suggest that education and employment may be inﬂuenced by ethnic identity. We therefore start with a conservative speciﬁcation that controls only for the gender of the child and the ethnicity of the mother. These variables are plausibly exogenous to parental ethnic identity, and may partly explain differences in cognitive test scores. Gender and ethnic differences in attainment emerge as early as the foundation stage of primary education.42 We next introduce controls that may partially be inﬂuenced by parental ethnic identity but are also important for children’s cognitive development. We label these variables our ‘main controls’. These include whether the mother is foreign born, whether she is a single parent, a quadratic term for her age at the time the child was born, and a linear term in the number of siblings in the household. Mothers who are born abroad often have different formative experiences, which may in turn inﬂuence their own parenting styles. Children of young mothers and those in single parent households typically fare worse than other children in both cognitive and behavioural development.43 Siblings may negatively affect child development as a result of increased competition for material resources and parental attention44 but may also have a positive inﬂuence on social, emotional and cognitive development.45 In this expanded speciﬁcation, we also control for cases where the child lives in a two-parent household, but the father did not respond to the identity questions in the MCS, and cases where the father responded as the main parent. Finally, we control for the household socio-economic circumstances most likely to be inﬂuenced by parental ethnic identity. We label these ‘additional controls’. © 2020 The Authors. Fiscal Studies published by John Wiley & Sons Ltd. on behalf of Institute for Fiscal Studies 3. Other controls These include parental education and employment status, household 12 Fiscal Studies TABLE 1 Summary statistics (1) (2) All mothers Mothers in couples Gender and ethnicity Girl 0.51 (0.50) 0.51 (0.50) Mother Indian 0.22 (0.42) 0.33 (0.47) Mother Pakistani or Bangladeshi 0.35 (0.48) 0.34 (0.47) Mother Black African or Black Caribbean 0.24 (0.43) 0.17 (0.38) Main controls Mother’s age at birth 28.28 (5.76) 28.51 (5.42) Single mother household 0.20 (0.40) 0.00 (0.00) Couple with incomplete father information 0.29 (0.46) 0.00 (0.00) Father is main respondent 0.03 (0.17) 0.06 (0.23) Number of siblings 1.55 (1.12) 1.56 (1.03) Foreign-born mother 0.45 (0.50) 0.50 (0.50) Foreign-born mother missing 0.16 (0.37) 0.14 (0.35) Additional controls Mother, low qualiﬁcation 0.39 (0.49) 0.36 (0.48) Mother, qualiﬁcation from abroad 0.08 (0.27) 0.09 (0.29) Father, low qualiﬁcation 0.20 (0.40) 0.24 (0.43) Father, qualiﬁcation from abroad 0.09 (0.29) 0.14 (0.35) Non-working mother 0.58 (0.49) 0.54 (0.50) Non-working father 0.09 (0.29) 0.10 (0.30) Family faces ﬁnancial constraints 0.26 (0.44) 0.18 (0.39) Family materially deprived 0.46 (0.50) 0.38 (0.49) Family in housing poverty 0.55 (0.50) 0.46 (0.50) Family below poverty line 0.51 (0.50) 0.39 (0.49) Family living in deprived area 0.49 (0.50) 0.40 (0.49) Mother has family networks 0.51 (0.50) 0.52 (0.50) Mother has friendship networks 0.70 (0.46) 0.72 (0.45) Foreign language spoken at home 0.42 (0.49) 0.46 (0.50) N 1,249 629 Note: Sample means and standard deviations (in parentheses). TABLE 1 Summary statistics N and neighbourhood deprivation, access to family and friendship networks in the local area, and whether a foreign language is the main language spoken at home. We explore heterogeneity in the impact of parental ethnic identity across several of these dimensions below. Table 1 shows summary statistics for the outcome variables, the ‘main controls’, and the ‘additional controls’. The table has a column for each of the two samples used in our analysis: the ‘All mothers’ sample and the ‘Mothers in couples’ sample. These samples differ in ethnic composition, and in the economic situation of households. © 2020 The Authors. Fiscal Studies published by John Wiley & Sons Ltd. on behalf of Institute for Fiscal Studies 3. Other controls The sample restricted to mothers in Parental ethnic identity and child test scores 13 TABLE 2 Summary statistics for test scores by maternal majority and minority identity (1) (2) Majority identity Minority identity Strongly disagree Maths score 0.288 (1.012) −0.023 (1.059) Spatial problem solving score 0.002 (0.871) 0.103 (0.793) Reading score 0.439 (0.974) 0.289 (1.075) N 42 37 Disagree Maths score −0.205 (1.185) −0.167 (0.950) Spatial problem solving score −0.521 (1.278) −0.317 (0.992) Reading score 0.267 (0.938) 0.133 (0.859) N 100 73 Neither agree nor disagree Maths score 0.022 (1.088) −0.107 (1.077) Spatial problem solving score −0.228 (1.029) −0.264 (1.045) Reading score 0.316 (0.956) 0.251 (0.989) N 323 252 Agree Maths score −0.186 (0.976) −0.123 (1.027) Spatial problem solving score −0.241 (0.976) −0.265 (1.085) Reading score 0.196 (0.992) 0.256 (0.973) N 473 523 Strongly agree Maths score −0.104 (1.043) −0.048 (1.071) Spatial problem solving score −0.287 (1.001) −0.288 (0.935) Reading score 0.228 (0.939) 0.257 (0.950) N 311 364 Note: Sample means and standard deviations (in parentheses). Outcome variables are standardised to have mean 0 and standard deviation of 1 within the main (i.e. majority and minority) MCS sample. TABLE 2 Note: Sample means and standard deviations (in parentheses). Outcome variables are standardised to have mean 0 and standard deviation of 1 within the main (i.e. majority and minority) MCS sample. couples with complete partner information has a higher proportion reporting their ethnicity as Indian, and a higher proportion born abroad. Families in this sample are also less likely to be deprived or to live in deprived neighbourhoods. There is no difference across the two samples in the mother’s age at the birth of the child, whether or not a foreign language is the main language spoken at home, or in the number of siblings. Table 2 shows summary statistics for all outcome variables by each maternal majority and minority identity category, and the number of respondents in each category. As we saw in Figure 2, the biggest response categories, for both majority and minority identity, are ‘Agree’ and ‘Strongly 14 Fiscal Studies Fiscal Studies agree’, and the category with the least number of respondents is ‘Strongly disagree’. The mean test scores in maths, spatial problem solving and reading do not show a clear pattern for majority and minority identity across all ﬁve responses in the Likert scale. However, the ‘Majority identity’ column does give a broad preview of the main ﬁnding of the paper – that children of mothers who either ‘Agree’ or ‘Disagree’ with the majority identity statement tend to do worse in cognitive tests than those in the ‘Neither agree nor disagree’ category. We examine this pattern in more detail in the next section. For completeness, summary statistics for controls across all parental minority and majority identity categories are presented in Tables A.2 and A.3 in the online Appendix. There are a few statistically signiﬁcant differences in characteristics across the categories of majority identity; for example, there are more Pakistani and Bangladeshi mothers who respond ‘Agree’ and ‘Strongly agree’ relative to ‘Neither agree or disagree’ and there are fewer Black African or Caribbean mothers who respond ‘Disagree’ and ‘Strongly disagree’ relative to ‘Neither agree or disagree’. For most of the other variables, there are few statistically signiﬁcant differences across categories of the majority or minority identity. © 2020 The Authors. Fiscal Studies published by John Wiley & Sons Ltd. on behalf of Institute for Fiscal Studies 2. Main results Table 3 presents our baseline results for the association between maternal identity and child test scores in maths, spatial problem solving and reading, across three groups of three columns. Results in the ﬁrst column of each group (columns 1, 4 and 7) come from regressions including only controls for the gender of the child and ethnicity of the mother, while the second column in each group (columns 2, 5 and 8) shows results from regressions including other demographic controls, as discussed above and listed as ‘main controls’ in Table 1. The ﬁnal column in each group (columns 3, 6 and 9) shows results from regressions including ‘additional controls’ as discussed above, and also in Table 1. All of these results use the full sample of mothers. Our results indicate that children of mothers who either ‘Agree’ or ‘Disagree’ that they hold a majority identity tend to perform worse than those in the neutral category across all three tests, although the differences are not always statistically signiﬁcant. Test scores in maths and spatial problem solving are 0.224 and 0.286 lower in the ‘Disagree’ group than in the neutral category after introducing all controls, and those in maths and reading are 0.213 and 0.126 lower in the ‘Agree’ group than in the neutral category. These negative estimates are of a similar size to those linked with the family being below the poverty line, and are larger than the estimates linked with living in material deprivation and in housing poverty (results showing the maths estimates for the full set of control variables are presented in Table A.4 in the online Appendix). Other estimates across the majority identity panel are generally of the same sign but not statistically signiﬁcant. The exceptional estimates in the majority identity panel are those associated with the ‘Strongly disagree’ category, which are positive but insigniﬁcantly different from the neutral category. This category is also an exception in the minority identity panel, where no estimates are statistically signiﬁcant except for those who ‘Strongly disagree’ that they hold a minority identity, and then only in one of the three cognitive tests. This group who ‘Strongly disagree’ scores 0.315 standard deviations higher than the neutral category in spatial problem solving. However, the ‘Strongly disagree’ category is small for both the majority and minority identity questions, with 42 and 37 respondents giving these responses, respectively (see Table 2). 1. Empirical model To examine the relationship between parental ethnic identity and cognitive test scores in ethnic minority children, we estimate the following equation using ordinary least squares (OLS): Yit = α + 4 j=1 β j1 ∗[Mother majority identityit−1 = j] + 4 j=1 γj1 ∗[Mother minority identityit−1 = j] + δXit + εit. (1) (1) Here, Yit denotes the relevant test score (maths, spatial problem solving, or reading) of child i at time t (age 7), Mother majority identityit−1 represents a series of dummies that take a value of 1 if maternal majority identity falls in category j, and Mother minority identityit−1 represents maternal minority identity at t −1 (age 5) in a similar manner. The four categories are ‘Strongly disagree’, ‘Disagree’, ‘Agree’ and ‘Strongly agree’, and our omitted category is ‘Neither agree nor disagree’. Hence, βj measures the difference in performance on each test (in standard deviations) between children whose mothers report a majority identity in category j and those whose mothers report Parental ethnic identity and child test scores 15 that they ‘Neither agree nor disagree’ with the identity statement. γ j represents the same for minority identity. We extend this speciﬁcation to include the father’s majority and minority identity for our sample of couples. In all cases, Xit is the vector of control variables discussed above, and εit is a random, normally distributed error term. © 2020 The Authors. Fiscal Studies published by John Wiley & Sons Ltd. on behalf of Institute for Fiscal Studies 2. Main results Therefore, we do not weight these estimates heavily in our interpretation of our results overall. 16 Fiscal Studies TABLE 3 Association between maternal identity and child test scores: baseline results (OLS) (1) (2) (3) (4) (5) (6) (7) (8) (9) Maths Maths Maths Spatial Spatial Spatial Reading Reading Reading Majority identity Strongly disagree 0.261 0.230 0.229 0.190 0.168 0.131 0.148 0.090 0.048 (0.172) (0.179) (0.179) (0.147) (0.149) (0.149) (0.162) (0.166) (0.163) Disagree −0.217* −0.222* −0.224* −0.275 −0.280 −0.286 −0.030 −0.074 −0.069 (0.130) (0.130) (0.130) (0.136) (0.138) (0.137) (0.108) (0.105) (0.104) Agree −0.182 −0.200* −0.213* −0.013 −0.027 −0.053 −0.131* −0.122* −0.126* (0.077) (0.076) (0.076) (0.075) (0.075) (0.075) (0.073) (0.072) (0.071) Strongly agree −0.103 −0.119 −0.120 −0.089 −0.087 −0.097 −0.086 −0.054 −0.051 (0.088) (0.087) (0.086) (0.083) (0.084) (0.084) (0.079) (0.079) (0.078) Minority identity Strongly disagree 0.062 0.012 0.045 0.351 0.300 0.315** 0.049 −0.006 0.006 (0.182) (0.182) (0.177) (0.143) (0.143) (0.144) (0.186) (0.184) (0.179) Disagree 0.007 0.031 0.034 −0.028 0.015 0.038 −0.079 −0.052 −0.061 (0.137) (0.136) (0.137) (0.134) (0.134) (0.134) (0.119) (0.116) (0.116) Agree 0.061 0.052 0.070 0.010 0.012 0.030 0.028 −0.004 0.004 (0.083) (0.082) (0.081) (0.083) (0.083) (0.082) (0.078) (0.076) (0.076) Strongly agree 0.056 0.067 0.085 0.014 0.025 0.037 0.009 −0.006 −0.002 (0.089) (0.088) (0.087) (0.084) (0.084) (0.084) (0.084) (0.083) (0.082) Gender and ethnicity Yes Yes Yes Yes Yes Yes Yes Yes Yes Main controls No Yes Yes No Yes Yes No Yes Yes Additional controls No No Yes No No Yes No No Yes N 1,249 1,249 1,249 1,249 1,249 1,249 1,249 1,249 1,249 Note: Standard errors in parentheses. Sample: all mothers. Base category for identity: Neither agree nor disagree. p < 0.1, p < 0.05, p < 0.01. © 2020 The Authors. Fiscal Studies published by John Wiley & Sons Ltd. on behalf of Institute for Fiscal Studies © 2020 The Authors. Fiscal Studies published by John Wiley & Sons Ltd. on behalf of Institute for Fiscal Studies 17 Parental ethnic identity and child test scores Results for the sample with information on father’s identity are presented in Table A.5 in the online Appendix. Note that the sample size shrinks substantially here, as not all families in the MCS have fathers present, and not all fathers who are present ﬁll out the ‘self-completion’ part of the survey in which the identity questions appear. 46See, for example, Schüller (2015). 47Speciﬁcations interacting ethnic identity with each ethnic group indicator give similar results. 48Schüller, 2015. 49Splitting the sample by gender does not reveal any substantial differences between boys and girls. © 2020 The Authors. Fiscal Studies published by John Wiley & Sons Ltd. on behalf of Institute for Fiscal Studies 2. Main results The negative coefﬁcients associated with mothers who ‘Disagree’ or ‘Agree’ that they hold a majority identity remain in maths and spatial problem solving, although they cease to be statistically distinct from the neutral category. There is a large and statistically signiﬁcant negative association between fathers who ‘Disagree’ that they have a minority identity and test scores in maths, and a positive association between fathers who ‘Agree’ that they hold a minority identity and test scores in spatial problem solving. These results are intriguing as, compared with our baseline results, they are more consistent with some results elsewhere in the literature.46 However, given the mixed results and small sample size, we leave analysis of the father’s ethnic identity here, and in subsequent analysis focus on the larger sample containing all mothers. The distinct cultures and immigration histories of different ethnic minority groups may affect the relationship between parental ethnic identity and children’s cognitive development. Given small sample sizes for different minority groups across the ﬁve response categories, we split the sample to compare estimates for the children of Black African and Caribbean mothers, Indian mothers and Pakistani/Bangladeshi mothers (Table 4).47 This exercise reveals that the negative associations we observe between maternal agreement or disagreement with the majority identity statement and cognitive test scores appear to be driven mostly by children of mothers from Black African and Caribbean backgrounds, and to some extent those from Pakistani and Bangladeshi backgrounds. This is an interesting result, given that language is often seen as an important factor linking ethnicity and identity,48 and yet Black African and Caribbean mothers are the mostly likely of these minority groups to speak English as their main language at home. However, again, the cell sizes are small, so we give this result less weight than our main results.49 Our baseline results suggest that there are systematic differences in test scores between children whose mothers ‘take a position’ on the majority identity, and children of mothers who report more neutral feelings towards it. This implies that it is taking a stance on the majority identity, rather than the stance taken, that matters for child outcomes, where both acceptance and rejection of the majority identity are negatively associated with test scores. While no other paper has found a similar result for ‘taking a stand’ on majority © 2020 The Authors. Fiscal Studies published by John Wiley & Sons Ltd. 2. Main results on behalf of Institute for Fiscal Studies 18 Fiscal Studies TABLE 4 Association between maternal identity and child test scores: ethnicity (OLS) Black Indian Pakistani/Bangladeshi (1) (2) (3) (4) (5) (6) (7) (8) (9) Maths Spatial Reading Maths Spatial Reading Maths Spatial Reading Majority identity Strongly disagree 0.314 0.262 0.232 −0.099 0.038 0.122 0.443 0.137 0.275 (0.347) (0.281) (0.305) (0.516) (0.240) (0.300) (0.318) (0.337) (0.353) Disagree −0.641* −0.522 −0.336 −0.012 −0.092 0.235 −0.439 −0.505 0.010 (0.238) (0.252) (0.216) (0.331) (0.322) (0.230) (0.196) (0.211) (0.179) Agree −0.493* 0.047 −0.304 −0.143 −0.073 0.045 −0.131 −0.157 −0.098 (0.144) (0.129) (0.145) (0.173) (0.157) (0.155) (0.138) (0.142) (0.124) Strongly agree −0.173 −0.108 −0.349 −0.176 0.063 0.273* −0.069 −0.194 0.056 (0.170) (0.172) (0.145) (0.190) (0.177) (0.154) (0.158) (0.158) (0.149) Minority identity Strongly disagree 0.020 0.054 0.220 −0.662 0.233 0.140 −0.026 0.364 −0.330 (0.386) (0.293) (0.357) (0.407) (0.345) (0.453) (0.313) (0.312) (0.332) Disagree 0.295 0.330 0.048 −1.021*** −0.078 −0.383 0.189 −0.143 −0.047 (0.241) (0.234) (0.211) (0.326) (0.248) (0.331) (0.230) (0.231) (0.171) Agree 0.249 −0.075 0.088 0.008 0.024 −0.170 −0.054 0.108 −0.101 (0.178) (0.172) (0.172) (0.201) (0.184) (0.174) (0.134) (0.138) (0.124) Strongly agree 0.160 0.009 0.178 0.173 −0.052 −0.215 −0.024 0.268* −0.127 (0.182) (0.173) (0.178) (0.205) (0.181) (0.175) (0.149) (0.145) (0.149) Gender and ethnicity Yes Yes Yes Yes Yes Yes Yes Yes Yes Main controls Yes Yes Yes Yes Yes Yes Yes Yes Yes Additional controls Yes Yes Yes Yes Yes Yes Yes Yes Yes N 303 303 303 280 280 280 443 443 443 Note: Standard errors in parentheses. Sample: all mothers. Base category for identity: Neither agree nor disagree. p < 0.1; p < 0.05; p < 0.01. © 2020 The Authors. Fiscal Studies published by John Wiley & Sons Ltd. on behalf of Institute for Fiscal Studies © 2020 The Authors. Fiscal Studies published by John Wiley & Sons Ltd. on behalf of Institute for Fiscal Studies Parental ethnic identity and child test scores 19 identity, there are contrasting results in the literature, with a strong minority identity having positive or negative implications in different national contexts and in different areas of economic life. Given the surprising nature of this result, we suggest two possible interpretations. The ﬁrst is that both parental adoption and active rejection of the majority identity genuinely affect children’s test scores in a negative way. 50See, for example, Phinney et al. (2001), Rumbaut (1994) and Tajfel and Turner (1979). 51Dickerson and Popli (2016) take a similar approach. 52For a full discussion of modelling parental investment, see Hernández-Alava and Popli (2017). 2. Main results ‘Taking a position’ on the majority identity could divert household resources from activities that are more beneﬁcial to a child’s academic development, or it could plausibly affect access to social support from family and friends. The second interpretation we suggest is that mothers adopt a position on the majority identity as a source of self-worth in response to challenging circumstances. This ‘protective’ role of identity has been emphasised in some of the sociological and social psychological literature in this area.50 In our analysis, such challenging circumstances may drive both maternal adoption of a position on the majority identity, and children’s lower test scores. This is therefore essentially an ‘omitted variables’ explanation for our result. We explore the evidence for these competing explanations in the next section. © 2020 The Authors. Fiscal Studies published by John Wiley & Sons Ltd. on behalf of Institute for Fiscal Studies © 2020 The Authors. Fiscal Studies published by John Wiley & Sons Ltd. on behalf of Institute for Fiscal Studies 1. The role of parental investments One reason for the negative association that we observe between maternal acceptance or rejection of the majority identity and child test scores could be differences in access to or use of productive inputs. ‘Taking a position’ on the majority identity could divert household resources away from the most productive parental investments. We examine this possibility by estimating latent factor scores based on parental investments measured when the child is aged 5, two years prior to the cognitive tests we use as outcomes.51 The MCS has detailed information on parental involvement with children, and we use this information to model parental investment as three different latent factors.52 The ﬁrst factor combines information on activities that parents undertake, which are directly related to schoolwork, and include information on how often parents help their children with reading, writing, maths, or painting. The second factor relates to the range of activities that parents carry out with their children, and the routines they establish. This includes how often they read to their children or take trips to the library, and whether they impose regular bedtimes, or monitor television watching. The third factor relates to parenting styles, and includes information on the frequency with which parents © 2020 The Authors. Fiscal Studies published by John Wiley & Sons Ltd. on behalf of Institute for Fiscal Studies 20 Fiscal Studies TABLE 5 TABLE 5 Association between parental identity and parental investments, and association between parental identity and child test scores (1) (2) (3) (4) (5) (6) PI1 PI2 PI3 Maths Spatial Reading Majority identity Strongly disagree 0.204 0.774 −0.145 0.181 0.107 −0.009 (0.198) (0.309) (0.179) (0.179) (0.150) (0.163) Disagree −0.039 0.048 −0.200 −0.224* −0.288 −0.071 (0.148) (0.198) (0.137) (0.129) (0.135) (0.103) Agree 0.108 −0.047 0.086 −0.209* −0.049 −0.120* (0.088) (0.135) (0.092) (0.074) (0.073) (0.069) Strongly agree 0.149 0.117 0.105 −0.124 −0.097 −0.054 (0.101) (0.153) (0.104) (0.084) (0.083) (0.076) Minority identity Strongly disagree 0.026 0.133 0.043 0.036 0.311** −0.003 (0.216) (0.311) (0.243) (0.175) (0.142) (0.171) Disagree 0.029 −0.020 0.034 0.040 0.042 −0.055 (0.153) (0.232) (0.164) (0.133) (0.131) (0.114) Agree −0.141 −0.210 −0.136 0.082 0.034 0.016 (0.094) (0.153) (0.099) (0.079) (0.081) (0.074) Strongly agree 0.048 −0.307* −0.082 0.106 0.048 0.024 (0.103) (0.164) (0.105) (0.086) (0.083) (0.080) Parental investments PI1 −0.025 −0.025 −0.037 (0.035) (0.034) (0.032) PI2 0.206*** 0.101* 0.234*** (0.055) (0.054) (0.053) PI3 0.015 −0.004 0.011 (0.034) (0.033) (0.031) N 1,249 1,249 1,242 1,249 1,249 1,249 Note: Standard errors in parentheses. Sample: all mothers. Base category for identity: Neither agree nor disagree. All controls used. p < 0.1; p < 0.05; p < 0.01. See Section IV for details of the empirical model. See Section III and Table 1 for details of the main control variables. The three latent factor scores capturing parental investment are referred to as PI1, PI2 and PI3. PI1 includes how often parents help their children with reading, writing, maths or painting; PI2 includes how often parents read to their children or take trips to the library, and whether they impose regular bedtimes or monitor television watching. PI3 includes the frequency with which parents smack, ‘tell off’, or shout at their children (PI3 is reverse coded). Association between parental identity and parental investments, and association between parental identity and child test scores smack, ‘tell off’, or shout at their children. We treat these ﬁnal three actions as parental disinvestment, and code accordingly. We standardise the three latent factor score to have a mean of zero and standard deviation of 1, and regress each of them on our measures of identity and main control variables. Results in columns 1–3 of Table 5 provide little evidence to suggest that ethnic identity inﬂuences parental investment. TABLE 5 The only signiﬁcant coefﬁcients Parental ethnic identity and child test scores 21 we ﬁnd are associated with the second latent factor. Mothers who report that they ‘Strongly disagree’ with the majority identity invest more in their children’s non-school activities and provide a more structured routine for their children. As we have noted, these mothers are very small in number. However, mothers who ‘Strongly agree’ with the minority identity provide less investment for their children along this dimension. It therefore seems unlikely that direct parental provision of inputs is a mechanism linking maternal identity with lower child test scores. y When we include these latent factor scores as controls in our baseline model (columns 4–6), the only signiﬁcant association we ﬁnd is with the second latent factor. Children with mothers who engage in more out-of-school activities, and provide a structured routine, do signiﬁcantly better in all three test scores. The estimated association with test scores and this particular latent factor is around the same size of that found in Hernández-Alava and Popli (2017), who use MCS data with both ethnic minority and majority children. Our estimates of interest do not change when we add these controls. While we cannot rule out the possibility that parental ethnic identity has an inﬂuence on parental investment, these results suggest it is not a decisive mechanism. Of course, it is possible that identity still affects household resource deployment more generally, and we therefore turn to examine household resources directly. © 2020 The Authors. Fiscal Studies published by John Wiley & Sons Ltd. on behalf of Institute for Fiscal Studies © 2020 The Authors. Fiscal Studies published by John Wiley & Sons Ltd. on behalf of Institute for Fiscal Studies 2. The role of poverty If ‘taking a position’ on the majority identity diverts household resources from more productive activities, or affects access to social support, the relationship between maternal identity and children’s cognitive outcomes could be mediated by the economic circumstances of the household. Families experiencing hardship may be more vulnerable to any negative effects of parental identity in these areas. Difﬁcult economic circumstances within the household could also cause mothers to adopt a position on the majority identity, as well as lowering child test scores, which would be consistent with an alternative explanation of the relationship we observe. We therefore investigate the association between maternal identity and child test scores by income poverty. Households are classiﬁed as income poor if their household equivalised income is below 60 per cent of contemporaneous median household equivalised income before housing costs, according to the most widely used deﬁnition in the UK (from the Child Poverty Act, 2010). Ethnic minority households are much more likely to be below the poverty line than majority households, and just over 50 per cent of our sample is classiﬁed as income poor. There is also substantial variation across ethnic minority groups. For example, 75 per cent of Pakistani or Bangladeshi households are classiﬁed as income poor, compared to 30 per cent of Indian households. 22 Fiscal Studies 3. The role of family networks Our results so far suggest that household poverty is important in shaping the association between maternal identity and child test scores. The absence or availability of local family networks may also be linked to resource constraints. Such networks can provide various resources to parents, including direct provision of ﬁnancial resources,54 or emotional and social support.55 Families may share useful information, such as how to navigate the health, welfare or education system, or provide a springboard for accessing wider community networks. Such networks may also enable parents to diversify limited resources between different types of investments. Without access to local family networks, parents are excluded from any such additional resources and the opportunity to diversify. A lack of family networks could also represent the kind of difﬁcult circumstances that would push mothers to adopt a position on the majority identity, which would be consistent with the alternative interpretation of our main results. Our results so far suggest that household poverty is important in shaping the association between maternal identity and child test scores. The absence or availability of local family networks may also be linked to resource constraints. Such networks can provide various resources to parents, including direct provision of ﬁnancial resources,54 or emotional and social support.55 direct provision of ﬁnancial resources,54 or emotional and social support.55 Families may share useful information, such as how to navigate the health, welfare or education system, or provide a springboard for accessing wider community networks. Such networks may also enable parents to diversify limited resources between different types of investments. Without access to local family networks, parents are excluded from any such additional resources and the opportunity to diversify. A lack of family networks could also represent the kind of difﬁcult circumstances that would push mothers to adopt a position on the majority identity, which would be consistent with the alternative interpretation of our main results. We investigate the role of family networks using responses to the question ‘Do you have other friends and family in the area?’.56 Parents may respond ‘Yes, friends’, ‘Yes, family’, ‘Yes, both’ or ‘No’. We use those that indicate ‘Yes, family’ and ‘Yes, both’ to represent family networks. According to this deﬁnition, just over 50 per cent of households in the sample have family who live locally. TABLE 6 TABLE 6 Association between maternal identity and child test scores for non-poor and poor mothers (OLS) Non–poor Poor (1) (2) (3) (4) (5) (6) Maths Spatial Reading Maths Spatial Reading Majority identity Strongly disagree 0.126 0.370 −0.082 0.654** 0.050 0.290 (0.219) (0.155) (0.203) (0.297) (0.291) (0.281) Disagree −0.127 0.008 −0.093 −0.327* −0.558* −0.040 (0.206) (0.214) (0.155) (0.171) (0.164) (0.148) Agree −0.146 0.090 −0.059 −0.280* −0.166 −0.176* (0.111) (0.101) (0.097) (0.104) (0.109) (0.105) Strongly agree 0.032 0.048 −0.052 −0.234 −0.192 −0.041 (0.131) (0.120) (0.109) (0.119) (0.118) (0.116) Minority identity Strongly disagree 0.140 0.190 0.054 −0.058 0.524 0.033 (0.221) (0.162) (0.215) (0.278) (0.266) (0.320) Disagree 0.065 0.001 −0.005 0.051 0.106 −0.121 (0.205) (0.181) (0.168) (0.187) (0.185) (0.168) Agree 0.211* 0.140 0.110 −0.026 −0.007 −0.084 (0.117) (0.112) (0.104) (0.117) (0.117) (0.114) Strongly agree 0.102 −0.104 0.101 0.113 0.224* −0.074 (0.124) (0.112) (0.110) (0.127) (0.122) (0.127) Gender and ethnicity Yes Yes Yes Yes Yes Yes Main controls Yes Yes Yes Yes Yes Yes Additional controls Yes Yes Yes Yes Yes Yes N 611 611 611 638 638 638 Note: Standard errors in parentheses. Sample: all mothers. Base category for identity: Neither agree nor disagree. p < 0.1; p < 0.05; *p <0.01. Association between maternal identity and child test scores for non-poor and poor mothers (OLS) Note: Standard errors in parentheses. Sample: all mothers. Base category for identity: Neither agree nor disagree. p < 0.1; p < 0.05; p <0.01. We present results for families above and below the poverty line in Table 6. These results show that the negative association between those who ‘Agree’ or ‘Disagree’ with the majority identity and child test scores is largely driven by children in poor families. For example, in maths, children in poor families where the mother agrees with the majority identity statement score 0.280 standard deviations below children of ‘neutral’ mothers in poor families, and those whose mothers disagree score 0.327 standard deviations below children of ‘neutral’ mothers in poor families. These differences are statistically signiﬁcant. Among non-poor families, the equivalent coefﬁcients are also negative, but much smaller in magnitude, and imprecisely estimated. Although we cannot reject the null of no difference in test score gaps across poor and non-poor families, these results are at least indicative of an inter-relationship Parental ethnic identity and child test scores 23 between parental identity and income poverty. TABLE 6 We observe a similar pattern of results for reading and spatial problem solving test scores.53 53We have also examined whether this heterogeneity by family poverty reﬂects the impact of neighbourhood poverty, through such factors as peer groups or school quality. We ﬁnd no clear results. 54Angelucci et al., 2010. © 2020 The Authors. Fiscal Studies published by John Wiley & Sons Ltd. on behalf of Institute for Fiscal Studies 53We have also examined whether this heterogeneity by family poverty reﬂects the impact of neighbourhood poverty, through such factors as peer groups or school quality. We ﬁnd no clear results. 54Angelucci et al., 2010. 55Burchinal, Follmer and Bryant, 1996; Bradley and Corwyn, 2002; Green, Furrer and McAllister, 2007; Stepick and Dutton Stepick, 2010; Haller, Portes and Lynch, 2011; Serrano-Villar, Huang and Calzada, 2017. 56Parents are ﬁrst asked ‘Are you friends with other parents in the area?’ followed by the question about family and friends. We have also examined the role of friendship networks but ﬁnd little evidence that this matters for shaping the effect of parental identity. 55Burchinal, Follmer and Bryant, 1996; Bradley and Corwyn, 2002; Green, Furrer and McAllister, 2007; Stepick and Dutton Stepick, 2010; Haller, Portes and Lynch, 2011; Serrano-Villar, Huang and Calzada, 2017. 56Parents are ﬁrst asked ‘Are you friends with other parents in the area?’ followed by the question about family and friends. We have also examined the role of friendship networks but ﬁnd little evidence that this matters for shaping the effect of parental identity. 2017. 56Parents are ﬁrst asked ‘Are you friends with other parents in the area?’ followed by the question about amily and friends. We have also examined the role of friendship networks but ﬁnd little evidence that this matters for shaping the effect of parental identity. g 55Burchinal, Follmer and Bryant, 1996; Bradley and Corwyn, 2002; Green, Furrer and McAllister, 2007; Stepick and Dutton Stepick, 2010; Haller, Portes and Lynch, 2011; Serrano-Villar, Huang and Calzada, 2017. TABLE 7 TABLE 7 Association between maternal identity and child test scores for mothers with and without family networks (OLS) Has family networks No family networks (1) (2) (3) (4) (5) (6) Maths Spatial Reading Maths Spatial Reading Majority identity Strongly disagree 0.051 0.192 0.353 0.391 0.063 −0.140 (0.259) (0.258) (0.220) (0.235) (0.193) (0.221) Disagree −0.150 −0.200 −0.145 −0.343 −0.389 −0.075 (0.206) (0.207) (0.148) (0.169) (0.181) (0.147) Agree −0.170 0.011 −0.108 −0.245 −0.102 −0.119 (0.108) (0.112) (0.095) (0.109) (0.103) (0.105) Strongly agree 0.046 −0.051 0.141 −0.307 −0.129 −0.268** (0.124) (0.123) (0.110) (0.123) (0.115) (0.112) Minority identity Strongly disagree 0.088 0.369 0.226 0.064 0.253 −0.084 (0.288) (0.252) (0.263) (0.230) (0.178) (0.252) Disagree 0.124 −0.044 0.040 −0.080 0.143 −0.141 (0.172) (0.182) (0.153) (0.218) (0.203) (0.187) Agree 0.011 −0.024 0.032 0.146 0.085 −0.001 (0.113) (0.120) (0.102) (0.118) (0.115) (0.114) Strongly agree 0.152 0.062 0.106 0.050 0.025 −0.030 (0.125) (0.123) (0.114) (0.124) (0.117) (0.121) Gender and ethnicity Yes Yes Yes Yes Yes Yes Main controls Yes Yes Yes Yes Yes Yes Additional controls Yes Yes Yes Yes Yes Yes N 640 640 640 609 609 609 Note: Standard errors in parentheses. Sample: all mothers. Base category for identity: Neither agree nor disagree. p < 0.1; p < 0.05; *p < 0.01. Association between maternal identity and child test scores for mothers with and without family networks (OLS) Note: Standard errors in parentheses. Sample: all mothers. Base category for identity: Neither agree nor disagree. p < 0.1; p < 0.05; *p < 0.01. estimated. Because almost half of mothers without family networks are not income poor, this ﬁnding is not simply a reﬂection of material poverty, and may reﬂect the broader constraints that having family in the local area can help to alleviate. Interestingly, access to family networks appears to have little independent association with test scores, operating only by modulating the associations with maternal majority identity (see Table A.4). © 2020 The Authors. Fiscal Studies published by John Wiley & Sons Ltd. on behalf of Institute for Fiscal Studies 3. The role of family networks This varies by ethnicity, with two-thirds of Pakistani or Bangladeshi households living close to family, compared to 40 per cent of Black or Mixed heritage households. We present results by access to local family networks in Table 7. These results suggest that the negative association between acceptance or rejection of the majority identity and child test scores is largely driven by households that do not have access to local family networks. Within this group, children of mothers who ‘Disagree’ with the majority statement score 0.343 standard deviations lower in maths and 0.389 lower in spatial problem solving, and children of mothers who ‘Agree’ score 0.245 lower in maths. The estimates for those who have access to family networks are smaller and imprecisely 24 Fiscal Studies 57Casey and Dustmann, 2010; Islam and Raschky, 2015. 58Nekby et al., 2009; Schüller, 2015. 59Battu et al., 2007; Battu and Zenou, 2010. 60Fryer and Torelli, 2010. © 2020 The Authors. Fiscal Studies published by John Wiley & Sons Ltd. on behalf of Institute for Fiscal Studies V. Discussion In this paper, we examine the relationship between parental ethnic identity and test scores in ethnic minority children. We employ separate measures of parental minority and majority identity, alongside tests of children’s cognitive Parental ethnic identity and child test scores 25 functioning in three key domains. We show that children whose mothers report either an adoption or an active rejection of the majority identity tend to score lower in cognitive tests at age 7, compared with those children whose mothers report neutral feelings about the majority identity. The results are driven by children from poor households, and by those from households who lack access to local family support networks. We ﬁnd no consistent differences in test scores according to the mother’s minority identity. While the overall character of these results is unusual, there has also been some variation in results elsewhere in the literature. Earlier studies generally ﬁnd the majority identity to be beneﬁcial to labour market outcomes for immigrants and ethnic minority individuals, while a minority identity is often found to be detrimental. However, two papers57 ﬁnd only weak effects of ethnic identity on labour market outcomes, and two other studies58 suggest that the strength of both minority and majority identity can be beneﬁcial for educational outcomes in some national settings. We could perhaps draw some parallels with papers suggesting that negative labour market outcomes are associated with rejection of the majority identity,59 and those suggesting that positive educational outcomes are associated with lower group acceptance.60 However, we do not know of another paper that shows both acceptance and rejection of the majority identity to have negative implications for labour market or educational outcomes. Therefore, we suggest two interpretations for our results. The ﬁrst possible interpretation of these results is that ‘taking a position’ on the majority identity somehow diverts family social or economic resources away from investments that would be more conducive to children’s cognitive development. In this interpretation, both accepting and actively rejecting the majority identity commits resources to less optimal activities. These activities need not be the same for those who ‘Agree’ and those who ‘Disagree’ with the majority identity statement – indeed they seem likely to be very different – but they must share the characteristic that they are less productive for child development than the alternatives. V. Discussion This ﬁrst interpretation would be consistent with some patterns of heterogeneity that we observe in the data: for example, that the result is driven largely by poor households and by those households that lack access to local family networks. Our examination of parental investment behaviour does not ﬁnd differences by maternal identity in our measures of this particular activity, but identity could affect household resource deployment more generally. If this interpretation is correct, then parental identity should be a matter of interest when considering the formation – but they must share the characteristic that they are less productive for child development than the alternatives. This ﬁrst interpretation would be consistent with some patterns of heterogeneity that we observe in the data: for example, that the result is driven largely by poor households and by those households that lack access to local family networks. Our examination of parental investment behaviour does not ﬁnd differences by maternal identity in our measures of this particular activity, but identity could affect household resource deployment more generally. If this interpretation is correct, then parental identity should be a matter of interest when considering the formation 26 Fiscal Studies Fiscal Studies of cognitive skills in early childhood, particularly in relation to ethnic minority children. However, given the unusual nature of these results, we are necessarily cautious in drawing strong policy conclusions. A second interpretation of our result is that mothers adopt a position on the majority identity as a source of self-worth in response to challenging circumstances, in line with the view of identity as a ‘protective’ device.61 These difﬁculties are then also reﬂected in children’s lower test scores. This interpretation is also consistent with the patterns of heterogeneity we observe in the data, as both household poverty and lack of access to local family networks could constitute the kind of challenging circumstances that would lead a mother to take a position on the majority identity. This interpretation does not imply a genuine relationship between parental ethnic identity and child test scores, but implies that the result is driven by omitted variables, inﬂuencing both parental identity and child outcomes. Ultimately, we cannot distinguish between these two competing explanations for why taking a stance on the majority identity matters in these circumstances. Finally, we note that our estimates are sometimes imprecise and unstable across speciﬁcations, perhaps due to measurement error in the identity variables. 61Tajfel and Turner, 1979; Rumbaut, 1994; Phinney et al., 2001. 62Nandi and Platt (2012) explore such measurement difﬁculties in relation to the newer ‘Understanding Society’ data set in the UK. 62Nandi and Platt (2012) explore such measurement difﬁculties in relation to the newer ‘Understanding Society’ data set in the UK. © 2020 The Authors. Fiscal Studies published by John Wiley & Sons Ltd. on behalf of Institute for Fiscal Studies 61Tajfel and Turner, 1979; Rumbaut, 1994; Phinney et al., 2001. 61Tajfel and Turner, 1979; Rumbaut, 1994; Phinney et al., 2001. References Aber, J. L., Bennett, N. G., Conley, D. C. and Li, J. (1997), ‘The effects of poverty on child health and development’, Annual Review of Public Health, vol. 18, pp. 463–83. Aber, J. L., Bennett, N. G., Conley, D. C. and Li, J. 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(2010), ‘Oppositional identities and employment for ethnic minorities: evidence from England’, Economic Journal, vol. 120, no. 542, pp. F52–F71. Bécares, L., Nazroo, J. and Kelly, Y. V. Discussion Having a richer measure of identity, derived from several identity- related questions, would go some way to addressing concerns about measurement error. Unfortunately, we do not have a way of testing such measurement questions with these data, so we leave this observation to be developed in future work.62 However, we regard this as a potentially important issue in the literature on the economics of identity, which predominantly relies on the kind of one-dimensional survey measures we use in this paper. If null or unusual results using such measures are less likely to reach publication stage, the literature may have given us an inaccurate perception of the reliability of these measures, or the range of possible outcomes they can produce. We noted in the introduction to this paper that the governments of several countries have responded to increased ethnic diversity by introducing education and citizenship policies that actively promote the majority identity, while discouraging the maintenance of separate minority identities. Neither of the two suggested interpretations of our results provides support for such policies. At best, it is unclear from these results whether promoting the majority identity will have an impact on children’s outcomes. Better measurement, and a better understanding of the mechanisms through which parental ethnic identity shapes childhood outcomes, is necessary to fully comprehend the impact of these policies on ethnic minority children. However, public resources are still invested in the promotion of the majority identity. © 2020 The Authors. Fiscal Studies published by John Wiley & Sons Ltd. on behalf of Institute for Fiscal Studies Parental ethnic identity and child test scores 27 Given what we know about the importance of material conditions in the early years, other issues, such as the high levels of income poverty observed in some ethnic minority groups, may be a more pressing policy concern. Supporting information Additional supporting information may be found online in the Supporting Information section at the end of the article. r Appendix © 2020 The Authors. Fiscal Studies published by John Wiley & Sons Ltd. on behalf of Institute for Fiscal Studies References (2015), ‘A longitudinal examination of maternal, family, and area-level experiences of racism on children’s socioemotional development: patterns and possible explanations’, Social Science & Medicine, vol. 142, pp. 128–35. Becker, G. S. (1981), A Treatise on the Family, Cambridge, MA: Harvard University Press. — and Tomes, N. 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https://openalex.org/W2318165324	https://dspace.palermo.edu/ojs/index.php/psicodebate/article/download/343/pdf, https://dialnet.unirioja.es/descarga/articulo/5645296.pdf	es		IVyF: Validez de un Instrumento de Medida de las Fortalezas del Carácter de la Clasificación de Peterson y Seligman (2004)	Revista psicodebate: psicología, cultura y sociedad/Psicodebate	2,015	cc-by	5,511	Psicodebate Vol. 15 Nº 2 \| Diciembre 2015 \| ISSN: 1515–2251 \| 99–122 IVyF: Validez de un Instrumento de Medida de las Fortalezas del Carácter de la Clasificación de Peterson y Seligman (2004) Alejandro César Cosentino1 y Alejandro Castro Solano2 Artículo Material original autorizado para la publicación en la revista Psicodebate. Facultad de Ciencias Sociales. Universidad de Palermo. Recibido 10-07-2015 \| Aceptado 14-08-2015 Resumen El IVyF (Strength of Character Inventory, en inglés) es un instrumento de PHGLFLyQ GH ODV IRUWDOH]DV GHO FDUiFWHU GH OD FODVL¿FDFLyQ GH 3HWHUVRQ \ Seligman (2004). A diferencia de otros cuestionarios homólogos para medir ODVIRUWDOH]DVGHHVDFODVL¿FDFLyQTXHHVWiQFRPSXHVWRVSRUGHFHQDVGHtWHPV HO,9\)HVXQLQVWUXPHQWRGHPHGLFLyQGHtWHPV(VWHHVWXGLRVHHQIRFyHQ la validez del IVyF. En primer lugar, el IVyF estuvo asociado a medidas de variables conceptualmente relevantes como satisfacción con la vida, los factores de personalidad del modelo de los Cinco Grandes, y deseabilidad social. En segundo lugar, se hallaron asociaciones con tamaños del efecto mayores a mediano entre autopuntuación y puntuación realizada por un observador. Los resultados de esta investigación han sido similares a los obtenidos con FXHVWLRQDULRVKRPyORJRV6HFRQFOX\HTXHHO,9\)SUHVHQWDQRVyORDGHFXDGD FRQ¿DELOLGDGVLQRWDPELpQDGHFXDGDYDOLGH] Palabras Clave:SVLFRORJtDSRVLWLYDYLUWXGHVWHVWYDOLGH] 1 Universidad de Palermo - Argentina; acosentino@outlook.com 2 Universidad de Palermo, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) - Argentina Cosentino & Castro Solano \| Psicodebate 15 (2) \| 99–122 Abstract The Strengths of Character Inventory (SCI; IVyF in Spanish) is a measurement instrument for the 24 character strengths from the Peterson and Seligman (2004) FKDUDFWHU FODVVL¿FDWLRQ 8QOLNH RWKHU KRPRORJRXV TXHVWLRQQDLUHV WR PHDVXUH WKH FKDUDFWHU VWUHQJWKV RI WKLV FODVVL¿FDWLRQ WKDW DUH FRPSRVHG RI GR]HQV RI items, the SCI is constituted by 24 items. This study focused on the validity of the SCI. Firstly, the SCI was associated with measures of conceptually relevant variables such as life satisfaction, personality from the Big Five model, and social desirability. Secondly, associations with more than medium effect size were found between self-rating and observer`s rating. The results of this research were similar WRWKRVHREWDLQHGZLWKKRPRORJRXVTXHVWLRQQDLUHV:HFRQFOXGHWKDW6&,SUHVHQWV not only acceptable reliability, but also acceptable validity. Keywords: positive psychology, virtues, test, validity. 100 Validez del IVyF /D 3VLFRORJtD 3RVLWLYD SXHGH FRQVLGHUDUVH FRQVWLWXLGD SRU WUHV iUHDV R pilares: la subjetiva, la individual y la grupal (Gable & Haidt, 2005; Seligman &VLNV]HQWPLKDO\L (O iUHD VXEMHWLYD VH UHODFLRQD FRQ HO HVWXGLR GH ODV H[SHULHQFLDV VXEMHWLYDV YDORUDGDV SRVLWLYDPHQWH HO iUHD LQGLYLGXDO TXH estudia los rasgos individuales positivos, como la capacidad de amar, el coraje, las habilidades interpersonales, la sensibilidad estética, la perseverancia, la FOHPHQFLDODRULJLQDOLGDGODHVSLULWXDOLGDG\ODVDELGXUtDHQUHVXPHQODVYLUWXGHV \IRUWDOH]DVGHOFDUiFWHU\¿QDOPHQWHHQHOiUHDJUXSDOVHLQYHVWLJDDORVJUXSRV humanos en relación con los aspectos positivos de los individuos, por ejemplo, a ODVLQVWLWXFLRQHVTXHLPSXOVDQDORVLQGLYLGXRVDVHUPHMRUHVFLXGDGDQRV Peterson y Seligman (2004) impulsaron el estudio de los rasgos individuales SRVLWLYRVRIRUWDOH]DVGHOFDUiFWHUFRQXQDPHWRGRORJtDFLHQWt¿FD(VWRVDXWRUHV KDQ FRQVLGHUDGR TXH FRQWDU FRQ XQD FODVL¿FDFLyQ GH ODV YLUWXGHV \ IRUWDOH]DV GHO FDUiFWHU FRQVWLWXtD XQ SDVR LPSRUWDQWH \ QHFHVDULR SDUD HO SURJUHVR GHO HVWXGLR FLHQWt¿FR HQ 3VLFRORJtD 3RVLWLYD \ VH WRPDURQ OD WDUHD GH GHVDUUROODU HVDFODVL¿FDFLyQ (OSDVRLQLFLDOHQHOFDPLQRGHGHVDUUROORGHVXFODVL¿FDFLyQ 'DKOVJDDUG Peterson, & Seligman, 2005), incluyó el examen de las respuestas sobre el FRPSRUWDPLHQWR PRUDOPHQWH EXHQR TXH VH FRQFLELHURQ HQ ODV WUDGLFLRQHV filosóficas y religiosas de impacto evidente y duradero en la civilización KXPDQD HO FRQIXFLDQLVPR \ WDRtVPR GH &KLQD HO EXGLVPR H KLQGXLVPR GHO VXU DVLiWLFR \ OD ¿ORVRItD DWHQLHQVH HO MXGDtVPR HO FULVWLDQLVPR \ HO LVODPLVPR GH 2FFLGHQWH 6HLV YLUWXGHV IXQGDPHQWDOHV VH UHSHWtDQ HQ HVDV tradiciones y esta convergencia sugirió un fundamento no arbitrario para la QXHYDFODVL¿FDFLyQHYLWDQGRTXHSUHVHQWDVHXQVHVJRKLVWyULFRRFXOWXUDO$¿Q GHJHQHUDUODVIRUWDOH]DVGHOFDUiFWHUSDUDLQFOXLUHQODFODVL¿FDFLyQXQJUXSRGH DFDGpPLFRVSURSXVRXQDOLVWDGHIRUWDOH]DVFDQGLGDWDVTXHIXHUH¿QDGDDWUDYpV GHXQDVHULHGHGHEDWHVOOHJDQGRDXQDFODVL¿FDFLyQGHYLUWXGHVTXHLQFOX\y fortalezas (Peterson & Seligman, 2004). (OFDUiFWHUHQODFODVL¿FDFLyQGH3HWHUVRQ\6HOLJPDQ LQFOX\HODVVHLV virtudes (las fortalezas del carácter correspondientes aparecen entre paréntesis): FRUDMH LQWHJULGDG SHUVLVWHQFLD YDOHQWtD YLWDOLGDG MXVWLFLD LPSDUFLDOLGDG compañerismo y liderazgo), humanidad (amor, bondad, inteligencia social), VDELGXUtD \ FRQRFLPLHQWR DSHUWXUD PHQWDO DPRU SRU HO VDEHU FUHDWLYLGDG curiosidad, perspectiva), templanza (autorregulación, clemencia, humildad, y prudencia) y trascendencia (admiración de la belleza y la excelencia, esperanza, HVSLULWXDOLGDGJUDWLWXG\KXPRU 1RREVWDQWH3HWHUVRQ\6HOLJPDQD¿UPDURQTXH HVWDFODVL¿FDFLyQHUDGHFDUiFWHUWHQWDWLYR\TXHSRGUtDVHUDOWHUDGDDFRQVHFXHQFLD GHOSURJUHVRHQHOHVWXGLRFLHQWt¿FR 101 Cosentino & Castro Solano \| Psicodebate 15 (2) \| 99–122 Instrumentos para la medición de las fortalezas del carácter de la clasificación de Peterson y Seligman (2004) +D\YDULRVLQVWUXPHQWRVTXHSXHGHQXWLOL]DUVHSDUDPHGLUODVIRUWDOH]DVGHO FDUiFWHUGHODFODVL¿FDFLyQGH3HWHUVRQ\6HOLJPDQ SDUDSREODFLyQDGXOWD Algunos de ellos se indican abajo. Values in Action Inventory of Strengths (VIA-IS) 3HWHUVRQ\6HOLJPDQ KDQGHVDUUROODGRHO9,$,6TXHHVXQFXHVWLRQDULR GH DXWRLQIRUPH GH tWHPV SDUD HYDOXDU ODV IRUWDOH]DV GHO FDUiFWHU VHJ~Q OD FODVL¿FDFLyQ 9DOXHV LQ$FWLRQ 9,$ HQ SREODFLyQ DGXOWD (VWH FXHVWLRQDULR SXHGH FRPSOHWDUVH OLEUHPHQWH RQOLQH GHVGH ¿QHV GH ²S HM KWWSZZZ YLDFKDUDFWHURUJ² \ KD\ WDPELpQ XQD YHUVLyQ GH 9,$,6 HQ IRUPDWR GH SDSHO y lápiz (Linley et al., 2007). Además, hay versiones disponibles del VIA-IS en HVSDxRO \ HQ RWURV LGLRPDV \ VH FRQWLQ~DQ GHVDUUROODQGR YHUVLRQHV HQ YDULRV idiomas más. Cuando se administra el VIA-IS, el entrevistado indica con cuánta IUHFXHQFLDVHSHUFLEHDVtPLVPRWHQLHQGRHVWDGRVUHSUHVHQWDWLYRVGHODVIRUWDOH]DV GHOFDUiFWHUXVDQGRXQDHVFDOD/LNHUWGHFLQFRSXQWRVTXHYDGHmuy parecido a mí a muy diferente a mí. International Personality Item Pool VIA revisado (IPIP-VIA-r) El International Personality Item Pool (IPIP) es un emprendimiento de FRODERUDFLyQ FLHQWt¿FD SDUD HO GHVDUUROOR GH PHGLFLRQHV GH OD SHUVRQDOLGDG \ RWUDV GLIHUHQFLDV LQGLYLGXDOHV TXH KD VLGR SUHVHQWDGR SRU SULPHUD YH] HQ HQODRFWDYD(XURSHDQ&RQIHUHQFHRQ3HUVRQDOLW\ ROGEHUJROGEHUJHW DO 6XVLWLRZHE KWWSLSLSRULRUJ HVWiGHVWLQDGRDSURSRUFLRQDUDFFHVR UiSLGR D PHGLFLRQHV GH GLIHUHQFLDV LQGLYLGXDOHV GH GRPLQLR S~EOLFR L H VLQ FRS\ULJKW 'HVGH HO VLWLR ZHE GHO ,3,3 SXHGHQ REWHQHUVH ORV tWHPV HQ LQJOpV constituyentes del instrumento IPIP-VIA-r para evaluar las 24 fortalezas del FDUiFWHUVLJXLHQGRODFODVL¿FDFLyQ9,$(OVLWLRSURYHHtWHPVDORV¿QHVGH HYDOXDUODVIRUWDOH]DVGHOFDUiFWHUGHODFODVL¿FDFLyQ9,$(MHPSORVGHtWHPV ²TXHVHWUDGXMHURQDOHVSDxRO²GHO,3,39,$UVRQsuelo obtener lo que quiero porque trabajo mucho para conseguirlo ²IRUWDOH]D SHUVLVWHQFLD² me pongo a pensar antes de hablar ²IRUWDOH]D SUXGHQFLD² me surgen ideas nuevas y diferentes²IRUWDOH]DFUHDWLYLGDG² IVyF (O ,9\) &RVHQWLQR &DVWUR 6RODQR E HV XQ LQYHQWDULR GH tWHPV GH párrafo bipolares de autopuntuación directa global para evaluar las 24 fortalezas 102 Validez del IVyF GHO FDUiFWHU GH OD FODVL¿FDFLyQ GH 3HWHUVRQ \ 6HOLJPDQ (O LQVWUXPHQWR FRQVWD GH tWHPV ELSRODUHV FRUUHVSRQGLHQGR FDGD tWHP D XQD IRUWDOH]D ORV FXDOHVVHUHVSRQGHQFRQXQDHVFDODWLSR/LNHUWTXHYDGH²Soy muy parecido a la primera persona² D ²Soy muy parecido a la segunda persona² /RV tWHPV\WLHQHQSXQWXDFLyQGLUHFWDHQWDQWR TXHWLHQHQSXQWXDFLyQUHYHUWLGDORVtWHPV (O ,9\) SXHGH VHU VROLFLWDGR D VXV DXWRUHV D ¿Q GH TXH SXHGD VHU XWLOL]DGR HQ investigaciones académicas, y será enviado en su formato digital.) 6H LQFOX\H XQ HMHPSOR GH tWHP JUDWLWXG GHO ,9\) (O SROR DXVHQFLD GH OD fortaleza del carácter gratitud está construido por el párrafo: “Son pocas las cosas por las cuales pueda sentirme agradecido. No me veo a mi mismo como XQDSHUVRQDDIRUWXQDGDSRUTXHQRFUHRTXHKD\DVLGRULFDPHQWHEHQGHFLGRHQOD YLGD$Vt TXH HYLGHQWHPHQWH QR WHQJR WRGRV ORV GtDV XQ SURIXQGR VHQWLPLHQWR GH JUDWLWXG SRU OR TXH PH WRFy YLYLU$GHPiV QR VLHQWR OD QHFHVLGDG GH GDUOH ODVJUDFLDVQLOHGHPXHVWURPLDJUDGHFLPLHQWRDODJHQWHTXHHVEXHQDFRQPLJR \ TXH VH SUHRFXSD SRU Pt´ (O SROR GH SUHVHQFLD GH JUDWLWXG HVWi FRQVWLWXLGR por el siguiente párrafo: “No son pocas las cosas por las cuales puedo sentirme DJUDGHFLGR0HYHRDPLPLVPRFRPRXQDSHUVRQDDIRUWXQDGDSRUTXHFUHRTXHKH VLGRULFDPHQWHEHQGHFLGRHQODYLGD$VtTXHHYLGHQWHPHQWHWHQJRWRGRVORVGtDV XQSURIXQGRVHQWLPLHQWRGHJUDWLWXGSRUORTXHPHWRFyYLYLU$GHPiVVLHQWROD QHFHVLGDGGHGDUOHODVJUDFLDV\OHGHPXHVWURPLDJUDGHFLPLHQWRDODJHQWHTXHHV EXHQDFRQPLJR\TXHVHSUHRFXSDSRUPt´ 6HKDQUHDOL]DGRDOJXQRVDQiOLVLVSVLFRPpWULFRVVREUHHO,9\)&RVHQWLQR KDPRVWUDGRTXHWRPDQGRFRPRSXQWRGHUHIHUHQFLDFXDWURDQiOLVLVSVLFRPpWULFRV VREUH SXQWXDFLRQHV SURGXFLGDV SRU LQVWUXPHQWRV TXH HYDO~DQ OD FODVL¿FDFLyQ VIA (Macdonald, Bore, & Munro, 2008; Peterson & Seligman, 2004; Peterson, 3DUN3ROH'¶$QGUHD 6HOLJPDQ³,3,3´ ODVFRQ¿DELOLGDGHV\ODYDOLGH] factorial del IVyF guardan similitud. Por una parte, el IVyF tiene una aceptable confiabilidad test-retest, DSUR[LPDGDPHQWHVHPDQDVGHVHSDUDFLyQWHPSRUDOHQWUHDGPLQLVWUDFLRQHV FRQ UVGHQWURGHOUDQJRGHM = .80, y el alfa de Cronbach sobre las puntuaciones GHORVtWHPVLQGLYLGXDOHVFRQVLGHUDQGRDHVWHDOIDFRPRXQLQGLFDGRUGHOJUDGR HQTXHHVWiQLQWHUUHODFLRQDGRV²LHFRYDUtDQ²ODVUHVSXHVWDVDORVtWHPVGDGDV SRU ORV SDUWLFLSDQWHV +HOPV HW DO IXH GH Į &RVHQWLQR /D FRQ¿DELOLGDGGHWLSRHVWDELOLGDGGHO,9\)HVDFHSWDEOH\VHPHMDQWHDODKDOODGDSDUD el VIA-IS (Peterson & Seligman, 2004; Ruch et al., 2010). Por otra parte, la cantidad y composición de los factores del IVyF es similar D ORV IDFWRUHV LQIRUPDGRV SRU 3HWHUVRQ \ 6HOLJPDQ TXH FRUUHVSRQGHQ D fortalezas interpersonales, fortalezas intelectuales, fortalezas de la restricción, 103 Cosentino & Castro Solano \| Psicodebate 15 (2) \| 99–122 IRUWDOH]DV HPRFLRQDOHV \ IRUWDOH]DV WHROyJLFDV &RVHQWLQR 6LQ HPEDUJR GHEH VHU GHVWDFDGR TXH OD HVWUXFWXUD IDFWRULDO GH OD FODVLILFDFLyQ 9,$ HV FRQWURYHUVLDO \D TXH HO UHVXOWDGR GH YDULRV DQiOLVLV IDFWRULDOHV KDQ PRVWUDGR YDULDFLRQHV HQ OD FRPSRVLFLyQ \ FDQWLGDG GH ORV IDFWRUHV H[WUDtGRV TXH generalizadamente no coinciden con la cantidad y composición de los factores teóricamente propuestos (e.g., Peterson & Seligman, 2004; Peterson et al., 2008; Macdonald et al., 2008; Ruch et al., 2010). La presente investigación El objetivo de esta investigación es mostrar análisis psicométricos del IVyF haciendo foco en diferentes tipos de validez. Tres relaciones son fundamentales para establecer la validez de este nuevo instrumento basado en las relaciones WHyULFDV\HPStULFDVHQWUHVDWLVIDFFLyQYLWDOSHUVRQDOLGDG\GHVHDELOLGDGVRFLDO 5HVSHFWRGHODVDWLVIDFFLyQFRQODYLGDVHKDSURSXHVWRTXHSRUGH¿QLFLyQODV fortalezas del carácter contribuyen a la realización, la satisfacción y la felicidad SHUVRQDOHQXQVHQWLGRDPSOLR 3DUN3HWHUVRQ 6HOLJPDQ3HWHUVRQ 6HOLJPDQ 5HVSHFWRGHODSHUVRQDOLGDG3HWHUVRQ\6HOLJPDQKDQD¿UPDGR TXHHVVXEVWDQFLDOODH[LVWHQFLDGHUHODFLRQHVHQWUHVXFODVL¿FDFLyQGHOFDUiFWHU\ el modelo de la personalidad Big Five, desarrollado a partir de la convergencia HQIDFWRUHVRGLPHQVLRQHVGHODVSDODEUDVUHIHULGDVDORVUDVJRVTXHVHKDOODQHQ HO OHQJXDMH ROGEHUJ \D TXH KDFH TXH VX FODVL¿FDFLyQ WHQJD VHQWLGR )LQDOPHQWH3HWHUVRQ\6HOLJPDQKDQD¿UPDGRTXHODVIRUWDOH]DVGHOFDUiFWHUVRQ socialmente deseables. Adicionalmente, se estudió la validez del IVyF en relación con un criterio REMHWLYR\HQUHODFLyQDXQFXHVWLRQDULRTXHHYDO~DIRUWDOH]DVGHOFDUiFWHU Estudio 1: Relaciones del IVyF con una Medición de Satisfacción Vital 3DUN HW DO KDQ KDOODGR TXH ODV IRUWDOH]DV HVWiQ DVRFLDGDV FRQ OD VDWLVIDFFLyQVXEMHWLYDFRQODYLGD\TXHODVIRUWDOH]DVGHOFDUiFWHUHVSHUDQ]D YLWDOLGDG JUDWLWXG DPRU \ FXULRVLGDG VRQ ODV TXH SUHVHQWDQ DVRFLDFLRQHV PiV DOWDV FRQ HVD YDULDEOH (VWRV DXWRUHV VXJLULHURQ TXH QR VH SRGUtD D¿UPDU TXH HOFDUiFWHUEXHQRSURYRFDHOEXHQYLYLUVLQRPiVELHQTXHODVDWLVIDFFLyQFRQ OD YLGD SRGUtD FRQVLGHUDUVH FRPR XQ DVSHFWR LQKHUHQWH DO EXHQ FDUiFWHU GHO PLVPRPRGRTXHODJUDFLDHVLQKHUHQWHDEDLODUELHQ\QRVXUHVXOWDGRRHIHFWR (Q FRQVHFXHQFLD VH HVSHUD TXH ODV SXQWXDFLRQHV GHO ,9\) VH DVRFLHQ FRQ ODV puntuaciones de satisfacción vital. 104 Validez del IVyF Participantes /DPXHVWUDGHSDUWLFLSDQWHVGH$UJHQWLQDHVWXYRLQWHJUDGDSRUYDURQHV\ 172 mujeres, n FRQXQDPHGLDGHHGDGGHDxRV'7 TXLHQHV IXHURQYROXQWDULRVTXHQRUHFLELHURQUHWULEXFLyQDOJXQDSRUVXSDUWLFLSDFLyQ Instrumentos IVyF. 6H XWLOL]y XQ LQYHQWDULR GH SDSHO \ OiSL] FRPSXHVWR SRU tWHPV GH DXWRSXQWXDFLyQ GLUHFWD JOREDO TXH HYDO~DQ ODV IRUWDOH]DV GHO FDUiFWHU VHJ~Q OD FODVL¿FDFLyQ 9,$ GH 3HWHUVRQ \ 6HOLJPDQ /D FRQ¿DELOLGDG GH WLSR FRQVLVWHQFLDLQWHUQDGHO,9\)HQHVWDPXHVWUDIXHGHĮ XWLOL]DQGRDOIDGH &URQEDFK FRQVLGHUDQGR D HVWH DOIDFRPR XQ LQGLFDGRUGHO JUDGR HQ TXH HVWiQ LQWHUUHODFLRQDGRV ²L H FRYDUtDQ² ODV UHVSXHVWDV D ORV tWHPV GDGDV SRU ORV SDUWLFLSDQWHV +HOPVHWDO Satisfaction with Life Scale (SWLS). Para medir el constructo satisfacción FRQ OD YLGD VH XWLOL]y OD DGDSWDFLyQ GH OD HVFDOD 6:/6 &DVWUR 6RODQR 'LHQHU(PPRQV/DUVHQ ULI¿Q TXHHYDO~DHOFRPSRQHQWHFRJQLWLYR GHOELHQHVWDU(OLQVWUXPHQWRHVWiFRPSXHVWRSRUtWHPVTXHVHUHVSRQGHQSRU PHGLRGHXQDHVFDODWLSR/LNHUWTXHYDGH²muy en desacuerdo²D²muy de acuerdo² HO LQYHQWDULR SURGXFH XQD SXQWXDFLyQ WRWDO FRUUHVSRQGLHQGR ODV puntuaciones más elevadas a mayor satisfacción con la vida. La consistencia LQWHUQDGHOD6:/6SDUDODPXHVWUDGHHVWHHVWXGLRHVGHĮ Procedimiento y análisis de datos /RVSDUWLFLSDQWHVFRPSOHWDURQHO,9\)\OD6:/6HQVXIRUPDWRGHSDSHO\ OiSL] (O DQiOLVLV GH GDWRV VH UHDOL]y SRU PHGLR GH OD REWHQFLyQ GH FRH¿FLHQWHV GH FRUUHODFLyQ VLPSOH HQWUH ODV SXQWXDFLRQHV GHO ,9\) \ GH OD 6:/6 \ GH FRH¿FLHQWHVGHFRUUHODFLyQSDUFLDOHQWUHODVSXQWXDFLRQHVGHHVRVLQVWUXPHQWRV mientras se controlaban los efectos de las variables sexo y edad. Resultados Todas las fortalezas del carácter se asociaron con satisfacción con la vida GH IRUPD HVWDGtVWLFDPHQWH VLJQL¿FDWLYD ²H[FHSWXDQGR KXPLOGDG² \ GLUHFWD Gratitud, curiosidad, vitalidad, esperanza, persistencia y amor presentaron asociaciones de tamaño superior a mediano con satisfacción con la vida, y estas relaciones se mantuvieron al controlar las variables edad y sexo; ver la Tabla 1. 105 Cosentino & Castro Solano \| Psicodebate 15 (2) \| 99–122 Tabla 1. Correlaciones entre Fortalezas del Carácter y Satisfacción con la Vida Fortaleza SWLSa SWLSb Gratitud .56 .57 Curiosidad .46 .46 Vitalidad .44 .44 Esperanza .40 .40 Persistencia .33 .33 Amor .31 .32 Bondad .28 .29 Perspectiva .30 .29 Liderazgo .28 .28 Autorregulación .26 .26 Valentía .26 .26 Integridad .24 .25 Creatividad .24 .24 Apertura mental .21 .21 Amor por saber .21 .21 Apreciación .19 .20 Inteligencia social .19 .19 Ciudadanía .15 .16 Prudencia .16 .16 Espiritualidad .14** .15* Humor .14** .15* Imparcialidad .14* .14* Clemencia .13* .13* Humildad -.07 -.07 Nota. SWLS = Satisfaction With Life Scale. Apreciación = apreciación de la belleza y la excelencia. a Correlación simple con el IVyF, n = 340. b Correlación parcial controlando sexo y edad con el IVyF, n = 340. p < .05, bilateral. p < .01, bilateral. 106 Validez del IVyF Estudio 2: Relaciones del IVyF con una Medición del Modelo de Personalidad de Cinco Factores El modelo de personalidad de cinco factores corresponde al procedimiento Op[LFR²lexical approachHQLQJOpV²TXHVHGHVDUUROOyDSDUWLUGHODFRQYHUJHQFLD HQIDFWRUHVRGLPHQVLRQHVGHODVSDODEUDVUHIHULGDVDORVUDVJRVTXHVHKDOODQHQ HOOHQJXDMH ROGEHUJ 3HWHUVRQ\6HOLJPDQFRQVLGHUDQTXHGHELGRDTXH H[LVWHQHQHOOp[LFRXQDJUDQFDQWLGDGGHWpUPLQRVUHIHULGRVDORVUDVJRVTXHVH DSOLFDQWDQWRDODVFDUDFWHUtVWLFDVPRUDOPHQWHYDORUDGDVFRPRDODVFDUDFWHUtVWLFDV GHSHUVRQDOLGDGQRGHEHUtDUHVXOWDUVRUSUHQGHQWHHQFRQWUDUYLQFXODFLRQHVHQWUHOD FODVL¿FDFLyQGHOFDUiFWHU\ORVIDFWRUHVGHO%LJ)LYH Peterson y Seligman (2004) han elaborado dos propuestas en relación con el YtQFXORHQWUHODSHUVRQDOLGDGVHJ~QHO%LJ)LYH\ORVUDVJRVGHOFDUiFWHU$PEDV SURSXHVWDV QR VH IXQGDPHQWDQ HQ DQiOLVLV HPStULFRV GH ODV UHODFLRQHV HQWUH GH ambos constructos. Siguiendo la primera propuesta de Peterson y Seligman (2004), los factores GHO%LJ)LYHTXHVHFRUUHVSRQGHQFRQODVIRUWDOH]DVGHOFDUiFWHUGHODFODVL¿FDFLyQ VIA son: a) responsabilidad con autorregulación y persistencia, b) agradabilidad con bondad y gratitud, c) apertura a la experiencia con curiosidad, creatividad y apreciación de la belleza, y d) extraversión con vitalidad y humor; en tanto el factor neuroticismo no se corresponde con ninguna fortaleza. También reconocen TXHORVLQGLYLGXRVHVSHUDQ]DGRVVHSXQW~DQEDMRHQQHXURWLFLVPR\TXHORVOtGHUHV DPHQXGRVHSXQW~DQDOWRHQH[WUDYHUVLyQ 6LJXLHQGR OD VHJXQGD SURSXHVWD GH 3HWHUVRQ \ 6HOLJPDQ HQ TXH VH correspondieron racionalmente los factores del Big Five con los agrupamientos de IRUWDOH]DVGHOFDUiFWHUH[WUDtGRVSRUXQDQiOLVLVIDFWRULDOH[SORUDWRULRORVIDFWRUHV GHSHUVRQDOLGDGVHFRUUHVSRQGHUtDQFRQHOFDUiFWHUGHOVLJXLHQWHPRGRD HOIDFWRU responsabilidad del Big Five con las fortalezas de la restricción, b) agradabilidad con las fortalezas interpersonales, c) apertura a la experiencia con las fortalezas LQWHOHFWXDOHV\G HORSXHVWRDOQHXURWLFLVPR²HVWDELOLGDGHPRFLRQDO²FRQODV IRUWDOH]DV HPRFLRQDOHV² )LQDOPHQWH ORV DXWRUHV GH OD FODVL¿FDFLyQ 9,$ QR YLQFXODURQQLQJXQDIRUWDOH]DGHOFDUiFWHUDOIDFWRUH[WUDYHUVLyQGHO%LJ)LYHDVt FRPRWDPSRFRYLQFXODURQQLQJXQDGLPHQVLyQGHODSHUVRQDOLGDGDOTXLQWRJUXSR GHIRUWDOH]DVH[WUDtGRHQHOPHQFLRQDGRDQiOLVLVIDFWRULDOODVIRUWDOH]DVWHROyJLFDV +D\XQHOHPHQWRHQFRP~QJHQHUDOHQWUHDPEDVSURSXHVWDVGHODUHODFLyQHQWUHHO %LJ)LYH\ODVIRUWDOH]DVGHODFODVL¿FDFLyQGHOFDUiFWHUGH3HWHUVRQ\6HOLJPDQ TXHHVTXHFDGDIDFWRUGHO%LJ)LYHVHFRUUHVSRQGHELXQtYRFDPHQWHFRQXQDIRUWDOH]DR grupo de fortalezas. Sin embargo, hay varias diferencias puntuales entre la primera y la segunda propuesta acerca de la relación entre el Big Five y las fortalezas del carácter: 107 Cosentino & Castro Solano \| Psicodebate 15 (2) \| 99–122 en la primera propuesta la autorregulación se vincula a la dimensión responsabilidad del Big Five, pero en la segunda a neuroticismo; humor se vincula a extraversión en la primera, a agradabilidad en la segunda; vitalidad se vincula a extraversión en la primera, DQHXURWLFLVPRHQODRWUDVHPHQFLRQDTXHORVOtGHUHVSXQW~DQDOWRHQH[WUDYHUVLyQ pero en la segunda propuesta liderazgo aparece vinculado a agradabilidad. Suponemos TXHODHYLGHQWHIDOWDGHH[DFWLWXGHQWUHODVSURSXHVWDVGH3HWHUVRQ\6HOLJPDQVREUH ODUHODFLyQHQWUHODVIRUWDOH]DV\HO%LJ)LYHVHGHEHHQSDUWHDTXHVXVSURSXHVWDVVH OLPLWDURQDPRVWUDUFLHUWDVFRUUHVSRQGHQFLDVTXHSDUHFtDQUDFLRQDOPHQWHVDWLVIDFWRULDV dicho de otro modo, sus propuestas no están basadas o corroboradas por un análisis HPStULFRGHODUHODFLyQHQWUHSHUVRQDOLGDG\FDUiFWHU Sin embargo, también hay convergencias en fortalezas del carácter entre las dos propuestas de Peterson y Seligman (2004): creatividad, curiosidad y apreciación de la belleza y la excelencia se vinculan al factor apertura a la experiencia; ERQGDGDOIDFWRUDJUDGDELOLGDG\WDPELpQVHSRGUtDOOHJDUDFRQVLGHUDUHVSHUDQ]D YLQFXODGDDOIDFWRUQHXURWLFLVPR²RVXLQYHUVRHVWDELOLGDGHPRFLRQDO²DSHVDU GHQRHVWDUH[SOtFLWDPHQWHSUHVHQWHHQODSULPHUDSURSXHVWD 3HWHUVRQ \ 3DUN KDQ LQIRUPDGR IUDJPHQWDULDPHQWH ²L H VLQ EULQGDU LQIRUPDFLyQ GHWDOODGD TXH SHUPLWD OD UHSOLFDFLyQ GH OD LQYHVWLJDFLyQ FRPR FRPSRVLFLyQ PXHVWUDO PRPHQWR \ IRUPD GH DGPLQLVWUDFLyQ HWF² UHVXOWDGRV HPStULFRV VREUH OD UHODFLyQ HQWUH ODV IRUWDOH]DV GHO FDUiFWHU \ HO 1(23, XQ LQVWUXPHQWRSDUDHYDOXDUHO%LJ)LYH'HELGRDTXHEULQGDDOJXQRVGDWRVHPStULFRV relevantes, a diferencia de las otras propuestas teóricas, se incluye el tamaño del HIHFWR GH ORV FRH¿FLHQWHV GH FRUUHODFLyQ &RKHQ FRPR LQGLFDGRU GHO JUDGR de asociación entre las variables: el factor apertura a la experiencia se asocia con las fortalezas del carácter apreciación de la belleza y la excelencia, curiosidad y amor por el saber con tamaños del efecto mayores a grandes; el factor agradabilidad con trabajo HQHTXLSRFRQWDPDxRGHOHIHFWRPHGLDQRDJUDQGH\HOIDFWRUUHVSRQVDELOLGDGFRQ persistencia, y autorregulación, con tamaños del efecto mayores a grandes. 0DFGRQDOGHWDO LQIRUPDQGHVXHVWXGLRHPStULFRGHVDUUROODGRSDUDYHUL¿FDU ODV UHODFLRQHV HQWUH ORV UDVJRV SRVLWLYRV GHO FDUiFWHU GH OD FODVL¿FDFLyQ9,$ \ ODV GLPHQVLRQHVGHODSHUVRQDOLGDGVHJ~QHO%LJ)LYH(QWUHRWURVKDOOD]JRVVHFRQ¿UPDOD LGHDGH3HWHUVRQ\6HOLJPDQ GHTXHH[LVWHQYLQFXODFLRQHVHQWUHHO%LJ)LYH\ODV IRUWDOH]DVGHOFDUiFWHU(OHVWXGLRHPStULFRGH0DFGRQDOGHWDOKDOOyTXHORVIDFWRUHV GHO%LJ)LYHHVWiQUHODFLRQDGRVFRQYDULDVIRUWDOH]DVGHOFDUiFWHUHVSHFt¿FDPHQWHHO IDFWRUH[WUDYHUVLyQFRQIRUWDOH]DVUHVSRQVDELOLGDGFRQDSHUWXUDDODH[SHULHQFLD FRQ DJUDGDELOLGDG FRQ \ QHXURWLFLVPR FRQ 1R REVWDQWH 0DFGRQDOG HW DO KDOODURQTXHKXERPiVGHXQSUHGLFWRUGHO%LJ)LYHSDUDODPD\RUtDGHODVIRUWDOH]DV GHOFDUiFWHUORFXDOVHJ~QHVWRVDXWRUHVSRQGUtDHQGXGDODSURSXHVWDGH3HWHUVRQ\ 6HOLJPDQTXHYLQFXODFDGDIRUWDOH]DGHOFDUiFWHUFRQXQVRORFRQVWUXFWRGHO%LJ)LYH 108 Validez del IVyF Participantes (QHVWHHVWXGLRKXERSDUWLFLSDQWHVGH$UJHQWLQDGHORVFXDOHVHUDQ PXMHUHV\HUDQYDURQHVFRQXQDPHGLDGHHGDGGHDxRV'7 ORV cuales aceptaron voluntariamente completar los instrumentos. Los participantes no recibieron retribución alguna por su inclusión en el estudio. Instrumentos IVyF. 6H XWLOL]y HO ,9\) SDUD HYDOXDU ODV IRUWDOH]DV GHO FDUiFWHU VHJ~Q ODV GH¿QLFLRQHV\FODVL¿FDFLyQGH3HWHUVRQ\6HOLJPDQ /DFRQ¿DELOLGDGGHO WLSRFRQVLVWHQFLDLQWHUQDSDUDHVWDPXHVWUDIXHGHĮ Big Five Inventory (BFI). Se utilizó la adaptación argentina (Castro Solano & &DVXOOR SDUDPHGLUODVGLPHQVLRQHVGHO%LJ)LYH(O%),TXHIXHGHVDUUROODGR por O. P. John para operacionalizar el modelo de los Cinco Grandes. El BFI mide los factores apertura a la experiencia, neuroticismo, extraversión, responsabilidad y DJUDGDELOLGDG&RQVWDGHtWHPVTXHVHUHVSRQGHQVHJ~QXQDHVFDODGHRSFLRQHV GHUHVSXHVWDTXHYDGH²completo desacuerdo²D²completo acuerdo²$ORV tWHPVORVDQWHFHGHXQDD¿UPDFLyQJHQHUDOYo me veo a mi mismo/a como alguien… TXHVHFRPELQDFRQODD¿UPDFLyQGHFDGDtWHPD¿QGHJHQHUDUFDGDRUDFLyQDVHU UHVSRQGLGD (MHPSORV GH tWHPV VRQ a quien le gusta hablar R TXH es curioso/a respecto a las cosas3RUVXVHVWXGLRVGHFRQ¿DELOLGDG\YDOLGH]VHFRQFOX\HTXH HO%),HVXQDPHGLGDYiOLGD\FRQ¿DEOH(QHVWDPXHVWUDSDUDFDGDIDFWRUODVDOIDV GH &URQEDFK IXHURQ H[WURYHUVLyQ DJUDGDELOLGDG UHVSRQVDELOLGDG QHXURWLFLVPRDSHUWXUDDODH[SHULHQFLDUHVSHFWLYDPHQWH Procedimiento y análisis de datos Los participantes completaron ambos instrumentos, IVyF y BFI, en formato de papel y lápiz. Los análisis de datos se realizaron correlacionando, mediante r de Pearson, las puntuaciones de las fortalezas del carácter medidas con el IVyF con las puntuaciones de las dimensiones de la personalidad del Big Five medida con HO%),/DVLJQL¿FDFLyQHVWDGtVWLFDVHFRQVLGHUyGHPRGRELODWHUDO Resultados En la Tabla 2 se muestran las correlaciones entre las puntuaciones de las fortalezas del carácter medidas con el IVyF y las puntuaciones de las dimensiones GHODSHUVRQDOLGDGVHJ~QHOPRGHORGHORV&LQFR)DFWRUHVPHGLGDFRQHO%),/RV DQiOLVLVHPStULFRVPXHVWUDQTXHORVUDVJRVGHSHUVRQDOLGDGHVWiQDVRFLDGRVDODV fortalezas del carácter. 109 Cosentino & Castro Solano \| Psicodebate 15 (2) \| 99–122 Tabla 2. Correlaciones entre Fortalezas del Carácter y Dimensiones de la Personalidad R A AE N E Amor .09 .22 .07 -.02 .24 Amor por saber .15 .08* .35 -.11 .05 Apertura Mental .27 .12 .14 -.15 -.08* Apreciación .18 .16 .35 -.06 .14 Autorregulación .34 .12 .06 -.17 -.00 Bondad .18 .47 .13 -.08* .16 Ciudadanía .12 .21 .04 -.09 .19 Clemencia .12 .38 .13 -.23** .00 Creatividad .09* .05 .57 -.13 .18 Curiosidad .21 .18 .29 -.25 .16 Esperanza .32 .22 .18 -.36 .13 Espiritualidad .24 .20 .03 -.03 .04 Gratitud .22 .29 .12 -.21 .15 Humildad .14 .25 -.16** -.09* -.29 Humor -.05 .10 .18 -.16 .36 Imparcialidad .12 .41 .15 -.21 .02 Integridad .31 .28 .07 -.14 -.04 Inteligencia Social .12 .15 .18 -.19 .48 Liderazgo .19 .17 .19 -.16 .25 Persistencia .59 .14 .06 -.12** .07* Perspectiva .19 .22 .20 -.22 .10 Prudencia .29 .32 .01 -.17 -.11 Valentía .13 -.01 .21 -.07 .30 Vitalidad .36 .21 .18 -.28 .26** Nota. Se destacan en negrita las correlaciones con tamaños del efecto superiores a mediano (r =.30; Cohen, 1992). Apreciación = apreciación de la belleza y la excelencia; R = responsabilidad; A. = agradabilidad; AE = apertura a la experiencia; N = neuroticismo; E = extraversión. * p < .05, bilateral. ** p < .01, bilateral. 110 Validez del IVyF Resumidamente, se presentan a continuación los resultados hallados en este HVWXGLR²HQWUHJXLRQHVVHGHWDOODQODVIRUWDOH]DVGHOFDUiFWHUTXHVHDVRFLDURQFRQ XQWDPDxRGHOHIHFWRPtQLPDPHQWHPHGLDQR &RKHQ FRQORVIDFWRUHVGHORV &LQFRUDQGHVRUGHQDGDVHQVXFHVLyQGHPRGRTXHHVWpQSULPHURODVIRUWDOH]DV GHOFDUiFWHUTXHWXYLHURQXQWDPDxRGHOHIHFWRPiVHOHYDGR²)XHURQGLUHFWDV todas las asociaciones de las fortalezas del carácter con el factor responsabilidad ²SHUVLVWHQFLD YLWDOLGDG DXWRUUHJXODFLyQ HVSHUDQ]D LQWHJULGDG² WRGDV ODV DVRFLDFLRQHV FRQ HO IDFWRU DJUDGDELOLGDG ²ERQGDG LPSDUFLDOLGDG FOHPHQFLD SUXGHQFLD² FDVL WRGDV ODV DVRFLDFLRQHV FRQ HO IDFWRU DSHUWXUD D OD H[SHULHQFLD ²FUHDWLYLGDGDPRUSRUHOVDEHU\DSUHFLDFLyQGHODEHOOH]D\ODH[FHOHQFLD²\ ODPD\RUtDGHODVDVRFLDFLRQHVFRQHOIDFWRUH[WUDYHUVLyQ¤²LQWHOLJHQFLDVRFLDO KXPRU \ YDOHQWtD$O FRQWUDULR IXHURQ LQYHUVDV WRGDV ODV DVRFLDFLRQHV KDOODGDV HQWUHODVIRUWDOH]DVGHOFDUiFWHU\ODGLPHQVLyQQHXURWLFLVPR²HVSHUDQ]D² Estudio 3: Relaciones del IVyF con una Medición de Deseabilidad Social /DGHVHDELOLGDGVRFLDOKDFHUHIHUHQFLDDTXHORVLQGLYLGXRVUHVSRQGHQ SHMDXQ cuestionario o en una entrevista) de un modo culturalmente apropiado y aceptable debido a su necesidad de obtener aceptación o aprobación de los demás (Crowne & 0DUORZH (QFRQVHFXHQFLDFXDQGRORVLQGLYLGXRVVHDWULEX\HQFDUDFWHUtVWLFDV FXOWXUDOPHQWHDSURSLDGDV\DFHSWDEOHV²LHVRFLDOPHQWHGHVHDEOHV²VHFRQMHWXUD la vinculación de sus respuestas con la deseabilidad social. 3HWHUVRQ \ 6HOLJPDQ KDQ D¿UPDGR TXH ODV IRUWDOH]DV GHO FDUiFWHU VRQ VRFLDOPHQWH GHVHDEOHV (VWD D¿UPDFLyQ SDUHFHUtD OOHYDU D OD FRQVHFXHQFLD OyJLFDGHTXHORVLQYHVWLJDGRUHVGHEHUtDQEXVFDU\KDOODUFRUUHODFLRQHVHQWUHODV IRUWDOH]DV\ODGHVHDELOLGDGVRFLDO&RQWUDULDPHQWHORVDXWRUHVGHODFODVL¿FDFLyQ 9,$ KDQ VRVWHQLGR TXH ODV IRUWDOH]DV GHO FDUiFWHU QR FRUUHODFLRQDQ FRQ OD GHVHDELOLGDGVRFLDO3HWHUVRQ\6HOLJPDQKDQPDQWHQLGRHVDD¿UPDFLyQVREUHXQ H[WUDFWRGHUHVXOWDGRV²LHUHVXOWDGRVTXHQRPXHVWUDQLQIRUPDFLyQGHWDOODGD TXHKDELOLWHODUHSOLFDFLyQGHOHVWXGLR²PRVWUDQGRDPRGRGHH[FHSFLyQTXHVROR GRVIRUWDOH]DVGHOFDUiFWHUSUHVHQWDURQDVRFLDFLRQHVFRQGHVHDELOLGDGVRFLDO²ODV cuales, sin embargo, presentaban una magnitud de correlación mediana o mayor a PHGLDQDHQWUHHVRVFRQVWUXFWRV &RKHQ 6H SURSRQH TXH KD\ XQ FLHUWR JUDGR GH LQFRQVLVWHQFLD HQ ODV SURSXHVWDV GH Peterson y Seligman (2004) sobre la vinculación entre las fortalezas del carácter \ODGHVHDELOLGDGVRFLDO3DUHFHUtDPiVFRQYHQLHQWHVXSRQHUTXHVLSRUGH¿QLFLyQ las fortalezas del carácter son socialmente deseables, entonces las fortalezas GHEHUtDQ WHQGHU HQ JHQHUDO D PRVWUDUVH HPStULFDPHQWH FRUUHODFLRQDGDV FRQ GHVHDELOLGDGVRFLDO(VWDFRQFOXVLyQTXHVHFRQVLGHUDPiVSDUVLPRQLRVDTXHODGH 111 Cosentino & Castro Solano \| Psicodebate 15 (2) \| 99–122 3HWHUVRQ \ 6HOLJPDQ SXHGH DSR\DUVH HPStULFDPHQWH HQ OR KDOODGR WDQWR SRU GRV LQYHVWLJDFLRQHVHPStULFDVLQGHSHQGLHQWHVTXHPRVWUDURQTXHPiVGHODPLWDGGHORV rasgos de carácter presentan asociaciones con la deseabilidad social (Macdonald HW DO 6DUURV &RRSHU FRPR SRU XQD LQYHVWLJDFLyQ UHDOL]DGD FRQ DGROHVFHQWHVTXHKDOOyXQUHVXOWDGRVHPHMDQWH 2VLQ (QRWURHVWXGLRVHKD KDOODGRTXHPiVGHXQWHUFLRGHODVIRUWDOH]DVFRUUHODFLRQDEDQFRQODVSXQWXDFLRQHV GHXQDHVFDODTXHIXHXVDGDSDUDHYDOXDUGHVHDELOLGDGVRFLDO 5XFKHWDO 3RUORWDQWRVHFRQVLGHUDTXHODSURSXHVWDTXHKDQVRVWHQLGRORVDXWRUHVGHOD FODVL¿FDFLyQ9,$FRQUHODFLyQDODYLQFXODFLyQHQWUHODVIRUWDOH]DVGHOFDUiFWHU y la deseabilidad social es criticable y no se halla apoyo sobre los hallazgos HPStULFRVPiVUHFLHQWHV Participantes (VWDPXHVWUDHVWXYRFRPSXHVWDSRUSDUWLFLSDQWHVGH$UJHQWLQDGHORVFXDOHV HUDQYDURQHV\HUDQPXMHUHV/DPHGLDGHHGDGIXHGHDxRVDT Instrumentos IVyF. 6H XWLOL]y HO ,9\) SDUD HYDOXDU ODV IRUWDOH]DV GHO FDUiFWHU VHJ~Q ODV GH¿QLFLRQHV\FODVL¿FDFLyQGH3HWHUVRQ\6HOLJPDQ Escala de Deseabilidad Social de Marlowe y Crowne (EDSCM). Para evaluar ODGHVHDELOLGDGVRFLDOVHXWLOL]yOD('6&0 &RVHQWLQR &DVWUR6RODQRD /D ('6&0 HV XQD DGDSWDFLyQ SDUD SREODFLyQ DUJHQWLQD GH OD HVFDOD FRPSOHWD 0DUORZH&URZQH6RFLDO'HVLUDELOLW\6FDOHTXHHVXQDXWRLQIRUPHGHVDUUROODGR SDUD HYDOXDU GHVHDELOLGDG VRFLDO LQGHSHQGLHQWHPHQWH GH SVLFRSDWRORJtD HQ VX IRUPDWR RULJLQDO GH SDSHO \ OiSL] &RQVWD GH tWHPV TXH VH FRQWHVWDQ SRU verdadero (V) o falso ) /D ('6&0 SURGXFH XQD VROD SXQWXDFLyQ WRWDO TXH VH REWLHQH GH VXPDU ODV SXQWXDFLRQHV GH FDGD tWHP GHELpQGRVH SUHYLDPHQWH LQYHUWLUODSXQWXDFLyQGHORVtWHPVGHSXQWXDFLyQUHYHUWLGD$PD\RUSXQWXDFLyQ PD\RUGHVHDELOLGDGVRFLDO/D('6&0FXHQWDFRQDGHFXDGDFRQVLVWHQFLDLQWHUQD YDOLGH]FRQUHODFLyQDODHVFDOD/GHO(\VHQFN3HUVRQDOLW\4XHVWLRQQDLUHYDOLGH] GLYHUJHQWHFRQODVHJXQGDHGLFLyQGHO,QYHQWDULRGH'HSUHVLyQGH%HFNYDOLGH]GH LQVWUXFFLRQHVGLIHUHQFLDOHV\YDOLGH]GHJUXSRVFRQRFLGRV¤ &RVHQWLQR &DVWUR 6RODQR /DFRQVLVWHQFLDLQWHUQDSDUDODVSXQWXDFLRQHVGHO('6&0GHODPXHVWUD GHOSUHVHQWHDQiOLVLVIXHGHĮ Procedimiento y análisis de datos /RVSDUWLFLSDQWHVFRPSOHWDURQORVLQVWUXPHQWRV,9\)\OD('6&0DPERVHQ su formato de papel y lápiz. Los análisis de datos se realizaron correlacionando, PHGLDQWHUGH3HDUVRQODVSXQWXDFLRQHVGHO,9\)TXHPLGHIRUWDOH]DVGHOFDUiFWHU 112 Validez del IVyF FRQODVSXQWXDFLRQHVGHOD('6&0TXHPLGHGHVHDELOLGDGVRFLDOLQGHSHQGLHQWH GHSVLFRSDWRORJtD/DVLJQL¿FDFLyQHVWDGtVWLFDVHFRQVLGHUyGHPRGRELODWHUDO Resultados Todas las fortalezas del carácter medidas con el IVyF se asociaron de forma SRVLWLYD\HVWDGtVWLFDPHQWHVLJQL¿FDWLYDFRQGHVHDELOLGDGVRFLDOHYDOXDGDDWUDYpV GHO('6&0 9HU7DEOD /RVUHVXOWDGRVGHHVWHHVWXGLRKDQUHVXOWDGRVLPLODUHVD ORVKDOODGRVHQODLQYHVWLJDFLyQGH0DFGRQDOGHWDO FRQXQDPXHVWUDGH HVWXGLDQWHVXQLYHUVLWDULRVTXHFRPSOHWDURQHO,3,39,$SDUDPHGLUIRUWDOH]DVGHO carácter, y una versión abreviada de la escala de deseabilidad social de Marlowe y &URZQH(VSDUWLFXODUPHQWHFRLQFLGHQWHHQWUHDPEDVLQYHVWLJDFLRQHVTXHFOHPHQFLD e imparcialidad presentaron las más elevadas correlaciones con deseabilidad social. Tabla 3. Correlaciones entre Fortalezas del Carácter y Deseabilidad Social Variable EDSCM Clemencia .39* Imparcialidad .36 Prudencia .35 Espiritualidad .31 Vitalidad .31 Gratitud .30 Integridad .30 Persistencia .29 Bondad .29 Perspectiva .29 Autorregulación .28 Esperanza .28 Inteligencia social .24 Humildad .23 Amor .22 Curiosidad .22 Ciudadanía .17 Apreciación .16 Apertura mental .16 113 Cosentino & Castro Solano \| Psicodebate 15 (2) \| 99–122 Tabla 3. Correlaciones entre Fortalezas del Carácter y Deseabilidad Social (continuación) Variable EDSCM Creatividad .14 Liderazgo .14 Humor .13 Amor por saber .12 Valentía .11 Nota. EDSCM = Escala de Deseabilidad Social de Crowne y Marlowe; Apreciación = apreciación de la belleza y la excelencia p < .01, bilateral. Estudio 4: Validez del IVyF en Relación con un Criterio Objetivo 3DUN \ 3HWHUVRQ KDQ SURSXHVWR TXH GHELGR D OD SRWHQFLDO GHVYHQWDMD SRUHOORVVXSXHVWDGHOYtQFXORHQWUHGHVHDELOLGDGVRFLDO\DXWRLQIRUPHGHUDVJRV SRVLWLYRVVHGHEHUtDREWHQHULQIRUPDFLyQGHP~OWLSOHVIXHQWHVSDUDODHYDOXDFLyQ GHODVIRUWDOH]DVGHOFDUiFWHUXQDGHODVFXDOHVVHUtDQORVREVHUYDGRUHVLQIRUPDQWHV L H IDPLOLDUHV DPLJRV R FRPSDxHURV TXH HYDO~HQ HO FDUiFWHU GH XQD SHUVRQD REMHWLYR(OSXQWRGHYLVWDGH3DUN\3HWHUVRQHVTXHHVDVP~OWLSOHVLQIRUPDFLRQHV REWHQLGDVVREUHODVIRUWDOH]DVGHOSDUWLFLSDQWHQRGHEHUtDQXWLOL]DUVHSDUDFUHDUXQ FRPSXHVWR~QLFRVLQRSDUDFUHDUXQDLPDJHQFRPSOHMDORVDXWRUHVOODPDQDHVWH procedimiento metodología 360 grados. Por su parte, Meyer et al. (2001) en relación con el autoinforme y al informe de ORVREVHUYDGRUHVKDQHQIDWL]DGRTXHFXDOTXLHUPpWRGR~QLFRGHPHGLFLyQSURYHH XQDUHSUHVHQWDFLyQSDUFLDORLQFRPSOHWDGHODFDUDFWHUtVWLFDTXHSUHWHQGHPHGLU \TXHODVGLIHUHQWHVIXHQWHVSURYHHQXQDLQIRUPDFLyQ~QLFD(QHVWHVHQWLGRVH KDOOyTXHORVDXWRLQIRUPHVGHSHUVRQDOLGDGSDUDDGXOWRVSUHVHQWDQGHSHTXHxDVD PRGHUDGDVDVRFLDFLRQHVFRQODVPLVPDVFDUDFWHUtVWLFDVPHGLGDVSRUREVHUYDGRUHV (Q 3VLFRORJtD 3RVLWLYD WDPELpQ KD VLGR DQDOL]DGD OD YLQFXODFLyQ HQWUH autopuntuaciones y puntuaciones de las evaluaciones de informantes. Un metaDQiOLVLVUHDOL]DGRVREUHHVWXGLRVDFHUFDGHOELHQHVWDU²TXHLQFOX\yORVFRQFHSWRV VDWLVIDFFLyQFRQODYLGDIHOLFLGDGDIHFWRSRVLWLYR\QHJDWLYR²KDOOyXQDPHGLD de correlaciones de r = .44 entre las puntuaciones de las autoevaluaciones y las HYDOXDFLRQHVGHORVLQIRUPDQWHV 6FKQHLGHU 6FKLPPDFN (VSHFtILFDPHQWH HQ UHODFLyQ FRQ OD PHGLFLyQ GH ORV UDVJRV SRVLWLYRV XQ HVWXGLR 5XFKHWDO XWLOL]DQGRODYHUVLyQDOHPDQDGHO9,$,6PRVWUyTXHOD DXWRHYDOXDFLyQ\ODHYDOXDFLyQUHDOL]DGDSRUREVHUYDGRUHVFRQYHUJtDQHQXQUDQJR 114 Validez del IVyF esperable, siendo la mediana de las 24 fortalezas del carácter de .40. Con relación al procedimiento de obtención de datos utilizado en el estudio de Ruch et al., la evaluación objetiva fue realizada en más del 50% de los casos por dos informantes. Los resultados de ese estudio mostraron la convergencia más elevada entre autopuntuación y puntuaciones de informante para la fortaleza religiosidad y la más EDMDSDUDLQWHJULGDG5XFKHWDOKDQVRVWHQLGRTXHODVFRQYHUJHQFLDVKDOODGDVHQVX investigación son comparables con las convergencias de las variables de personalidad, \ VRQ PiV HOHYDGDV TXH ODV FRQYHUJHQFLDV KDOODGDV SDUD OD YHUVLyQ GHO9,$,6 GH ((88HQWUHODVQRPLQDFLRQHVGHIRUWDOH]DVGHOFDUiFWHUUHDOL]DGDSRURWURV²DPLJRV \IDPLOLDUHV²\ODHVFDODFRUUHVSRQGLHQWHUV 3HWHUVRQ 3DUN Participantes 'RV PXHVWUDV GH SDUWLFLSDQWHV YROXQWDULRV GH$UJHQWLQD VH XWLOL]DURQ SDUD HVWH análisis: una muestra de sujetos observados y una muestra de sujetos observadores. La PXHVWUDGHORVSDUWLFLSDQWHVTXHFRQVWLWX\yHOFRQMXQWRGHVXMHWRVREVHUYDGRVHVWXYR FRQVWLWXLGDSRUSHUVRQDVPXMHUHV\KRPEUHVFRQXQSURPHGLRGHHGDGGH DxRVDT /DPXHVWUDTXHFRQVWLWX\yHOFRQMXQWRGHVXMHWRVREVHUYDGRUHV IXHGHSHUVRQDVPXMHUHV\YDURQHVFX\DHGDGSURPHGLRIXHGHDxRV '7 /RVREVHUYDGRUHVFRQRFtDQDORVVXMHWRVREVHUYDGRVGHVGHKDFtDXQDPHGLD GHDxRV'7 \ORVYtQFXORVVRFLDOHVFRQHOORVHUDQHQVXPD\RUtDFHUFDQRV ²QRYLD]JRRDPLVWDG²n RIDPLOLDUHVn HQWDQWRTXHXQDPLQRUtDGH SDUWLFLSDQWHVPDQWHQtDUHODFLRQHVODERUDOHVn 1LQJXQRGHORVSDUWLFLSDQWHVTXH fueron voluntarios, recibió retribución alguna por su participación. Instrumentos 6H XWLOL]y HO ,9\) SDUD TXH ORV SDUWLFLSDQWHV REVHUYDGRV DXWRLQIRUPHQ VXV IRUWDOH]DVGHOFDUiFWHUVHJ~QODFODVL¿FDFLyQ\GH¿QLFLRQHVGH3HWHUVRQ\6HOLJPDQ /DFRQVLVWHQFLDLQWHUQDGHHVWDPXHVWUDIXHGHĮ 6H XWLOL]y HO ,9\) HQ IRUPDWR LQIRUPDQWH SDUD TXH ORV SDUWLFLSDQWHV TXH constituyeron la muestra de observadores informen los rasgos positivos de los observados. El IVyF en formato informante es idéntico al IVyF original, excepto TXHODVLQVWUXFFLRQHV\ORVtWHPVHVWiQDGDSWDGRVGHPRGRWDOTXHVHUH¿HUHQDRWUD SHUVRQDHQYH]GHDXQRPLVPR(OFiOFXORGHFRQVLVWHQFLDLQWHUQDVREUHORVtWHPV GHO,9\)HQIRUPDWRLQIRUPDQWHGLRFRPRUHVXOWDGRXQĮ SDUDHVWDPXHVWUD Procedimiento y análisis de datos Para evitar la maximización de las asociaciones entre las puntuaciones provenientes de los observadores y de los observados, se eligió un procedimiento conservador consistente en utilizar un solo observador por sujeto, en lugar de 115 Cosentino & Castro Solano \| Psicodebate 15 (2) \| 99–122 varios observadores por sujeto (Meyer et al., 2001). En consecuencia, se utilizaron las puntuaciones aportadas por pares de participantes observadores-observados. $¿QGHUHVSRQGHUVHJ~QORVLQVWUXPHQWRVVHVLJXLyHOVLJXLHQWHSURFHGLPLHQWR XQ SDUWLFLSDQWH UHFLEtD XQD EDWHUtD FRPSXHVWD SRU ORV GRV LQVWUXPHQWRV mencionados en el apartado Instrumentos, en su formato de papel y lápiz, más XQD LQVWUXFFLyQ HVFULWD (VWD LQVWUXFFLyQ VROLFLWDED DO SDUWLFLSDQWH TXH FRPSOHWH HO,9\)²TXHGDQGRFRQVWLWXLGRFRPRVXMHWRREVHUYDGR²\TXHHOLJLHVHDXQD SHUVRQD ²IDPLOLDU SDUHMD R DPLJRD TXH FRQRFLHVH DO VXMHWR REVHUYDGR GHVGH KDFtD PiV GH XQ DxR² SDUD TXH FRPSOHWH HO ,9\) HQ IRUPDWR LQIRUPDQWH ² FXPSOLHQGRHVWD~OWLPDSHUVRQDHOSDSHOGHVXMHWRREVHUYDGRU² Se utilizaron las correlaciones r de Pearson para evaluar el grado de asociación entre las puntuaciones del IVyF y las puntuaciones del IVyF en formato LQIRUPDQWH/DVLJQL¿FDFLyQHVWDGtVWLFDVHFRQVLGHUyGHIRUPDELODWHUDO Resultados Las correlaciones entre las puntuaciones del autoinforme de fortalezas del carácter y las puntuaciones de las fortalezas provenientes del IVyF en formato LQIRUPDQWHSUHVHQWDURQXQDPHGLD\XQDPHGLDQDGHUHVSHFWLYDPHQWHFRQXQ UDQJRTXHVHH[WHQGLyGHVGHXQPi[LPRGHFRUUHODFLyQGHFRUUHVSRQGLHQWHD ODIRUWDOH]DHVSLULWXDOLGDGDXQPtQLPRGHFRUUHODFLyQGHFRUUHVSRQGLHQWHDOD IRUWDOH]DLQWHJULGDG YHU7DEOD /RVYDORUHVTXHVHKDOODURQHQHVWDLQYHVWLJDFLyQ VRQVLPLODUHVDODYH]TXHDOJRVXSHULRUHVDORVKDOODGRVSRUODLQYHVWLJDFLyQGH Ruch et al. (2010). Particularmente, ambos resultados coinciden en las fortalezas GHOFDUiFWHUTXHPXHVWUDQFRQYHUJHQFLDVPi[LPDV²HVSLULWXDOLGDG\DPRUSRUHO VDEHU² \ PtQLPD ²LQWHJULGDG² &RQ UHODFLyQ DO ,9\) ODV FRQYHUJHQFLDV PiV bajas entre las puntuaciones de las autoevaluaciones y las evaluaciones hechas por personas conocidas se encuentran en el rango de valores esperables, si se considera como punto de referencia a los valores correspondientes a las convergencias halladas para los constructos de personalidad (Meyer et al., 2001). Tabla 4. Correlaciones entre el Autoinforme y el Informe de Observador Escala del IVyF IVyF Espiritualidad .75 Amor por el Saber .58 Humildad .56 Amor .55 116 Validez del IVyF Tabla 4. Correlaciones entre el Autoinforme y el Informe de Observador Escala del IVyF IVyF Autorregulación .55 Ciudadanía .55 Valentía .54 Persistencia .54 Apreciación .53 Prudencia .51 Imparcialidad .51 Bondad .51 Apertura Mental .48 Gratitud .48 Clemencia .48 Curiosidad .48 Liderazgo .48 Creatividad .44 Esperanza .40 Inteligencia Social .39 Perspectiva .37 Vitalidad .37 Humor .36 Integridad .35 Nota. Apreciación = apreciación de la belleza y la excelencia. Discusión Las asociaciones halladas entre las fortalezas del carácter medidas con el ,9\)\ODVYDULDEOHVTXHIRUPDQSDUWHGHODUHGFRQFHSWXDOGHOFDUiFWHUFRPRHV conceptualizado por Peterson y Seligman (2004) son semejantes a lo encontrado en otras investigaciones. En relación con la satisfacción con la vida, los resultados de este estudio FRLQFLGHQGHIRUPDVXVWDQFLDOFRQORVKDOODGRVSRUODLQYHVWLJDFLyQGH3DUNHWDO (2004). Las siete correlaciones parciales más elevadas con satisfacción con la vida ²IRUWDOH]DV GHO FDUiFWHU JUDWLWXG FXULRVLGDG YLWDOLGDG HVSHUDQ]D SHUVLVWHQFLD DPRU\SHUVSHFWLYD²VRQODVPLVPDVSDUDDPEDVLQYHVWLJDFLRQHV En relación con las asociaciones halladas entre las fortalezas del carácter y OD SHUVRQDOLGDG VHJ~Q HO 0RGHOR GH ORV &LQFR UDQGHV ORV UHVXOWDGRV GH HVWD investigación vuelven a respaldar la propuesta por Peterson y Seligman (2004) de 117 Cosentino & Castro Solano \| Psicodebate 15 (2) \| 99–122 TXHODVIRUWDOH]DVGHOFDUiFWHUHVWiQDVRFLDGDVDORVUDVJRVGHSHUVRQDOLGDGGHO%LJ )LYH6LJXLHQGRVXDUJXPHQWDFLyQORVKDOOD]JRVHPStULFRVGHHVWDLQYHVWLJDFLyQ YXHOYHQDPRVWUDUTXHODFODVL¿FDFLyQGHUDVJRVSRVLWLYRV9,$WLHQHVHQWLGR /RVUHVXOWDGRVGHODLQYHVWLJDFLyQHPStULFDVRQPX\VLPLODUHVDORVUHVXOWDGRV HPStULFRV GH OD UHODFLyQ HQWUH ODV IRUWDOH]DV GHO FDUiFWHU GH OD FODVL¿FDFLyQ9,$ \ HO %LJ )LYH PRVWUDGRV SRU 3HWHUVRQ \ 3DUN \ D ORV UHVXOWDGRV KDOODGRV por Macdonald et al. (2008). En particular, todas las asociaciones informadas por 3HWHUVRQ\3DUNHQWUHODVIRUWDOH]DVGHOFDUiFWHU\HO%LJ)LYHVHKDOODURQHQHVWD LQYHVWLJDFLyQHPStULFD$GHPiVHQJHQHUDOFDGDJUXSRGHIRUWDOH]DVGHOFDUiFWHUTXH se hallaron vinculadas a cada factor del Big Five en la investigación de Macdonald et DO VHHQFRQWUDURQWDPELpQYLQFXODGDVHQHVWDLQYHVWLJDFLyQHPStULFD Por su parte, varias fortalezas del carácter están asociadas a más de un factor GHO%LJ)LYHDOLJXDOTXHORKDOODGRHQHOHVWXGLRGH0DFGRQDOGHWDO (Q este sentido, los resultados respaldan la sugerencia de los autores mencionados SUHYLDPHQWHGHTXHODSURSXHVWDGH3HWHUVRQ\6HOLJPDQGHTXHFDGDIRUWDOH]DGHO FDUiFWHUHVWDUtDUHSUHVHQWDGDSRUXQFRQVWUXFWRGHO%LJ)LYHHVWiSXHVWDHQFXHVWLyQ 6LQHPEDUJRHOH[DPHQGHFLHUWRVSXQWRVHQFRP~QHQWUHORVKDOOD]JRVGHOHVWXGLR GH 0DFGRQDOG HW DO \ HVWD LQYHVWLJDFLyQ SDUHFHUtD VRVWHQHU SDUFLDOPHQWH OD LGHD GH 3HWHUVRQ \ 6HOLJPDQ GH YLQFXODFLyQ ELXQtYRFD HQWUH IDFWRUHV GHO %LJ )LYH \ fortalezas del carácter. Al restringir el examen de los resultados de esta investigación \GHODLQYHVWLJDFLyQHPStULFDGH0DFGRQDOGHWDODODVYLQFXODFLRQHVPiVIXHUWHV entre los factores del Big Five y las fortalezas del carácter, surgen coincidencias sugerentes: el factor responsabilidad aparece vinculado con persistencia, vitalidad y autorregulación; el factor agradabilidad con bondad, clemencia e imparcialidad; el factor apertura a la experiencia con las fortalezas apreciación de la belleza y la excelencia, amor por el saber y curiosidad; el factor extraversión con humor, LQWHOLJHQFLD VRFLDO \ FLXGDGDQtD \ HO IDFWRU QHXURWLFLVPR FRQ HVSHUDQ]D (VWH análisis acotado de las relaciones entre Big Five y fortalezas del carácter muestra a diferentes factores del Big Five vinculados a diferentes fortalezas. Con relación a la deseabilidad social, los resultados de las investigaciones GH 0DFGRQDOG HW DO 6DUURV \ &RRSHU 2VLQ \ 5XFK HW DO DGHPiV GH ORV UHVXOWDGRV GH ORV DQiOLVLV GH HVWH HVWXGLR PXHVWUDQ TXH QR SXHGH VRVWHQHUVH OD D¿UPDFLyQ GH IDOWD GH DVRFLDFLyQ HQWUH ODV IRUWDOH]DV GHO carácter y deseabilidad social sugerida por Peterson y Seligman (2004). Más bien, UHFLEHUHVSDOGRHPStULFRHOUD]RQDPLHQWRGHTXHVLODVIRUWDOH]DVGHOFDUiFWHUVRQ VRFLDOPHQWHGHVHDEOHV²FRPRSURSXVLHURQ3HWHUVRQ\6HOLJPDQ²ODVSXQWXDFLRQHV de fortalezas deben tender, en general, a asociarse a deseabilidad social. Con relación a la validez relacionada con observadores objetivos, los resultados LQGLFDQTXHODVSXQWXDFLRQHVGHO,9\)FRQYHUJHQFRQODVHYDOXDFLRQHVUHDOL]DGDV 118 Validez del IVyF por informantes dentro del rango esperable para mediciones comparables de personalidad (Meyer et al., 2001). Además, el resultado de la convergencia del IVyF es similar al hallado para el VIA-IS (Ruch et al., 2010). 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En este sentido, futuros HVWXGLRV SRGUtDQ GHVDUUROODUVH FRQ PXHVWUDV GH SREODFLyQ JHQHUDO UXUDO R GH pueblos originarios rurales argentinos. &URQEDFK SURSXVRTXHODYDOLGDFLyQHVXQSURFHVRODUJR\WDOYH]VLQ ¿Q 6LJXLHQGR HVWH FRQFHSWR VH GHEHQ UHFRQRFHU OLPLWDFLRQHV TXH DSXQWHQ D IXWXURVHVWXGLRV3RUHMHPSORXQHVWXGLRSRGUtDDQDOL]DUODYDOLGH]FRQYHUJHQWH entre el IVyF y otras mediciones de fortalezas del carácter, como el del VIA-IS R HO ,3,39,$U$GHPiV SRGUtD LQYHVWLJDUVH OD YDOLGH] GH JUXSRV FRQRFLGRV para cada fortaleza del carácter, p. ej., comparando artistas y no artistas para la fortaleza del carácter apreciación de la belleza y la excelencia, los religiosos y los no religiosos para la fortaleza del carácter espiritualidad, etc. Finalmente, se han propuesto algunos métodos alternativos para calcular la FRQ¿DELOLGDG GH ORV LQVWUXPHQWRV FRQ XQ VROR tWHP SRU YDULDEOH FRPR XWLOL]DU OD IyUPXOD SDUD FRUUHFFLyQ GH OD DWHQXDFLyQ S HM:DQRXV 5HLFKHUV +XG\ KDFHUDQiOLVLVIDFWRULDOLQFRUSRUDQGRORVtWHPVGHORVLQVWUXPHQWRVODUJRV KRPyORJRV S HM 'HQLVVHQ HHQHQ 6HOIKRXW 9DQ$NHQ :DQRXV +XG\ :RRGV +DPSVRQ R XVDU PRGHODFLyQ OLQHDO MHUiUTXLFD LQFRUSRUDQGR HYDOXDFLRQHV KHFKDV SRU GLYHUVRV REVHUYDGRUHV 'HQLVVHQ HW DO (Q FRQVHFXHQFLD IXWXURV HVWXGLRV SRGUtDQ IRFDOL]DUVH HQ UHDOL]DU RWURV SURFHGLPLHQWRVSDUDHOFiOFXORGHODFRQ¿DELOLGDGGHO,9\) 119 Cosentino & Castro Solano \| Psicodebate 15 (2) \| 99–122 Referencias Castro Solano, A. (2000). Estilos de personalidad, objetivos de vida y satisfacción vital. Un estudio comparativo con adolescentes argentinos 7HVLV'RFWRUDO Universidad Complutense de Madrid, España. Castro Solano, A., & Casullo, M. M. (2001). Rasgos de personalidad, bienestar psicológico y rendimiento académico en adolescentes argentinos. ,QWHUGLVFLSOLQDULD5HYLVWDGH3VLFRORJtD\&LHQFLDV$¿QHV ± &RKHQ - $ SRZHU SULPHU 3V\FKRORJLFDO %XOOHWLQ ± GRL &RVHQWLQR $ & Evaluación de las virtudes y fortalezas humanas en población de habla hispana.3VLFRGHEDWH± Cosentino, A. C., & Castro Solano, A. (2008a). Adaptación y validación argentina GHOD0DUORZH&URZQH6RFLDO'HVLUDELOLW\6FDOHInterdisciplinaria Revista de 3VLFRORJtD\&LHQFLDV$¿QHV ± Cosentino, A. C., & Castro Solano, A. (2008b). Inventario de virtudes y fortalezas. Manuscrito sin publicar. &URQEDFK/- &RQVWUXFWYDOLGDWLRQDIWHUWKLUW\\HDUV(Q5(/LQQ (G ,QWHOOLJHQFH0HDVXUHPHQWWKHRU\DQGSXEOLFSROLF\ SS± 8UEDQD University of Illinois Press. &URZQH'3 0DUORZH' The approval motive: Studies in evaluative dependence.1HZ<RUN1<86-RKQ:LOH\ 6RQV 'DKOVJDDUG.3HWHUVRQ& 6HOLJPDQ0(3 6KDUHGYLUWXH7KH convergence of valued human strengths across culture and history. Review of HQHUDO3V\FKRORJ\ ±GRL 'HQLVVHQ --$ HHQHQ 5 6HOI KRXW 0 9DQ $NHQ 0$* 6LQJOHLWHPELJ¿YHUDWLQJVLQDVRFLDOQHWZRUNGHVLJQEuropean Journal of 3HUVRQDOLW\ 'LHQHU((PPRQV5$/DUVHQ5- ULI¿Q6 7KH6DWLVIDFWLRQ :LWK/LIH6FDOHJournal of Personality Assessment, 49 ±GRL VMSDB 120 Validez del IVyF DEOH6/ +DLGW- :KDW DQGZK\ LVSRVLWLYHSV\FKRORJ\"Review of General Psychology, 9 ±GRL ROGEHUJ/5 7KHVWUXFWXUHRISKHQRW\SLFSHUVRQDOLW\WUDLWVAmerican 3V\FKRORJLVW ±GRL; ROGEHUJ/5 $EURDGEDQGZLGWKSXEOLFGRPDLQSHUVRQDOLW\LQYHQWRU\ PHDVXULQJWKHORZHUOHYHOIDFHWVRIVHYHUDO¿YHIDFWRUPRGHOV(Q,0HUYLHOGH ,'HDU\)'H)UX\W )2VWHQGRUI (GV 3HUVRQDOLW\SV\FKRORJ\LQ(XURSH 9ROSS± 7LOEXUJ3DtVHV%DMRV7LOEXUJ8QLYHUVLW\3UHVV ROGEHUJ/5-RKQVRQ-$(EHU+:+RJDQ5$VKWRQ0&5REHUW& RXJK+* 7KH,QWHUQDWLRQDO3HUVRQDOLW\,WHP3RRODQGWKHIXWXUH of public-domain personality measures. Journal of Research in Personality, 40 ±GRLMMUS +HOPV - ( +HQ]H . 7 6DVV 7 / 0LIVXG 9 $ 7UHDWLQJ &URQEDFK¶V $OSKD 5HOLDELOLW\ &RHI¿FLHQWV DV 'DWD LQ &RXQVHOLQJ 5HVHDUFK The Counseling Psychologist, 34 ±GRL /LQOH\3$0DOWE\-:RRG$0-RVHSK6+DUULQJWRQ63HWHUVRQ&« 6HOLJPDQ0(3 &KDUDFWHUVWUHQJWKVLQWKH8QLWHG.LQJGRP7KH VIA Inventory of Strengths. Personality and Individual Differences, 43(2), ±GRLMSDLG 0DFGRQDOG&%RUH0 0XQUR' 9DOXHVLQDFWLRQVFDOHDQGWKH%LJ 5: An empirical indication of structure. Journal of Research in Personality, ±GRLMMUS 0H\HU-)LQQ6((\GH/'.D\0RUHODQG./'LHV55 «5HHG0 3V\FKRORJLFDOWHVWLQJDQGSV\FKRORJLFDODVVHVVPHQW A review of evidence and issues. $PHULFDQ 3V\FKRORJLVW ± GRL; 2VLQ ( 1 6RFLDO GHVLUDELOLW\ LQ SRVLWLYH SV\FKRORJ\ ELDV RU GHVLUDEOH VRFLDOLW\"(Q7)UHLUH (G Understanding positive life: Research and practice on positive psychology SS± /LVERD3RUWXJDO&OLPHSVL(GLWRUHV 3DUN1 3HWHUVRQ& 0HWKRGRORJLFDOLVVXHVLQSRVLWLYHSV\FKRORJ\DQG WKHDVVHVVPHQWRIFKDUDFWHUVWUHQJWKV(Q$'2QJ 0+0YDQ (GV Oxford handbook of methods in positive psychology SS± 1HZ<RUN NY, US: Oxford University Press. 121 Cosentino & Castro Solano \| Psicodebate 15 (2) \| 99–122 3DUN 1 3HWHUVRQ & 6HOLJPDQ 0 ( 3 6WUHQJWKV RI FKDUDFWHU DQG ZHOOEHLQJ -RXUQDO RI 6RFLDO DQG &OLQLFDO 3V\FKRORJ\ ± GRLMVFS 3HWHUVRQ & 3DUN 1 &ODVVL¿FDWLRQ DQG 0HDVXUHPHQW RI &KDUDFWHU Strengths: Implications for Practice. En P. A. Linley S. Joseph (Ed.), Positive psychology in practice SS± +RERNHQ1-86-RKQ:LOH\ 6RQV Inc. Peterson, C., & Seligman, M. E. P. (2004). Character strengths and virtues: A KDQGERRNDQGFODVVL¿FDWLRQ1HZ<RUN1<862[IRUG8QLYHUVLW\3UHVV 3HWHUVRQ & 3DUN 1 3ROH 1 '¶$QGUHD : 6HOLJPDQ 0 ( 3 Strengths of character and posttraumatic growth. Journal of Traumatic Stress, ±GRLMWV 5XFK:3UR\HU57+DU]HU&3DUN13HWHUVRQ& 6HOLJPDQ0(3 Values in Action Inventory of Strengths (VIA-IS): Adaptation and validation of the German version and the development of a peer-rating form. Journal of ,QGLYLGXDO'LIIHUHQFHV ±GRLD 6DUURV-& &RRSHU%. %XLOGLQJFKDUDFWHU$OHDGHUVKLSHVVHQWLDO Journal of Business and Psychology ± GRLV 6FKQHLGHU / 6FKLPPDFN 8 6HOILQIRUPDQW DJUHHPHQW LQ ZHOO being ratings: A meta-analysis. Social Indicators Research, 94 ± GRLV\ 6HOLJPDQ 0 ( 3 &VLNV]HQWPLKDO\L 0 3RVLWLYH SV\FKRORJ\ An introduction. $PHULFDQ 3V\FKRORJLVW ± GRL ; :DQRXV -3 +XG\ 0- 6LQJOHLWHP UHOLDELOLW\ $ UHSOLFDWLRQ DQG extension. 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https://openalex.org/W4213043068	http://deturope.eu/doi/10.32725/det.2015.008.pdf	Croatian	null	Food Product Processing in the Frame of Farming-Practice in Hungary, Opportunities in Serbia	Deturope	2,015	cc-by	3,669	Apstrakt Prerada poljoprivrednih proizvoda u okviru poljoprivrednog gazdinstva u Sbiji u pravnom smislu nije precizno definisana. U ovom radu predstavljena je praksa prerade poljopivrednih proizvoda u Mađarskoj u okviru domaćinstva i analizirani su zakonski propisi Republike Srbije koji se odnose na ove mogućnosti. Kao rezultat rada autori su predložili moguće korake koji mogu pomoći u regulisanju ove oblasti. Ključne reči: Tradicionalni proizvođač, proizvodnja u okviru domaćinstva, sledljivost Abstract Food product processing in the frame of farming in legal aspect does not precisely defined in Serbia. In this study is introduced Hungarian agricultural product processing in registered farms/househols, and then analyzed the legislations in Republic of Serbia related to this issue. As a result of study, the authors recommended some possible steps for regulation of this domain. p p g Keywords: primary producer, production within the household, traceability. DETUROPE – THE CENTRAL EUROPEAN JOURNAL OF REGIONAL DEVELOPMENT AND TOURISM Vol. 7 Issue 1 2015 ISSN 1821-2506 DETUROPE – THE CENTRAL EUROPEAN JOURNAL OF REGIONAL DEVELOPMENT AND TOURISM Vol. 7 Issue 1 2015 ISSN 1821-2506 Hajnalka KOVÁCS SÁRKÁNYa, Vilmos KOVÁCSa a University of Debrecen, AGTC, Hankóczy Jenő Doctoral School of Crop, Horticulture and Food Sciences,Böszörményi út 138, 4015 Debrecen, phone: +381(0) 63460 797, +381(0) 63 580 729, e- mail: hajnalka.kovac@gmail.com, vilmos.kovac@gmail.com Cite this article: Kovács Sárkány, H., Kovács, V. (2015). Prerada prehrambenih proizvoda u okviru gazdinstva - praksa u Mađarskoj, mogućnosti u Srbiji. Deturope, 7, 1: 110-119 CILJ RADA Cilj rada je analiza zakonskih odredaba vezanih za proizvodnju prehrambenih proizvoda u okviru domaćinstva. Analizama zakonskih odredaba Mađarske želimo da damo uvid u praksu Mađarske. Takođe, želimo da predstavimo zakonske propise iz 2014. godine koji su na snazi u Republici Srbiji i koji se odnose na preradu poljoprivrednih proizvoda u okviru domaćinstva. Kao zaključak ovog rada, daćemo sugestije za mogućnosti prerade poljoprivrednih proizvoda, koje ne bi otežavale proizvodnju proizvođačima, bile bi lako primenjive, a u mnogome bi pomogle nadležnim institucijama u toku kontrole i oporezivanja delatnosti. Cilj rada je analiza zakonskih odredaba vezanih za proizvodnju prehrambenih proizvoda u okviru domaćinstva. Analizama zakonskih odredaba Mađarske želimo da damo uvid u praksu Mađarske. Takođe, želimo da predstavimo zakonske propise iz 2014. godine koji su na snazi u Republici Srbiji i koji se odnose na preradu poljoprivrednih proizvoda u okviru domaćinstva. Kao zaključak ovog rada, daćemo sugestije za mogućnosti prerade poljoprivrednih proizvoda, koje ne bi otežavale proizvodnju proizvođačima, bile bi lako primenjive, a u mnogome bi pomogle nadležnim institucijama u toku kontrole i oporezivanja delatnosti. UVOD U poslednje vreme se sve više posvećuje pažnja specifičnim prehrambenim proizvodima karakterističnim za određeno podneblje. Osnovno pitanje u Srbiji je koji proizvodi i na koji način se mogu prerađivati u okviru poljoprivrednog gazdinstva, da bi se mogli prodavati na tržištu. Za uporedjivanja koristiće se iskustva iz Mađarske gde registrovani poljoprivredni proizvođači, imaju pravo da prerađuju i puštaju u promet proizvode koji potiču iz sopstvene proizvodnje. 110 Sárkány, H., Kovács, V. Zakonski okvir u Mađarskoj Legitimacija i pravila za dozvolu za prodaju Svaki poljoprivredni proizvođač u Mađarskoj, ukoliko želi da svoje primarne proizvode ili prerađevine tih proizvoda prodaje potrošačima, mora da poseduje važeću, takozvanu Poljoprivrednu legitimaciju - "Őstermelői igazolvány". Da bi dobio legitimaciju, poljoprivredni proizvođač/poljoprivredno gazdinstvo treba da se registruje kod Nacionalne Agrarnoekonomske Komore (Nemzeti Agrárgazdasági Kamara) na osnovu Zakona o Mađarskoj komori za agrar, prehrambeno ekonomsku oblast i za ruralni razvoj (2012. évi CXXVI. Törvény). Članstvo u komori je obaveza i za organizacije-kompanije koje se bave proizvodnjom ili preradom poljoprivrednih proizvoda. Ukoliko je ovakva organizacija već član neke druge komore, na primer, Veterinarske komore ili Komore za Zaštitu bilja, nije u obavezi da bude i član pomenute komore. Proizvođačima poljoprivrednih proizvoda i proizvoda izrađenih u okviru domaćinstva se izdaje Poljoprivredna legitimacija na osnovu odredaba Regulative Vlade o Poljoprivrednim legitimacijama (228/1996. (XII. 26.). Svakom tradicionalnom proizvođaču dodeljuje se jedinstveni registracioni broj. Legitimacija se izdaje na maksimalno tri godine, a na zahtev poljoprivrednog proizvođača, s tim, da se za svaku godinu posebno izdaje formular - dodatak legitimaciji, u kom se vodi evidencija o prihodima gazdinstva. Na osnovu Zakona o ličnom dohotku građana (1995. évi CXVII. törvény), svaki poljoprivredni proizvođač i prerađivač u domaćinstvu (u daljem tekstu tradicionalni 111 Sárkány, H., Kovács, V. proizvođač), je u obavezi da prilikom prodaje proizvoda, kupcu izda priznanicu. Izdavanje priznanice se strogo kontroliše od strane Nacionalne uprave za oporezivanje i carinjenje. Prihod ispod 600.000 HUF (cca. 2.000,00 EUR) se ne oporezuje. Poljoprivredno gazdinstvo, čiji su članovi supružnici, od kojih je jedno u radnom odnosu kod drugogog poslodavca, ima pravo na dodatni prihod od 600.000 HUF koji se ne oporezuje. Pravo na Poljoprivrednu legitimaciju imaju samo oni članovi domaćinstva koji su u direktnoj rodbinskoj vezi (otac, majka, sin, ćerka) i imaju istu adresu stanovanja. Ukoliko poljoprivredni proizvođač nema stalno zaposlenje, poljoprivredna proizvodnja se automatski tretira kao glavna delatnost i tada plaća doprinose. Obaveza plaćanja doprinosa odnosi se i na članove gazdinstva koji su upisani na Legitimaciji gazdinstva pri registraciji, a nisu u radnom odnosu (izuzev penzionera). Pravo da prodaju proizvode imaju samo oni članovi gazdinstva koji su upisani u Legitimaciji gazdinstva. Svake godine tradiocionalni proizvođači moraju prijaviti komori svoje prihode. Predstavnik komore pregledava dodatak legitimaciije i na osnovu visine neto prihoda određuje naknadu za komoru. Minimalan iznos je 2.000 HUF (cca. 6,5 EUR) koji se plaća ako neto prihod tradiocionalnog proizvođača ne prekorači 600.000 HUF. Zakonski okvir u Mađarskoj Prihode proizvođača predstavnik komore kontroliše i kod Nacionalne uprave za oporezivanje i carinjenje (Nemzeti adó- és vámhivatal). Pravila u vezi prodaje Prva zakonska regulativa u Mađarskoj koja se odnosi na tradicionalne proizvođače datira iz 2006. godine: Regulativa 14/2006. (II. 16.) o uslovima proizvodnje, prerade i prodaje proizvoda proizvedenih na gazdinstvu. Uzimajući u obzir zahteve potrošača i na inicijativu pedeset i dve nevladine organizacije ova regulativa pod oznakom "52/2010. (IV. 30.) FVM" je izmenjena 2010. godine.Izdata je još jedna regulativa, koja definiše uslove vezane za proizvodnju i prodju u okvru domaćinstva. Ova regulativa (4/2010. (VII.5.) VM) sadrži detalje u vezi poslovanja tradicionalnih proizvođača. Regulative se temelje na Zakonu o snabdevačkom lancu hrane i službenom nadzoru u kom je predviđeno objedinjeno osnivanje i organizovanje kontrole lanca ishrane. U uredbi 52/2010 (IV. 30.) FVM predviđena su detaljna pravila koja se odnose na tradicionalne proizvođače. (4/2010. (VII.5.) VM) Tradicionalnim proizvođačima se smatraju ona lica, koja neposredno prodavaju proizvode koje su sami proizveli ili sakupili u krugu od 40 km od 112 Sárkány, H., Kovács, V. Sárkány, H., Kovács, V. mesta gazdinstva. Kupci mogu biti svi potrošači, uključujući maloprodajne i ugostiteljske objekte. Proizvode biljnog porekla i med, tradicionalni proizvođač ima pravo da prodaje na sopstvenom gazdinstvu, na celoj teritoriji Mađarske na pijacama, vašarima, i na svim odobrenim privremenim mestima za maloprodaju. Proizvode animalnog porekla tradicionalni proizvođač ima pravo da prodaje na sopstvenom gazdinstvu, u Budimpešti i na pijacama, vašarima, i na svim odobrenim privremenim mestima za maloprodaju u krugu udaljenom 40 km od mesta gazdinstva. Proizvođač mora posedovati Veterinarsko uverenje kojim se potvrđuje adekvatno zdravstveno stanje životinja. Pregled mesa od strane nadležnog lica je obavezan, ukoliko se meso ne prodaje direktno sa gazdinstva, nego se njime snabdeva ugostiteljski objekat ili maloprodaja. Sveže meso mora da potiče od životinja iz sopstvenog stada, da životinje budu zaklane na klanici koja je odobrena od strane nadležnih tela. Ovakvo meso moguće je prodavati isključivo na gazdinstvu. Klanje pilića i kunića može se vršiti i na gazdinstvu, ali je potrebno obavestiti nadležno telo. Kontrola proizvodnje proizvoda animalnog porekla kao i proizvoda biljnog porekla se vrši na osnovu procene rizika. Prerađivači proizvoda animalnog porekla mogu da očekuju godišnju kontrolu od strane inspekcijskih organa. Regulativa određuje maksimalne količine proizvoda koje tradicionalni proizvođač može da prodaje. Sárkány, H., Kovács, V. Ukoliko se proizvod prodaje zapakovan, u tom slučaju proizvod mora da je označen deklaracijom. Na deklaraciji pored podataka o imenu tradicionalnog proizvođača, adrese, naziva proizvoda, roka trajanja, temperture skladištenja, ukoliko je to primenjivo i težini, moraju da budu i navedene sve odredbe pravilnika o deklarisanju ukoliko se proizvod prodaje u maloprodajnim objektima. Regulativa određuje i bliže uslove vezane za uslove proizvodnje. Pravila u vezi prodaje U nastavku izdvajamo primere: - nedeljno šest, a godišnje maksimalno 72 komada svinja, ovaca, koza - nedeljno maksimalno 200 kokošaka, 100 ćuraka - može proizvesti nedeljno 70 kg, a godišnje 2600 kg prerađevina od mesa - godišnje 20 000 kg proizvoda biljnog porekla - nedeljno 150, a godišnje 5200 kg termički obrađenih proizvoda biljnog porekla. - nedeljno 150, a godišnje 5200 kg termički obrađenih proizvoda biljnog porekla. Tradicionalni proizvođač ima pravo i da pruža usluge i to dimljenje, sušenje, mlevenje mesa, pripremanje hleba, ceđenje ulja, ceđenje i pasterizacija sokova, itd. Tradicionalni proizvođač ima pravo i da pruža usluge i to dimljenje, sušenje, mlevenje mesa, pripremanje hleba, ceđenje ulja, ceđenje i pasterizacija sokova, itd. Pored obaveznog primenjivanja sistema sledljivosti proizvoda, tradicionalni proizvođači, za prerađevine, moraju izraditi specifikacije proizvoda. Pored toga moraju voditi ecidenciju o proizvedenoj količini, datumu proizvodnje i o tome gde i koliko je prodato tog proizvoda. Ova evidencija mora da bude na mestu prodaje i čuva se dve godine. 113 Zakonske regulative u Srbiji Slika u vezi zakonskih regulativa u Srbiji nije jednoznačna. Poljoprivredni proizođači upisuju se u Registar poljoprivrednih gazdinstva, na osnovu Pravilnika o upisu u registar poljoprivrednih gazdinstava i obnovi registracije, kao i o uslovima za pasivan status poljoprivrednog gazdinstva (2013), (registracija nije obavezna), međutim prilikom godišnje registracije upisuje se i vodi se samo evidencija o vrsti biljnih kultura i o vrsti stočnog fonda. Fizičko lice upisano u Registar poljoprivrednih gazdinstava shodno odredbama Uredbe o registru poljoprivrednih gazdinstava (2008) automatski postaje upisano u Centralni registar. Nije precizno definisano da li, šta i gde mogu prodavati registrovani poljoprivredni proizvođači primarnih proizvoda i/ili njihovih prerađevina. U definisanju ove problematike pomažu pravilnici koji se odnose na pijačnu prodaju. U Pravilniku o bližim uslovima koji obezbeđuju higijensko postupanje sa životnim namirnicama i mogućnost zdravstvenog nadzora nad prometom van prostorija određenih za prodaju (1976) koji je na snazi od 1976. Godine, definisano je da na pijačnim tezgama pored prodaje voća i povrća, mleka i mlečnih proizvoda, jaja i meda, prodaje se i testenina. U Pravilniku o minimalnim tehničkim uslovima za obavljanje prometa robe i vršenje usluga u prometu roba (1996) definisano je da "na zelenim pijacama, na otvorenim pijačnim tezgama i sličnim objektima obavlja se promet na malo: poljoprivredno- prehrambenih proizvoda (svežeg i sušenog voća, povrća, šumskih plodova i jaja)". Pijace kao prostor za prodaju proizvoda prepoznaje i Pravilnik o veterinarsko- sanitarnim uslovima u objektima za prodaju proizvoda životinjskog porekla van poslovnih prostorija (1994). Pod objektima za prodaju proizvoda životinjskog porekla, smatraju se stalni objekti na pijacama i privremeni objekti na vašarima, ulicama, izletištima i sl. 114 Sárkány, H., Kovács, V. Fizičko lice koje proizvodi mleko i proizvode od mleka u domaćinstvu koji su namenjeni za javnu potrošnju mogu da se prodaju na mestu proizvodnje ili pijacama samo ako potiču iz objekata koji su upisani u Registar objekata, odnosno u Registar odobrenih objekata." Trenutnu situaciju u Srbiji najbolje predstavlja "Pijačni red" Grada Kragujevca (Odluka o pijacama, 1998) u kom se dozvoljava prodaja na tezgama primarnih poljoprivredno- prehrembenih proizvoda, u paviljonima za prodaju mlečnih proizvoda i to: mlečni 115 Sárkány, H., Kovács, V. proizvodi (sir i kajmak), testenine, zaklana živina po zakonom propisanim uslovima, sušeni mesni proizvodi i prerađevine od mesa sa uverenjem o transportu i deklaracijom. "Poljoprivredno-prehrambene proizvode na tezgama na zelenim pijacama mogu prodavati individualni poljoprivredni proizvođači i članovi porodičnog domaćinstva. Konzumna jaja, mogu prodavati lica koja poseduju, važeću sanitarnu knjižicu, ovlašćenje za prodaju, validne deklaracije, važeću analizu, transportni list (u slučaju da dolaze iz druge opštine), ukoliko se prodaju tuđi proizvodi moraju imati registrovan STR (Samostalna trgovinska radnja). Mlečne proizvode (sir i kajmak) mogu prodavati individualni poljoprivredni proizvođači, uz dokaz da se bave proizvodnjom. Prodaja mlečnih proizvoda vrši se iz rashladnih vitrina u paviljonima za prodaju mlečnih proizvoda. Prodavci mlečnih proizvoda su obavezni da poseduju mlečnu kartu, sanitarnu knjižicu, rešenje o registrovanom objektu , transportni list (ukoliko dolaze iz druge opštine) radnu uniformu (beli mantil ili bluzu, kecelju, narukvice, kapu ili maramu). Hleb i peciva, meso i mesne prerađevine, mleko i mlečne proizvode, testenine mogu prodavati preduzetnici (pravna lica) i to na prostorima namenjenim za ovu vrstu prodaje (lokali, adaptirana pijačna mesta), uz priloženo rešenje o registraciji Preduzeća ili STR-a. Poljoprivredno-prehrambeni proizvodi koji se prodaju na pijačnim tezgama, namenjeni ljudskoj ishrani prodaju se, po pravilu u neprerađenom stanju." Preuzimanjem evropskih regulativa izdat je Pravilnik o veterinarsko-sanitarnim uslovima, odnosno opštim i posebnim uslovima za higijenu hrane životinjskog porekla, kao i o uslovima higijene hrane životinjskog porekla (2011) u kojem se propisuju bliži veterinarsko-sanitarni uslovi, koji moraju da ispunjavaju objekti u kojima se obavlja delatnost klanja životinja. Ovaj pravilnik prepoznaje objekat na gazdinstvu za proizvodnju mleka i kolostruma; odreuđuje i uslove u pogledu higijene klanja na gazdinstvu. Sárkány, H., Kovács, V. Po Zakonu o trgovini (2010), pijačna prodaja obuhvata prodaju robe naročito na tezgama, boksovima ili posebnim prodajnim objektima i to svežih poljoprivrednih i prehrambenih proizvoda i proizvoda domaće radinosti i zanatskih proizvoda. Zakon ne specificira šta se podrazumeva pod proizvodima domaće radinosti i zanatskih proizvoda, ali daje definiciju proizvođača: "proizvođač je pravno lice, preduzetnik ili fizičko lice koje izrađuje proizvod ili se u tom svojstvu predstavlja stavljanjem na proizvod svog poslovnog imena, imena ili naziva, žiga ili druge prepoznatljive oznake ili načina". Pored uslova za prodaju potrebno je uzeti u obzir i regulative vezane za bezbednost i sledljivost hrane. U Pravilniku o uslovima higijene hrane (2010 - primenjuje se od juna 2011. godine) bliže se propisuju uslovi higijene hrane za sve subjekte u poslovanju hranom u svim fazama proizvodnje, prerade i prometa hrane koji su pod njihovom kontrolom uključujući i propise vezane za objekat. Ipak, odredbe pravilnika ne primenjuju se na: "direktno snabdevanje malim količinama primarnih proizvoda kojima proizvođač snabdeva krajnjeg potrošača ili lokalni objekat u maloprodaji koji direktno snabdeva krajnjeg potrošača". Pravilnik se poziva na poseban propis, koji se odnosi na ove slučajeve, a koji još nije izdat. Nadležni organ za kontrolu je Ministarstvo poljoprivrede i zaštite životne sredine, odnosno Ministarstvo zdravlja Republike Srbije. Zakonom o bezbednosti hrane (2009) uređuju se opšti uslovi za bezbednost hrane i hrane za životinje, obaveze i odgovornosti subjekata u poslovanju hranom i stočnom hranom, sistem brzog obaveštavanja i uzbunjivanja, hitne mere i upravljanje kriznim situacijama, higijena i kvalitet hrane i hrane za životinje. Odredbe ovog zakona se ne odnose na primarnu proizvodnju hrane, pripremu, rukovanje, odnosno skladištenje hrane za potrebe sopstvenog domaćinstva, kao i na hranu za životinje koje ne služe za proizvodnju hrane. To bi značilo da se odnosi na proizvodnju hrane koju vrši tradicionalni proizvođač. Zakon o veterinarstvu (2009) jasno određuje sledeće: "Izvan odobrene klanice može da se obavlja i klanje svinja, ovaca, koza, živine i kunića ako su namenjeni za upotrebu u domaćinstvu. ZAKLJUČAK Prerada poljoprivrednih proizvoda u okviru registrovanog gazdinstva omogućena je u Mađarskoj ukoliko prerađivač zadovoljava minimalne higijenske zahteve. Praksa iz Mađarske može nam služiti kao dobar primer. Prerada poljoprivrednih proizvoda u okviru registrovanog domaćinstva u Srbiji još nije dovoljno pravno precizirana. Kao prvi i osnovni korak, pored formiranja odgovarajućih zakonskih odredaba, potrebna je evidencija onih poljoprivrednih gazdinstva na kojima se 116 Sárkány, H., Kovács, V. vrši prerada poljoprivrednih proizvoda. To bi se moglo uraditi prilikom registracije poljoprivrednog gazdinstva. Kontrola od strane ispekcijskih organa mogla bi da se vrši i na mestu prodaje. Sa ovim potezom i nadležna tela imala bi uvid u trenutnu situaciju. vrši prerada poljoprivrednih proizvoda. To bi se moglo uraditi prilikom registracije poljoprivrednog gazdinstva. Kontrola od strane ispekcijskih organa mogla bi da se vrši i na mestu prodaje. Sa ovim potezom i nadležna tela imala bi uvid u trenutnu situaciju. Svakako potrebno je formirati zakonske odredbe vezane za uslove proizvodnje u okviru registrovanog poljoprivrednog gazdinstva. Kao rezultat ovog postupka pojaviće se i potreba inspekcijske kontrole ovih proizvoda. Takođe potrebno je definisanje poreskih obaveza ovih proizvođača. Uvođenjem obaveznog izdavanja priznanica na pijacaman i na svim ostalim mestima prodaje inspekcija bi imala uvid u sledljivost, u količine i u vrste prodate robe. Smatramo da bez stroge kontrole izdavanja priznanica sistem ne bi funkcionisao. SUMMARY Practice from Hungary might be served as a good example Practice from Hungary might be served as a good example. As a first and basic step, besides forming of the appropriate legislations, it is necessary to acquire recordings related to such agricultural farms on which perform processing of agricultural products. This could be done during the registration process of the agricultural farms. Control by the inspection authority would be placed at sale outlets. Owing to this move, authority would be had insight into the current situation. Also, there is a need to define tax liability for these manufacturers. Owing to the introduction of mandatory issuance of receipts on the marketplaces and all other places of vending, inspection would be had insight into traceability, into the amount and type of sold products. Of course, without strict control, the issuance of receipts system would not be working. SUMMARY The household food processing laws and requirements are confirmed by appropriate legislations in Hungary. In order to someone to be qualified for food sale in the frame of household farming, it has to possess „Primary producer license“. The license could be acquired through the registration process at the National Chamber of Agriculture. With this the producer will be become the member of the National Chamber of Agriculture, which involves yearly members’ fee what should be paid. The Primary producer license is issued for 3 years at most, but the inserts are issued on yearly basis related to it, so the producer has to record its incomes of the farm, on the basis of issued receipts. The receipts issued are controlled by the National Tax and Custom House. The farm should not have to pay taxes on income under 2,000 EUR. Facilitating both the husband and the wife may register themselves as members of the farm, as long as they permanent employees. In this case they have to pay tax on income above 4,000 EUR. As the representatives of the farm in the sale outlets only relatives of the registered farm owner with the same address are allowed to present. As long as the farm owner is unemployed, so the farming will be its main activity automatically by the farm registration, therefore compulsory contributions should be paid. The Chamber membership fee is determined in accordance with the income of the farm on the basis of insert data. The minimal membership fee is 6.5 EUR. nsert data. The minimal membership fee is 6.5 EUR. The farms or estates their products entitled to vend typically in Budapest and at most 40 km radius from the resident. Actual legislations exactly determined such quantities, which registered farms are allowed to produc on weekly or yearly level. Authority can trace the vended quantities according to the insert data. y y y y q g The legislations are not unequivocal in Serbia related to the household processing of the agricultural products; the legislations just tangentially mentioned the range of these products. These legislations are mostly concerns on the market, on food safety and on veterinary. The milk products household processing make an exception from this, since this issue is regulated by law. There is a need for full law regulation related to the household food processing in Serbia. 117 117 Sárkány, H., Kovács, V. Zakon o bezbednosti hrane ("Sl. glasnik RS", br. 41/2009) LITERATURA 14/2006. (II. 16.) FVM-EüM-ICsSzEM együttes rendelet a kistermelői élelmiszer- termelés, -előállítás és -értékesítés feltételeiről - Objedinjena regulativa o uslovima proizvodnje, prerade i prodaje proizvoda izrađenim u od strane tradioinalnih proizvođača p 1995. évi CXVII. törvény a személyi jövedelemadóról- Zakon o ličnom dohotku 2008. évi XLVI. törvény az élelmiszerláncról és hatósági felügyeletéről- Zakon o lancu hrane i službenom nadzoru É 2012. évi CXXVI. Törvény a Magyar Agrár-, Élelmiszergazdasági és Vidékfejlesztési Kamaráról-Zakon o Mađarskoj agrarno, prehrambeno ekonomskoj i komri za ruralni razvoj j 228/1996. (XII. 26.) Kormány rendelet a mezőgazdasági őstermelői igazolványról- Regulativa Vlade o Poljoprivrednim legitimacijama 4/2010 (VII.5) VM rendelet a kistermelői élelmiszer-termelés, -előállítás és -értékesítés feltételeiről- Regulativa o uslovima proizvodnje, prerade i prodaje proizvoda izrađenih od strane tradioinalnih proizvođača 52/2010 (IV.30.) FVM rendelet a kistermelői élelmiszer-termelés, -előállítás és – értékesítés feltételeiről- Regulativa o uslovima proizvodnje, prerade i prodaje proizvoda izrađenh od strane tradioinalnih proizvođača Odluka o pijacama ("Sl.list Grada Kragujevca" br.4/98, 2/99, 35/08 i 5/09) član 45 stav 2 tačka 15 i član 73 Statuta Preduzeća, Upravni odbor JKP "Gradske tržnice", na sednici održanoj dana 28. 04. 2010. godine, Pijačni red, http://www.trznicekg.rs/PDF%20documents/Pijacni%20red.pdf Pristupljeno: 06.07.2014. Pravilnik o upisu u registar poljoprivrednih gazdinstava i obnovi registracije, kao i o uslovima za pasivan status poljoprivrednog gazdinstva ("Sl. glasnik RS", br. 17/2013) Pravilnik o minimalnim tehničkim uslovima za obavljanje prometa robe i vršenje usluga u prometu robe ("Sl. glasnik RS", br. 47/96, 22/97, 6/99, 99/2005, 100/2007, 98/2009 i 62/2011 - dr. pravilnik) Pravilnik o bližim uslovima koji obezbeđuju higijensko postupanje sa životnim namirnicama i mogućnost zdravstvenog nadzora nad prometom van prostorija određenih za prodaju ("Sl. glasnik SRS", br. 25/76) Pravilnik o veterinarsko-sanitarnim uslovima u objektima za prodaju proizvoda životinjskog porekla van poslovnih prostorija ("Sl. glasnik RS", br. 22/94) Pravilnik o uslovima higijene hrane ("Sl. glasnik RS", br. 73/2010) (primennjuje se od juna 2011. godine.) Pravilnik o veterinarsko-sanitarnim uslovima, odnosno opštim i posebnim uslovima za higijenu hrane životinjskog porekla, kao i o uslovima higijene hrane životinjskog porekla ("Sl. glasnik RS", br. 25/2011 i 27/2014) Tájékoztató a kistermelők élelmiszer-előállítással kapcsolatos lehetőségeiről- Informator za tradicionalne proizvođače o mogućnostima prerade hrane. http://elelmiszerlanc.kormany.hu/download/3/7e/50000/Kistermel%C5%91i%20t% C3%A1j%C3%A9koztat%C3%B3%202013.pdf Pristupljeno:06.07. 2014. Uredba o registru poljoprivrednih gazdinstava ("Službeni glasnik RS", br. 119/08, 21/09 i 36/09) Uredba o registru poljoprivrednih gazdinstava ("Službeni glasnik RS", br. 119/08, 21/09 i 36/09) Zakon o bezbednosti hrane ("Sl. glasnik RS", br. 41/2009) 118 Sárkány, H., Kovács, V. Zakon o trgovini ("Sl. glasnik RS", br. 53/2010 i 10/2013) Zakon o veterinarstvu ("Sl. glasnik RS", br. 91/2005, 30/2010 i 93/2012) Sárkány, H., Kovács, V. Zakon o trgovini ("Sl. glasnik RS", br. 53/2010 i 10/2013) Zakon o veterinarstvu ("Sl. glasnik RS", br. 91/2005, 30/2010 i 93/2012) Zakon o trgovini ("Sl. glasnik RS", br. 53/2010 i 10/2013) Zakon o veterinarstvu ("Sl. glasnik RS", br. 91/2005, 30/2010 i 93/2012) Zakon o trgovini ("Sl. glasnik RS", br. 53/2010 i 10/2013) Zakon o veterinarstvu ("Sl. glasnik RS", br. 91/2005, 30/2010 i 93/2012) 119
https://openalex.org/W3211555049	https://digital.csic.es/bitstream/10261/256657/1/Journal%20of%20Cancer%20Metastasis%20and%20Treatment_Garc%c3%ada-Pardo_2021.pdf	English	null	Regulation and function of angiogenic factors in chronic lymphocytic leukemia	null	2,021	cc-by	19,226	Angeles García-Pardo, Javier Redondo-Muñoz Department of Molecular Biomedicine, Centro de Investigaciones Biológicas Margarita Salas, Consejo Superior de Investigaciones Científicas, Madrid 280140, Spain. Correspondence to: Dr. Angeles García-Pardo, Centro de Investigaciones Biológicas Margarita Salas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, Madrid 28040, Spain. E-mail: gelivejer@gmail.com; Dr. Javier Redondo-Muñoz, Correspondence to: Dr. Angeles García-Pardo, Centro de Investigaciones Biológicas Margarita Salas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, Madrid 28040, Spain. E-mail: gelivejer@gmail.com; Dr. Javier Redondo-Muñoz, Department of Molecular Biomedicine, Centro de Investigaciones Biológicas Margarita Salas, Ramiro de Maeztu 9, Madrid 28040 Spain E-mail: javier redondo@cib csic es Investigaciones Científicas, Ramiro de Maeztu 9, Madrid 28040, Spain. E-mail: gelivejer@gmail.com; Dr. Javier Redondo-Muñoz, Department of Molecular Biomedicine, Centro de Investigaciones Biológicas Margarita Salas, Ramiro de Maeztu 9, Madrid 28040, Spain. E-mail: javier.redondo@cib.csic.es Department of Molecular Biomedicine, Centro de Investigaciones Biológicas Margarita Salas, Ramiro de Maeztu 9, M 8040, Spain. E-mail: javier.redondo@cib.csic.es How to cite this article: García-Pardo A, Redondo-Muñoz J. Regulation and function of angiogenic factors in chronic lymphocytic leukemia. J Cancer Metastasis Treat 2021;7:62. https://dx.doi.org/10.20517/2394-4722.2021.103 Received: 28 Apr 2021 First Decision: 9 Jul 2021 Revised: 16 Jul 2021 Accepted: 18 Oct 2021 Published: 5 Nov 2021 Academic Editors: Lucio Miele, Ribatti Domenico, Dominique Bonnet Copy Editor: Yue-Yue Zhang Production Editor: Yue-Yue Zhang García-Pardo et al. J Cancer Metastasis Treat 2021;7:62 DOI: 10.20517/2394-4722.2021.103 García-Pardo et al. J Cancer Metastasis Treat 2021;7:62 DOI: 10.20517/2394-4722.2021.103 Journal of Cancer Metastasis and Treatment Open Access © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, sharing, adaptation, distribution and reproduction in any medium or format, for any purpose, even commercially, 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. INTRODUCTION Chronic lymphocytic leukemia (CLL), the most common leukemia in Western countries, is characterized by the clonal proliferation and accumulation of CD5+ B lymphocytes in the peripheral blood and lymphoid organs[1-4]. The degree of organ infiltration serves to classify CLL into different clinical stages, following the criteria established by Rai (stages 0-IV)[5] and Binet (stages A-C)[6]. According to these criteria, the CLL stage 0/A represents low-risk, I-II/B is an intermediate-risk, and III-IV/C represents high-risk. CLL staging is also useful to determine the need for treatment. Current therapies for CLL include the combination fludarabine- cyclophosphamide-rituximab, as well as inhibitors of the B-cell receptor (BCR) signaling pathway, such as ibrutinib/acalabrutinib (Bruton’s tyrosine kinase inhibitors) and idelalisib (phosphatidylinositol 3-kinase-δ inhibitor)[7,8]. An increasing number of CLL cases are now been treated with the Bcl-2 antagonist venetoclax, either as monotherapy or combined with other therapeutic agents[7,8]. Although many patients respond to treatment and some achieve remission, CLL remains an incurable disease. Clinically, CLL is very heterogenous, with good or poor prognosis mostly determined by the presence of specific markers, particularly mutated (M-CLL) or unmutated (U-CLL) immunoglobulin heavy-chain variable region (IGVH)[1-4]. U-CLL originates from B cells that have not experienced the germinal center and represents an aggressive disease, whereas M-CLL originates from germinal center-differentiated B cells and usually represents a mild form of the malignancy[9]. Other established prognostic markers for CLL are CD38 and CD49d (the α subunit of the α4β1 integrin), whose elevated expression on CLL cells (> 30%) is associated with a poor outcome[10,11]. CLL tumors also accumulate multiple gene mutations and/or deletions, such as those affecting the p53 protein, whose loss of function is related to resistance to chemotherapy[4]. Whole-genome sequencing analyses have identified genes that are recurrently mutated in CLL, including notch1 (NOTCH1) and myeloid differentiation primary response gene 88 (MYD88)[12]. NOTCH1 mutations are mainly detected in U-CLL cases, while MYD88 mutations are predominant in M-CLL[12]. NOTCH1 mutations are also linked to poor CLL prognosis and high CD49d expression[13]. Recurrent mutations have also been found in genes affecting crucial signaling pathways in CLL, such as those induced by the BCR, NF-κB transcription factor, or MAPK-ERK system[4]. Epigenomic changes are also frequent in CLL, with different patterns in U-CLL and M-CLL cases[4]. CLL progression is determined by the infiltration of bone marrow and secondary lymphoid organs by the malignant cells. Abstract Progression of chronic lymphocytic leukemia (CLL) is determined by the localization of malignant cells in lymphoid tissues, where they receive growth and survival signals. CLL cells produce angiogenic factors that are regulated by internal and external stimuli and whose levels vary according to the clinical stage of the disease. Stromal cellular and molecular components in CLL niches disturb the balance of pro- and antiangiogenic molecules in CLL cells and induce an angiogenic switch. Additionally, CLL cells also influence the behavior of microenvironmental cells, inducing endothelial cell proliferation and increasing the angiogenic capacity of macrophages, neutrophils, and other cells present in CLL niches. As a result of these reciprocal functional interactions, bone marrow angiogenesis is frequently increased in CLL and has been proposed as a prognostic marker in early disease. Besides their role in regulating angiogenesis, angiogenic factors are also involved in CLL cell migration and survival, all contributing to disease progression. Angiogenic factors, particularly vascular endothelial growth factor, have therefore been attractive therapeutic targets in CLL and many clinical trials were established in the past years. However, the results of these trials reveal that anti-angiogenic therapies alone are not as efficient as expected and should rather be used in combination with other treatments. © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, sharing, adaptation, distribution and reproduction in any medium or format, for any purpose, even commercially, 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. www.jcmtjournal.com © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, sharing, adaptation, distribution and reproduction in any medium or format, for any purpose, even commercially, 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. www.jcmtjournal.com García-Pardo et al. J Cancer Metastasis Treat 2021;7:62 https://dx.doi.org/10.20517/2394-4722.2021.103 Page 2 of 22 Keywords: Chronic lymphocytic leukemia, angiogenic factors, CLL microenvironment, CLL migration and survival, antiangiogenic therapy Keywords: Chronic lymphocytic leukemia, angiogenic factors, CLL microenvironment, CLL migration and survival, antiangiogenic therapy CLL CELLS PRODUCE PRO- AND ANTIANGIOGENIC FACTORS AND THEIR RECE Angiogenic factors produced by CLL cells Previous excellent reviews provide a detailed characterization of the angiogenic factors present in CLL and their possible role in the disease[20,21,34,35]. The present review expands and updates the reported studies. CLL cells spontaneously synthesize and secrete pro- and antiangiogenic molecules, including basic fibroblast growth factor (bFGF)[21,34-36], vascular endothelial growth factor (VEGF)[36-38], platelet-derived growth factor (PDGF)[39], thrombospondin-1 (TSP-1)[36], angiopoietin-2 (Ang-2)[36], and matrix metalloproteinase-9 (MMP-9)[40-42] [Figure 1]. The secreted factors are found in the conditioned media of cultured CLL cells as well as in the plasma/serum and urine of CLL patients, and their levels vary during the course of the disease. Several groups have analyzed samples from CLL patients by enzyme-like immunoassays and have demonstrated that elevated levels of bFGF in lymphocytes and plasma correlate with advanced stages of the disease (Rai stage III/IV)[43-45]. In another study, bFGF levels in urine were also higher in CLL patients than in controls, but they did not significantly correlate with the clinical stage[22]. VEGF is clearly the most studied angiogenic factor in CLL. The human VEGF family comprises five members: VEGF-A, VEGF-B, VEGF-C, VEGF-D, and placental growth factor[46-48]. VEGF-A is the best characterized and comprises five different isoforms (VEGF121, VEGF145, VEGF165, VEGF189, and VEGF206) that arise by alternative splicing of the VEGF gene[46-49]. Using ELISA, Western blot, and RT-PCR analyses of concentrated CLL cell culture media, Chen et al.[38] demonstrated the presence of VEGF121 and VEGF165, with molecular weights of 28 and 42 kDa, respectively. VEGF165 is the predominant form in CLL and the best studied in terms of expression, regulation and signaling; we refer to this isoform as VEGF throughout this review. A correlation was found between elevated levels of serum VEGF in early CLL stages (Rai I/II) and the risk of CLL progression/progression-free survival, supporting the role of VEGF as a prognostic marker in this disease[26,50,51]. The amount of plasma PDGF was also higher in CLL patients than in controls and strongly correlated with the levels of VEGF and with advanced stages (Rai II-IV) and poor prognosis markers (ZAP- 70 or CD38 positive)[52]. High concentrations of Ang-2 mRNA and plasma protein were also found in several cohorts of CLL patients, with a significant correlation with an aggressive phenotype (unmutated IGHV, CD38 positive), advanced Binet stages (B-C), and shorter survival[53-55]. Similarly, the intracellular and serum concentrations of MMP-9 were higher in CLL than in normal lymphocytes[40-42]. INTRODUCTION This process is mostly mediated by the α4β1 integrin and the chemokine/receptor axes CXCL12/CXCR4 and CCL21/CCR7[14,15]. MMP-9, CD44, particularly the CD44v6 variant, and CD38 have also be shown to play roles in CLL cell migration to tissues[15-17]. Localization in lymphoid niches is beneficial for CLL cells as they receive proliferation and survival signals, which contribute to disease development[18]. Angiogenesis, the development of new vessels from pre-existing ones, is another feature associated to CLL progression[19-21]. Increased angiogenesis has been observed in the bone marrow and lymph nodes of CLL patients[22-24]. The microvessel density in the bone marrow positively correlates with the clinical stage of CLL[22] and has also been associated with a dysregulation of angiogenic factors[25]. Additionally, increased bone marrow angiogenesis in CLL has been suggested as a possible prognostic marker to determine the risk of progression in early disease[26,27]. Page 3 of 22 García-Pardo et al. J Cancer Metastasis Treat 2021;7:62 https://dx.doi.org/10.20517/2394-4722.2021.103 The angiogenic status of CLL tissues is influenced by cells present in the microenvironment, including CLL cells. Indeed, CLL cells are known to establish reciprocal interactions with stromal cellular components[28-30]. These interactions affect cellular functions and modify the CLL cell gene expression pattern, resulting in the so-called “angiogenic switch”[31-33]. This review describes the angiogenic factors produced by CLL cells, their regulation by internal or external factors, and their function in angiogenesis and other cellular processes. We also summarize the anti-angiogenic therapies that have been evaluated in CLL and the results obtained. CLL CELLS PRODUCE PRO- AND ANTIANGIOGENIC FACTORS AND THEIR RECE CLL cells are also able to recruit and induce a proangiogenic phenotype in immune cells by secreting soluble factors, such as GM/CSF (granulocyte-macrophage colony stimulating factor), CCL3 (C-C Motif Chemokine Ligand 3), CCL4 (C-C Motif Chemokine Ligand 4), and NAMPT (Nicotinamide phosphoribosyltransferase). Both CLL and stromal cells also release exosomes, whose cargo (proteins and miRNAs) allows neovascularization and angiogenesis. CLL: Chronic lymphocytic leukemia; PDGF: platelet-derived growth factor; VEGF: vascular endothelial growth factor; ANG2: angiopoietin-2; TSP-1: thrombospondin-1; MMP-9: matrix metalloproteinase-9; bFGF: basic fibroblast growth factor. Figure 1. Angiogenesis mediators in CLL niches. In lymphoid tissues, CLL cells establish bidirectional interactions with immune and resident cells. Crosstalk with stromal cells induces an angiogenic switch in CLL cells. Conversely, CLL cells secrete the indicated angiogenic factors, which influence the behavior of immune and resident cells to favor angiogenesis. CLL cells are also able to recruit and induce a proangiogenic phenotype in immune cells by secreting soluble factors, such as GM/CSF (granulocyte-macrophage colony stimulating factor), CCL3 (C-C Motif Chemokine Ligand 3), CCL4 (C-C Motif Chemokine Ligand 4), and NAMPT (Nicotinamide phosphoribosyltransferase). Both CLL and stromal cells also release exosomes, whose cargo (proteins and miRNAs) allows neovascularization and angiogenesis. CLL: Chronic lymphocytic leukemia; PDGF: platelet-derived growth factor; VEGF: vascular endothelial growth factor; ANG2: angiopoietin-2; TSP-1: thrombospondin-1; MMP-9: matrix metalloproteinase-9; bFGF: basic fibroblast growth factor. Expression of angiogenic factor receptors in CLL cells bFGF receptors Besides producing angiogenic/angiostatic molecules, CLL cells also express some of the receptors for these factors. The family of bFGF receptors comprises four members, FGFR1-4, which are present in nearly all hematopoietic cells and mediate multiple physiological processes. In an initial study, Rosenwald et al.[59] performed gene expression analyses on CLL cells and identified only the FGFR1 transcript, with higher expression in unmutated IgVH cases. However, using Western blotting, immunoprecipitation, and flow cytometry analyses, Sinha et al.[60] demonstrated that CLL cells expressed elevated levels of FGFR3. Low expression of FGFR1, -2, and -4 was also detected but their levels were similar to those observed on normal B-cells. Additional RT-PCR analyses confirmed the predominant expression of FGFR3[60]. Constitutive phosphorylation at Y653/654 tyrosine residues was also observed in this study, suggesting an active signaling role for FGFR3 in CLL. CLL CELLS PRODUCE PRO- AND ANTIANGIOGENIC FACTORS AND THEIR RECE These elevated MMP-9 levels were detectable at early CLL stages[40] and correlated with the risk of disease progression[56,57]. In contrast to the above-mentioned factors, the levels of the antiangiogenic molecule TSP-1[58], both mRNA and protein, were higher in low-risk CLL patients (Rai I/II) than in high-risk patients (Rai stage > II)[36]. The same pattern was observed when TSP-1 was quantitated in the conditioned medium of CLL cells cultured for 24 h[36]. These studies indicate that expression and secretion of pro- and antiangiogenic molecules is an active process in CLL, with a clear proangiogenic switch as disease progresses (see below). Quantitation of these factors, however, is not done routinely in the clinic when diagnosing and staging CLL, and their amounts are usually determined as complementary indicators or for specific studies. Accordingly, the levels of angiogenic factors are not commonly included among the criteria used to decide the initiation of Page 4 of García-Pardo et al. J Cancer Metastasis Treat 2021;7:62 https://dx.doi.org/10.20517/2394-4722.2021.103 22 Page 4 of 22 García-Pardo et al. J Cancer Metastasis Treat 2021;7:62 https://dx.doi.org/10.20517/2394-4722.2021.103 Figure 1. Angiogenesis mediators in CLL niches. In lymphoid tissues, CLL cells establish bidirectional interactions with immune and resident cells. Crosstalk with stromal cells induces an angiogenic switch in CLL cells. Conversely, CLL cells secrete the indicated angiogenic factors, which influence the behavior of immune and resident cells to favor angiogenesis. CLL cells are also able to recruit and induce a proangiogenic phenotype in immune cells by secreting soluble factors, such as GM/CSF (granulocyte-macrophage colony stimulating factor), CCL3 (C-C Motif Chemokine Ligand 3), CCL4 (C-C Motif Chemokine Ligand 4), and NAMPT (Nicotinamide phosphoribosyltransferase). Both CLL and stromal cells also release exosomes, whose cargo (proteins and miRNAs) allows neovascularization and angiogenesis. CLL: Chronic lymphocytic leukemia; PDGF: platelet-derived growth factor; VEGF: vascular endothelial growth factor; ANG2: angiopoietin-2; TSP-1: thrombospondin-1; MMP-9: matrix metalloproteinase-9; bFGF: basic fibroblast growth factor. Figure 1. Angiogenesis mediators in CLL niches. In lymphoid tissues, CLL cells establish bidirectional interactions with immune and resident cells. Crosstalk with stromal cells induces an angiogenic switch in CLL cells. Conversely, CLL cells secrete the indicated angiogenic factors, which influence the behavior of immune and resident cells to favor angiogenesis. VEGF receptors Initial analyses on a cohort of 216 CLL patients by Western blot showed the consistent expression of VEGFR2 protein, while VEGFR2 was not detected in control samples[61]. Using ribonuclease protection and RT-PCR analyses on CLL cells from 30 patients, Kay et al.[36] demonstrated the presence of mRNA for Page 5 of García-Pardo et al. J Cancer Metastasis Treat 2021;7:62 https://dx.doi.org/10.20517/2394-4722.2021.103 22 García-Pardo et al. J Cancer Metastasis Treat 2021;7:62 https://dx.doi.org/10.20517/2394-4722.2021.103 Page 5 of 22 VEGF receptor 1 (VEGFR1) and VEGF receptor 2 (VEGFR2). Expression of all three tyrosine kinase VEGF receptors (VEGFR1, VEGFR2, and VEGFR3) at the CLL cell surface was also demonstrated by flow cytometry (13 patient samples) and immunocytochemical (27 patient samples) analyses[62]. The non- enzymatic VEGF receptor neuropilin-1 was also shown to be present in CLL cells, at both mRNA and protein levels, and with a higher expression than in control B-cells[63,64]. VEGF binds to VEGFR1 and VEGFR2 but not to VEGFR3, whose ligands are VEGF-C and -D[46-49]. Although the binding affinity of VEGF to VEGFR1 and VEGFR2 is high, VEGFR2 is the main receptor responsible for delivering intracellular signaling upon interaction with VEGF[20,61]. Therefore, most studies on the CLL system have focused on the functional consequences of the VEGF/VEGFR2 interaction. The levels of expression of VEGFR2 in CLL cells are variable. Ferrajoli et al.[61] showed that elevated levels of VEGFR2 protein, measured in cell extracts, correlated with shorter survival. An association between high VEGFR2 expression and advanced Rai stages (III/IV) was also observed in that study, although it was not statistically significant. Conversely, we analyzed the constitutive VEGFR2 expression on 38 CLL samples by flow cytometry and did not find a correlation with Rai/Binet stages[65]. Despite this, high expression of VEGFR2 appears to be a characteristic of CLL cells, suggesting an active role of this receptor in the pathology of the disease. Ang-2 and TSP-1 receptors Ang-2 and TSP-1 receptors The receptor for Ang-2 is Tie-2, one of the two members (Tie-1 and Tie-2) of the Tie receptor tyrosine kinase family[66]. While expression of Tie-1 in CLL cells was early recognized[36,67,68], the expression of Tie-2 on these cells has been controversial. Analyses by PCR and flow cytometry on nine plasma CLL samples failed to show Tie-2 expression, while Tie-2 was present on endothelial cells used as control[69]. Treatment of these CLL cells with various stimuli (CD40L, hypoxia, and stromal/endothelial cells) did not induce Tie-2 expression. VEGF receptors In the same study, bone marrow-derived CLL cells expressed Tie-2, detected by PCR and flow cytometry, suggesting a role for the microenvironment in Tie-2 expression on CLL[69]. Aguirre Palma et al.[68] did not find a constitutive expression of Tie-2 by PCR analyses of peripheral blood CLL cells. However, they observed a transient expression of Tie-2 upon stimulation of these cells with Ang-2. Another study found that a small percentage of peripheral blood CLL cells expressed Tie-2 at the cell surface[70]. Because Tie-2 undergoes rapid internalization upon activation[71] these authors performed intracellular staining and found a moderate positive expression of Tie-2 receptor. CLL cells infiltrating lymph nodes also expressed Tie-2. The presence of Tie-2 was further confirmed by Western blotting and PCR analyses[70]. It therefore appears that expression of the Tie-2 receptor in CLL cells is subjected to modulation by several external factors. Two main TSP-1 receptors, CD36 and CD47, are expressed by CLL cells. CD36 expression was initially studied by Rutella et al.[72] on 24 CLL samples. Using flow cytometry and immunofluorescence analyses, they found variable expression of CD36 in most CLL cases. Elevated CD36 expression was associated with advanced disease (Rai stage III/IV, organ infiltration)[72]. CD47 was also shown to be present in CLL cells and to mediate several functions upon binding to TSP-1, including cell death and cytoskeleton reorganization[73,74]. p Although MMP-9 is mainly a secreted protease found in soluble form in serum and cell conditioned media, we and others have consistently found MMP-9 at the CLL cell surface[40-42,75]. Using immunoprecipitation with anti-MMP-9 antibodies, function blocking antibodies, siRNA transfection, and immunofluorescence Page 6 of García-Pardo et al. J Cancer Metastasis Treat 2021;7:62 https://dx.doi.org/10.20517/2394-4722.2021.103 22 García-Pardo et al. J Cancer Metastasis Treat 2021;7:62 https://dx.doi.org/10.20517/2394-4722.2021.103 analyses, our group demonstrated that binding of MMP-9 to CLL cells is mediated by a docking complex consisting of α4β1 integrin and a 190 kDa CD44v isoform[75]. Interestingly, silencing either α4β1 integrin or 190 kDa CD44 with specific siRNAs reduced the amount of cell surface-bound MMP-9 and increased the levels of secreted MMP-9, indicating that there is a dynamic traffic between soluble and cell-bound MMP-9 in CLL. Perhaps this could explain why we could not find in our studies a clear association between the expression of membrane-bound MMP-9 and the clinical stage of the disease[76-79]. REGULATION OF ANGIOGENIC FACTORS IN CLL Regulation by conditions of the microenvironment hypoxia Regulation by conditions of the microenvironment - hypoxia Regulation by conditions of the microenvironment hypoxia The constitutive production of angiogenic molecules and their receptors by CLL cells is subjected to regulation by several stimuli. Because progression of CLL is characterized by the migration and localization of malignant cells in lymphoid tissues[18,30], these stimuli are mainly provided by the microenvironment of these tissues. Hypoxia, a common condition in human bone marrow and other tissues, is a key regulator of VEGF in physiological and pathological conditions[20,47,49]. Indeed, hypoxia stabilizes the hypoxia-inducible factor-1α (HIF-1α), a major transcription factor for VEGF, allowing its binding to specific elements in the VEGF promoter and increasing VEGF synthesis and secretion[47,49]. Several authors have shown that, upon a hypoxic stimulus, cultured CLL cells increase their VEGF production, at both mRNA and protein levels[36,38]. Moreover, we and others demonstrated by immunohistochemical analyses that VEGF is present in the bone marrow and lymph nodes of CLL patients, and at higher expression than in normal tissues[38,65]. Kay et al.[36] found that culturing CLL cells under hypoxic conditions also reduced the levels of TSP-1 produced by these cells, thus favoring an angiogenic switch. The levels of PDGF and Ang-2 in CLL cells were also shown to be upregulated by hypoxia[36,52,83]. VEGF receptors The ability to bind and retain MMP-9 at their surface appears to be characteristic of CLL cells as it was not observed in normal B- cells[75]. α4β1 integrin is present in approximately 40% of CLL cases, and there is now extensive evidence showing that the α subunit of this integrin, also known as CD49d, is a strong independent prognostic marker in CLL, associated with an aggressive disease[10,80]. Our studies have shown that, as a receptor for MMP-9, α4β1 integrin contributes to disease progression by regulating CLL cell migration and survival[77-79,81]. This contribution was recently confirmed based on a bimodal pattern of expression of CD49d in CLL cells[82]. CD44 (standard and variant forms) is expressed at high levels in CLL cells and has also been proposed as a prognostic marker, particularly due to its role in CLL cell migration and organ localization[17]. Regulation by stromal cells - induction of a proangiogenic phenotype on CLL cells Upon migration and localization in lymphoid tissues, CLL cells interact with surrounding stromal cells, establishing an active crosstalk that provides survival and proliferation signals to the malignant cells. The molecular bases of these interactions have been recapitulated in excellent previous reviews[30,84] and involve direct cell-cell contact via adhesion molecules, release of soluble factors such as chemokines, or material exchange via extracellular vesicles [Figure 1]. One important consequence of these cellular interactions is the modification of the gene expression profile of CLL cells, mainly resulting in the upregulation of anti- apoptotic molecules (Bcl-2, Bcl-xL, Mcl-1, and XIAP) and the activation of survival signaling pathways (PI3-K/NF-κB and Notch)[30]. Additionally, the balance of pro- and antiangiogenic factors on CLL cells is also affected by the contact with stromal cells. Kay et al.[31] showed that culturing CLL cells on primary bone marrow-derived stromal cells dramatically increased the secretion of bFGF and reduced the levels of the antiangiogenic molecule TSP-1. In another study, Edelmann et al.[32] performed gene microarray analyses on CLL cells that had been co-cultured with the murine bone marrow fibroblast cell line M2-10B4, either in direct cell-cell contact or separated by Transwell inserts. They found that direct contact with the fibroblastic cells significantly increased VEGF expression at both gene and protein levels and decreased TSP-1 Page 7 of García-Pardo et al. J Cancer Metastasis Treat 2021;7:62 https://dx.doi.org/10.20517/2394-4722.2021.103 22 Page 7 of 22 García-Pardo et al. J Cancer Metastasis Treat 2021;7:62 https://dx.doi.org/10.20517/2394-4722.2021.103 expression. Consistent with this decrease, there was a correlation between advanced CLL (Binet stages B and C) and low TSP-1 plasma levels, in agreement with the studies mentioned above. Additionally, MMP-9 was also dramatically upregulated (25.9-fold change) upon CLL cell contact with the fibroblastic cells, compared with freshly isolated CLL cells[32], a fact also confirmed by Schulz et al.[85]. In line with this upregulation, we showed that culturing CLL cells on primary stromal cells (derived from a CLL patient) for 48 h increases the amount of MMP-9 bound to the CLL cell surface by two-fold[81]. Moreover, CLL cells isolated from the bone marrow or lymph nodes of patients consistently showed higher levels of surface-bound MMP-9 than their peripheral blood counterparts, confirming the increased production of MMP-9 in a pathophysiological context[81]. This MMP-9, which is produced by the CLL cells as well as by stromal cells, likely contributes to the increased angiogenic status observed in CLL tissues (see below). Regulation by stromal cells - induction of a proangiogenic phenotype on CLL cells Endothelial cells are also an important stromal component of lymphoid tissues [Figure 1]. Maffei et al.[33] showed that physical interaction of CLL cells with human umbilical vein endothelial cells protected CLL cells from spontaneous and drug-induced apoptosis. This interaction was mediated by β1 and β2 integrins, and it also resulted in the modulation of 1944 genes, 1217 upregulated and 727 downregulated, determined by gene microarray analyses[33]. Many of the upregulated genes were related to angiogenesis and included the Ang-2 receptors Tie-1 and Tie-2, VEGFC, TSP-1, MMP-2, and MMP-14, while VEGFR3 was downregulated. The C-C motif chemokine-2 (CCL2), which is known to regulate angiogenesis and recruit monocytes/macrophages to tissues[86], was also significantly upregulated in CLL cells upon coculture with endothelial cells[33]. It can be concluded that, in lymphoid tissues, particularly in the bone marrow, these CLL-produced factors disturb the angiogenic balance and contribute to disease progression. Autocrine and inter-regulation among angiogenic factors CONTRIBUTION OF CLL CELLS TO THE ANGIOGENIC STATUS OF LYMPHOID TISSUES Functional effects on endothelial cells The above-mentioned studies have clearly established that the molecular and cellular components of the microenvironment in CLL tissues modulate the gene expression pattern of CLL cells, inducing a pro- survival and proangiogenic phenotype in the malignant cells. It is also known that modulation of angiogenic molecules in CLL has functional consequences in the CLL niches [Figure 1]. Chen et al.[38] showed that the conditioned medium (CM) of CLL cells cultured for 24 h contained VEGF and induced proliferation of endothelial cells. Moreover, this CM also induced moderate in vitro angiogenesis, and this effect was prevented by a neutralizing anti-VEGF antibody[38]. It was later demonstrated that the CM of cultured CLL cells also contained Ang-2, and the levels of this protein correlated with the degree of bone marrow vascularization of the patients studied[84]. These authors also showed that both Ang-2 and VEGF were responsible for the CM-induced tube formation by endothelial cells on Matrigel matrices, as neutralizing antibodies to either factor diminished the angiogenic effect[83]. In both studies, a hypoxic stimulus further increased the functional effects of the CLL cell CM on endothelial cells. Additionally, we showed that the CM of CLL cells that had been incubated with MMP-9 for 24 h had a significantly higher effect on endothelial cell proliferation compared to the CM of CLL cells incubated on 0.5% BSA[79]. This is likely due to the MMP-9-induced production of proangiogenic factors (MMP-9 and VEGF) by CLL cells mentioned above. Other levels of regulation - epigenetic and gene polymorphism Other levels of regulation - epigenetic and gene polymorphism Angiogenic molecules in CLL cells can also be regulated through other molecular mechanisms, including gene polymorphisms and epigenetic alterations. The genomic polymorphism in VEGF-A of CLL cells has been extensively studied. According to Lozano-Santos et al.[93], a specific VEGF haplotype correlates with shorter survival in CLL patients. Furthermore, patients who carry this haplotype in combination with other clinical features, such as negative CD38 expression or early age at diagnosis, show a poor survival ratio[93]. Other studies have also indicated that VEGF polymorphism might be relevant as a genetic risk and adverse survival marker in CLL[94,95]. On the other hand, epigenetic profiles might serve to stratify patients and identify different molecular pathways involved in CLL progression. For example, the expression of the Ang-2 gene promoter in CLL cells is highly dependent on the DNA methylation status[96]. This methylation is regulated by VEGF, which promotes Ang-2 expression and neovascularization[96]. In agreement with the notion that DNA methylation regulates critical steps during CLL angiogenesis, it has been reported that Ang-2 expression is also regulated by the tumor suppressor gene microcephalin[97]. Furthermore, other angiogenic molecules such as endothelin-1 are also regulated by DNA methylation in CLL cells[98]. Autocrine and inter-regulation among angiogenic factors The fact that CLL cells express angiogenic factors and their receptors supports the existence of autocrine and crosstalk regulations of the expression and function of these factors. These regulations may take place on circulating CLL cells as well as in CLL cells located in niches. On isolated CLL cells, Bauvois et al.[40] showed that antibodies to VEGF decreased MMP-9 expression, suggesting a link between both proteins. We showed that binding of VEGF to its VEGFR2 receptor in CLL cells downregulates the expression of MMP-9 as well as the migration of these cells through Matrigel or endothelial cells[87]. Regulation of MMP-9 by the VEGF/VEGFR2 axis was at the transcriptional level and was mediated by the phosphorylation and translocation to the nucleus of the signal transducer and activator of transcription 1 (STAT1)[87], a factor known to suppress MMP-9 gene transcription in response to interferons in several cell systems[88]. Using immunofluorescence analyses, our group also demonstrated that MMP-9 is present in the bone marrow and lymph nodes of CLL patients, in partial association with the macrophages in these tissues[79]. We studied whether, as a component of the CLL microenvironment, MMP-9 affected the CLL cell angiogenic pattern. Culturing CLL cells on immobilized MMP-9 for 24 h increased the expression of MMP-9 and VEGF and reduced the expression of the angiostatic molecules TSP-1 and Ang-2, all at the gene and protein levels[79], establishing that isolated MMP-9 is able to induce a proangiogenic profile in CLL cells. The fact that MMP-9 interaction with its receptors in CLL cells increases its own production may represent an autocrine positive feedback loop, similar to that described for VEGF in these cells[20]. Further mechanistic studies demonstrated that downregulation of TSP-1 by MMP-9 involved α4β1 integrin (MMP-9 receptor), Src kinase family activity, and the STAT3 transcription factor[79]. The fact that STAT3 is also a transcription factor for VEGF, regulates the expression and degradation of HIF-1α, and is a key factor in angiogenesis[89-92] strongly suggests that upregulation of VEGF by MMP-9 is also mediated by STAT3 activation. Page 8 of 22 García-Pardo et al. J Cancer Metastasis Treat 2021;7:62 https://dx.doi.org/10.20517/2394-4722.2021.103 Functional effects on macrophages Macrophages are also important components of tissue microenvironments. In CLL, the specific population of leukemia-associated macrophages, known as nurse-like cells, plays a fundamental role in inducing and maintaining CLL cell proliferation and survival[29,100,101]. The important function of tumor-associated macrophages in angiogenesis is also well documented[102,103]. Macrophages can be activated in vitro by specific stimuli towards two different functional phenotypes: M1 and M2[104]. M1 macrophages are pro- inflammatory and induce strong antitumor immune responses by producing relevant specific molecules and cytotoxic factors. In contrast, M2 macrophages are anti-inflammatory and secrete chemokines/cytokines that suppress immune responses and support tumor expansion[105,106]. In vivo evidence indicates that tumor- associated macrophages are mostly polarized towards the M2-like phenotype and that tumor cells contribute to this polarization[107]. In the case of CLL, Audrito et al.[108] showed that activated CLL cells induced monocyte differentiation to M2 macrophages, and this was mediated by the nicotinamide phosphoribosyltransferase enzyme produced by CLL cells [Figure 1]. These M2 macrophages sustained CLL cell survival and reduced T-cell proliferation, thus favoring disease progression[108]. In another study, the high-mobility group protein B-1 produced by CLL cells was shown to differentiate nurse-like cells to M2- polarized macrophages, concomitant with a STAT3 and NF-κB activation on both cell types[109]. The hypoxic conditions found in CLL niches also indirectly induced M2 macrophage polarization via increase production of adenosine[29]. Polarization of macrophages towards the M2 subtype also has important consequences for angiogenesis. Zajac et al.[110] showed that M2 macrophages have higher proangiogenic capacity than the M1 subtype, due to the increased production of MMP-9 free of tissue inhibitor of metalloproteinases-1 (TIMP-1), the specific MMP-9 inhibitor. Indeed, these authors showed that M2 macrophages shutdown their TIMP-1 gene expression, a fact that was also demonstrated with murine bone marrow-derived macrophages. The importance of MMP-9 for M2 macrophage angiogenic function was further demonstrated by showing that MMP-9-null M2 macrophages were not angiogenic, despite their downregulation of the TIMP-1 gene[110]. Moreover, the same group showed that neutrophils, which do not produce TIMP-1[111], are the major source of proangiogenic MMP-9 in tumor tissues[112]. MMP-9 is present in CLL tissues, where it is produced by the mentioned stromal cellular components as well as by CLL cells. Functional effects on macrophages Our group demonstrated that the CM of 24 h CLL cell cultures induced a proangiogenic profile in both M1 and M2 macrophages after 7 days of culture, as determined by the increased expression of MMP-9 in these cells, at both mRNA and protein levels[79]. Although we did not test whether the MMP-9 produced by M2 macrophages was more angiogenic than that produced by M1 macrophages, this study constitutes another example of how CLL cells modulate the angiogenic profile of microenvironmental cells in CLL tissues. Functional effects on mesenchymal stromal cells Besides affecting endothelial cell behavior, CLL cells also influence the function of other cells present in the microenvironment of CLL niches[29]. An active crosstalk between CLL and mesenchymal stromal cells (MSC), resulting in malignant cell survival and changes in the pattern of cytokine production by MSC, has been reported[99]. Moreover, the CM of CLL cells was shown to drive migration and proliferation of MSC[52]. This effect was mediated by the interaction of the PDGF present in the CM of CLL cells with its PDGFR in mesenchymal cells, which resulted in activation of the receptor and induction of Akt phosphorylation. Binding of the CLL-derived PDGF to PDFGR also induced the production of VEGF (but not bFGF or TSP-1) by mesenchymal cells through a PI3-K-dependent mechanism[52]. This is a clear example of how CLL cells actively modulate their microenvironment, inducing an angiogenic switch that is permissive for CLL progression. García-Pardo et al. J Cancer Metastasis Treat 2021;7:62 https://dx.doi.org/10.20517/2394-4722.2021.103 Functional effects of CLL cell-derived exosomes Functional effects of CLL cell-derived exosomes Exosomes are small extracellular vesicles secreted by normal and malignant cells via exocytosis in response to multiple physiological and pathological conditions[118]. Exosomes contain proteins, DNA, mRNAs and non-coding RNAs (such as miRNAs) that play fundamental roles in cell-cell interactions and tumor- induced microenvironment modifications[119]. Exosomes released by CLL cells were shown to induce the transition of mesenchymal stromal cells to cancer-associated fibroblasts, which support the growth and survival of malignant cells[120]. These exosomes also impacted on endothelial cells increasing angiogenesis in CLL tissues[120] and modulated intracellular signaling pathways on stromal cells, all favoring disease progression[121,122]. Analysis of the proteomic and miRNA profiles from exosomes produced by CLL cells revealed the presence of several cell surface proteins (CD9, CD37, CD53, CD63, and CD82) and miRNAs, including miR-21, miR-146a, miR-29 family, miR-150, miR-155, and miR-223[120,123] [Table 1]. The expression of some of these miRNAs increases upon BCR activation and has been associated with CLL pathogenesis[123]. Moreover, the expression levels of miR-155 in plasma exosomes were proposed to be a biomarker for the risk of progression from monoclonal B-cell lymphocytosis to CLL, as well as for the identification of CLL patients who may show resistance to therapy[124]. In another study, Farahani et al.[125] showed that exosomes released by CLL cells altered the transcriptome of the stromal cell line HS-5, a fact that may influence the pro- survival signals induced by these cells which favor CLL progression[86]. CLL cell-derived exosomes also contain proteins that affect the molecular pathways of recipient cells, including those involved in angiogenesis. For instance, they contain the small calcium-binding protein S100-A9, which regulates the PI3-K/Akt/NF-κB signaling pathway, VEGF production, and inflammatory processes[126]. Another protein present in CLL exosomes is Axl, a receptor tyrosine kinase, which induces stromal cell activation, VEGF production, and CLL progression[121]. Moreover, the chloride intracellular channel 1 protein, also found in CLL exosomes, was recently shown to induce endothelial cell proliferation and angiogenesis through upregulation of β1 integrin and the MAPK/ERK signaling pathway[127]. It has also been reported that extracellular vesicles from bone marrow stromal cells modify the gene expression pattern in CLL cells, affecting BCR activation and other processes[128]. Uptake of these vesicles also rescued CLL cells from apoptosis and enhanced their migratory capacity in response to the CXCL12 chemokine[128]. Functional effects of CLL cell-derived exosomes Altogether, extracellular vesicles produced by CLL or stromal cells constitute another important way of tumor-microenvironment communication in tissues, with consequences on many processes including angiogenesis in CLL niches. These vesicles thus represent potential therapeutic targets, and several clinical trials are currently addressing this possibility[30]. OTHER FUNCTIONS OF ANGIOGENIC FACTORS IN CLL Besides contributing to the observed increased angiogenesis in CLL niches, several angiogenic factors produced by CLL cells are also able to perform other functions that directly affect disease progression. The best studied functions are the role of these factors in CLL cell migration and survival. Functional effects on neutrophils The role of neutrophils in tumor initiation and angiogenesis is well recognized[113]. In solid tumors, such as melanoma, hepatocellular carcinoma, and breast carcinoma, the recruitment of proangiogenic neutrophils triggers STAT3 activation and the production of angiogenic molecules, including VEGF and MMP-9[114]. There is also a proangiogenic subpopulation of neutrophils, which express high levels of α4β1 integrin, CXCR4, and MMP-9 and are recruited to hypoxic tumor sites[115]. Indeed, as mentioned above, inflammatory neutrophils are the major producers of angiogenic MMP-9 in tumor tissues[112]. In the case of CLL, neutrophils were shown to be activated, expressing high levels of CD54 and presenting some functional defects[116]. Podaza et al.[117] showed that CLL cells induced neutrophil survival and their reprogramming to an immunosuppressive phenotype. In turn, neutrophils were shown to affect CLL cell activation and survival through the induction of extracellular traps[117], thus establishing a reciprocal cellular interaction that supports the proangiogenic function of neutrophils in CLL niches. Page 10 of García-Pardo et al. J Cancer Metastasis Treat 2021;7:62 https://dx.doi.org/10.20517/2394-4722.2021.103 22 García-Pardo et al. J Cancer Metastasis Treat 2021;7:62 https://dx.doi.org/10.20517/2394-4722.2021.103 miRNAs The table shows for each cargo the producer cell type (CLL or stromal cells) and the cell function regulated. CLL: Chronic lymphocytic leukemia; CLIC1: chloride Intracellular Channel 1; HLA-DR: human leukocyte antigen- DR; ITGA4: alpha 4 integrin. The table shows for each cargo the producer cell type (CLL or stromal cells) and the cell function regulated. CLL: Chronic lymphocytic leukemia; CLIC1: chloride Intracellular Channel 1; HLA-DR: human leukocyte antigen- DR; ITGA4: alpha 4 integrin. relevant in the migration of CLL cells to lymph nodes, as it was preferentially observed in patients with lymphadenopathy[129]. Another example of VEGF involvement in CLL cell migration is the previously mentioned study[88], in which we showed that binding of exogeneous VEGF to VEGFR2 reduces CLL cell migration in a dose-dependent manner. This effect was mediated by the VEGF/VEGFR2-induced downregulation of MMP-9[88]. relevant in the migration of CLL cells to lymph nodes, as it was preferentially observed in patients with lymphadenopathy[129]. Another example of VEGF involvement in CLL cell migration is the previously mentioned study[88], in which we showed that binding of exogeneous VEGF to VEGFR2 reduces CLL cell migration in a dose-dependent manner. This effect was mediated by the VEGF/VEGFR2-induced downregulation of MMP-9[88]. Function of angiogenic factors in CLL cell migration VEGF A role of VEGF in CLL cell migration was first inferred by the fact that blocking autocrine VEGF or the VEGFR2 kinase activity reduced the chemokine-induced CLL cell migration through endothelium[129]. This study also showed that chemokine activation of αLβ2 integrin and subsequent transendothelial migration was defective in CLL cells and that stimulation with autocrine VEGF and α4β1 integrin overcame this defect and restored cell motility. Regulation of cell motility by these two proteins appears to be particularly Page 11 of García-Pardo et al. J Cancer Metastasis Treat 2021;7:62 https://dx.doi.org/10.20517/2394-4722.2021.103 22 Table 1. Molecular components of stromal and CLL-derived exosomes Cargo Producer cell Cell function Ref. Proteins S100-A9 CLL CLL progression [126] CLIC1 CLL Angiogenesis [127] CD19, CD20 CD37, CD53, and CD82 CLL Stromal cell activation [120] Axl Stromal and CLL VEGF production, stromal cell activation [121] HLA-DR, ITGA4, Lyn, CD81 and CD37 CLL Stromal cell proliferation [125] CD9, CD63, and CD37 CLL CLL activation [123] miRNAs miR-155 CLL CLL progression [124] miR-29 family, miR-150, miR-155, and miR-630 CLL CLL activation [123] miR-21, miR-155, miR-146a, miR-148a, and let-7g CLL Stromal cell activation [120] miR-202-3p, miR-29a, miR-26, let-7g, and miR-21 CLL Stromal cell proliferation [125] Not identified Stromal Gene expression, CLL migration, CLL survival [128] The table shows for each cargo the producer cell type (CLL or stromal cells) and the cell function regulated. CLL: Chronic lymphocytic leukemia; CLIC1: chloride Intracellular Channel 1; HLA-DR: human leukocyte antigen- DR; ITGA4: alpha 4 integrin. Table 1. Molecular components of stromal and CLL-derived exosomes Function of angiogenic factors in CLL cell survival/apoptosis Function of angiogenic factors in CLL cell survival/apoptosis Function of angiogenic factors in CLL cell survival/apoptosis VEGF Binding of many angiogenic factors to their respective receptors in CLL cells are known to induce signaling pathways that lead to apoptosis resistance and cell survival[20,21,34,35]. It was first demonstrated that culturing CLL cells with VEGF for 24 h significantly decreased spontaneous and chrorambucil-induced apoptosis, and this involved upregulation of the anti-apoptotic molecules myeloid cell leukemia-1 (Mcl-1) and X- linked inhibitor of apoptosis protein (XIAP) via activation of the transcription factor STAT3[134,135]. These authors also showed that the VEGFR1 and VEGFR2 receptors are constitutively phosphorylated in CLL cells and that tyrosine kinase inhibitors or anti-VEGF antibodies inhibited this phosphorylation and STAT3 activation, reduced Mcl-1 and XIAP levels, and induced apoptosis[134,135]. These studies substantiate the presence of a VEGF/VEGFR autocrine pathway that supports CLL cell survival and demonstrate that blocking this pathway results in CLL cell apoptosis. Interestingly, it was further demonstrated that this internal autocrine VEGF survival loop particularly operates in CLL cells expressing the prognostic marker CD38, thus introducing a different VEGF behavior in CD38+ and CD38- CLL cells[136]. Autocrine VEGF was also shown to mediate the survival effect of CD40 ligand (CD154), a molecule expressed on activated T cells, monocytes, macrophages, and other components of the microenvironment[137]. These authors demonstrated that the anti-apoptotic effect of CD154 required the cooperative signaling provided by both CD40 and VEGFR1, 2, resulting in NF-κB activation and upregulation of the protein survivin[137]. Functional cooperation of VEGF receptors and other cell surface molecules, such as integrins, has been clearly established in the context of endothelial cells and tumor angiogenesis[138]. In CLL cells, we demonstrated that the α4β1 integrin is associated with VEGFR2 and modulates VEGF functions on these cells, including the survival effect induced by exogenous VEGF[65]. MMP-9 It is well established that MMPs facilitate cell migration by degrading basement membranes and extracellular matrices, as well as by releasing matrix-bound growth factors and chemokines[130]. It is also well established that many MMPs may display non-catalytic activities, mostly by localizing at the cell surface, either via transmembrane domains (MT-MMPs) or by binding to specific cell surface receptors[131]. MMP-9 is the main MMP expressed by CLL cells and localizes at the cell surface by binding to the α4β1 integrin/CD44v complex[75]. Our group showed that CLL migration in vitro as well as in vivo homing to bone marrow and spleen requires optimal MMP-9 expression, and that above these optimal levels migration is inhibited[75,77]. This was demonstrated using the CLL-derived MEC-1 cell line stably transfected with MMP-9 or empty vector as control, as well as primary CLL cells previously incubated with MMP-9[77]. Inhibition of CLL cell migration by elevated concentrations of MMP-9 was partly due to the downregulation of migration regulatory pathways such as those involving the GTPase RhoA and the kinases Akt, ERK, and FAK, together with the concomitant upregulation of p190RhoGAP (RhoA inhibitor) and PTEN (Akt/ERK/FAK inhibitor)[77]. Moreover, a proteolytically inactive MMP-9 mutant had a partial migration inhibitory effect, indicating that both catalytic and non-catalytic MMP-9 functions were involved[132]. Notably, the dual regulatory role of MMP-9 likely operates in vivo since: (1) CLL cell interaction with stroma increases cell-bound MMP-9[32,81,85]; (2) CLL cells from lymphoid tissues express more MMP-9 than their peripheral blood counterparts[81]; and (3) MMP-9 is present in CLL tissues[79]. Upregulation of MMP-9 in CLL cells and tissues is also induced by α4β1 integrin ligation, chemokine (CXCL12 and CCL21)-receptor interactions, or CD38 interactions[15,42,133]. Elevated levels of MMP-9 in lymphoid tissues would therefore favor the retention of CLL cells in these niches and contribute to disease progression. Page 12 of García-Pardo et al. J Cancer Metastasis Treat 2021;7:62 https://dx.doi.org/10.20517/2394-4722.2021.103 22 García-Pardo et al. J Cancer Metastasis Treat 2021;7:62 https://dx.doi.org/10.20517/2394-4722.2021.10 bFGF In initial studies, it was shown that elevated intracellular levels of bFGF correlated with CLL cell resistance to fludarabine in vitro and that addition of bFGF delayed apoptosis in fludarabine-treated cells[44]. Using CLL-derived cell lines and primary CLL cells, it was also demonstrated that bFGF delayed fludarabine- induced apoptosis by upregulating Bcl-2, at both mRNA and protein levels[139]. Bcl-2 expression was also shown to positively correlate with the levels of bFGF in serum in the 85 patients studied[140]. In another study, Romanov et al.[141] reported that bFGF suppressed p53 activation in cultured CLL cells exposed to ionizing radiation, mainly by upregulating the p53-inhibitory protein MDM2 (mouse double minute 2). bFGF may thus induce CLL cell survival by several mechanisms. Moreover, the survival activity induced by bFGF interaction with its FGFR3 receptor in CLL cells can be potentiated by the tyrosine kinase Axl, which forms a complex with FGFR3 and modulates its signaling pathway[60]. Ang-2 and TSP-1 g It was initially reported that Ang-2 induces CLL cell survival after 24 h but that, at longer times, Ang-2 has a pro-apoptotic activity[68]. This was attributed to the transient modulation of the Tie-2 receptor, as well as of caspases and ATP content[68]. However, another study has shown that the Ang-2/Tie-2 axis induces CLL cell survival even after 96 h of treatment[70]. These authors also showed that Tie-2 engagement by Ang-2 activated the PI3-K-Akt signaling pathway, and this was abolished by a Tie-2 kinase inhibitor, which, in fact, induced apoptosis[70]. This study therefore supports an autocrine role of Ang-2 which affects CLL cell survival. In contrast to the described pro-survival function of some angiogenic factors, binding of TSP-1 to its receptor CD47 was shown to induce apoptosis in CLL cells[73]. The mechanism involved in this action was Page 13 of García-Pardo et al. J Cancer Metastasis Treat 2021;7:62 https://dx.doi.org/10.20517/2394-4722.2021.103 22 Page 13 of 22 independent of caspase activation and was not overcome by survival factors such as IL-4 or γ-interferon. It was later demonstrated that induction of apoptosis by TSP-1-CD47 interaction was mediated by cytoskeleton reorganization, mainly involving the small GTP binding protein Cdc42 and the Wiskott- Aldrich Syndrome protein signaling pathway[74]. Another study has shown that peptides derived from the C- terminal domain of TSP-1 targeted CD47 and efficiently induced apoptosis of CLL cells[142]. bFGF This apoptotic effect involved activation of phospholipase C γ-1 and was also observed in vivo when injecting these peptides in a CLL xenograft murine model[142]. MMP-9 A role for MMP-9 in CLL cell survival was first demonstrated in co-cultures of CLL and bone marrow stromal cells[143]. These authors showed that blocking the constitutive MMP-9 activity with neutralizing antibodies suppressed the anti-apoptotic effect of stromal cells. We reported that culturing CLL cells on immobilized or soluble MMP-9 prevented their spontaneous apoptosis[81]. The MMP-9 effect was dose- dependent and persisted for the seven days of the assay. Importantly, induction of cell survival did not involve the proteolytic activity of MMP-9 but was mediated by MMP-9 binding to α4β1 integrin and CD44v at the CLL cell surface. Indeed, a catalytically inactive MMP-9 mutant or the isolated hemopexin domain of MMP-9 fully reproduced the survival-supporting function of the molecule. This study further showed that MMP-9 (or its hemopexin domain) binding to these receptors induces an intracellular signaling pathway consisting in Lyn activation, STAT3 phosphorylation, and Mcl-1 upregulation. This pathway was observed in all CLL cases studied and was active in CLL lymphoid tissues[81]. Besides preventing spontaneous apoptosis, MMP-9 also protected CLL cells from the apoptotic effect of cytotoxic drugs, such as fludarabine or arsenic trioxide[76]. This study showed that both of these compounds transcriptionally upregulated MMP-9, which mainly localized at the CLL cell surface. Blocking MMP-9 with antibodies reduced the protective effect of stromal cells to the apoptotic function of arsenic trioxide or fludarabine. Additionally, MMP-9 induced CLL cell drug resistance by modulating the balance of anti- and pro- apoptotic proteins of the Bcl-2 family towards an anti-apoptotic pattern[76]. Induction of cell survival therefore represents another contribution of MMP-9 to CLL progression. Other possible roles of angiogenic factors in CLL During the course of CLL, approximately 2%-10% of patients develop an aggressive lymphoma, known as Richter’s transformation or Richter’s syndrome[144]. In the CLL phase, risk factors for Richter’s transformation include an advanced clinical stage, presence of poor prognostic markers (unmutated IGVH, CD38, and CD49d), and genetic aberrations/mutations of specific genes (TP53, NOTCH1, and MYC)[144]. Other factors, however, may certainly contribute to the CLL-lymphoma transformation. A recent study used patient-derived samples and murine animal models and elegantly demonstrated that activation of Akt triggers Richter transformation via induction of Notch1 signaling[145]. Although the possible involvement of angiogenic factors in Richter’s transformation has not been directly addressed, it is well known that activation of the PI3-K/Akt pathway is a major signaling event upon binding of several angiogenic factors (VEGF, bFGF, PDGF, and Ang-2) to their respective receptors[48,49,52,70]. It is therefore possible that these factors, which, as explained above, are present at elevated levels in advanced CLL, contribute to Richter’s transformation by activating Akt. Page 14 of García-Pardo et al. J Cancer Metastasis Treat 2021;7:62 https://dx.doi.org/10.20517/2394-4722.2021.103 22 Page 14 of 22 Endothelin receptor inhibitor CLL cells express high levels of endothelin receptors, which regulate CLL cell-microenvironment interactions and angiogenesis[98]. Maffei et al.[150] reported that inhibiting endothelin receptors with macitentan impaired CLL cell survival, migration, and proliferation. Macitentan also reduced VEGF expression by decreasing HIF-1α accumulation, thus directly affecting angiogenesis[150]. ANTIANGIOGENIC THERAPY IN CLL Despite the development of many new therapies in the last decades targeting specific pathways or molecules, CLL remains an incurable disease. Angiogenesis is a common process in cancer, which helps the growth and dissemination of malignant cells. As in other tumors, angiogenesis provides CLL cells with nutrients and survival signals to facilitate disease progression[20-22]. Targeting angiogenesis thus seems a useful therapeutic strategy against CLL [Table 2]. This section summarizes the results obtained with these antiangiogenic treatments in CLL. Fludarabine Fludarabine is a purine analogue and a frontline treatment in CLL, alone or combined with other compounds such as rituximab and/or cyclophosphamide[1,7,8,146]. The antiangiogenic role of fludarabine was first assessed by Molica et al.[147] who measured the microvessel density in the bone marrow samples of CLL patients and found that this density decreased after fludarabine treatment. The same authors later evaluated the effect of combining fludarabine with alemtuzumab (anti-CD52 antibody) and found an even more significant reduction of angiogenesis in the bone marrow of CLL patients who had received this treatment[148]. In another study, the combination of fludarabine with the γ-secretase inhibitor PF-03084014 resulted in a synergistic induction of apoptosis and the impairment of angiogenesis and cell migration[149]. Importantly, these synergistic effects were specific for Notch1-mutated CLL cells, known to represent a high-risk form of the disease[149]. Lenalidomide The use of inhibitors of the transcription factor NF-κB for the treatment of hematological tumors has been proposed for a long time. Thalidomide was a first-generation drug which downmodulated the levels of angiogenic molecules, such as tumor necrosis factor-α and VEGF[151,152]. Indeed, these two angiogenic factors were downregulated in CLL patients treated with thalidomide in combination with fludarabine[153]. Lenalidomide is a thalidomide analog frequently used as a therapeutic agent in hematological malignancies, including multiple myeloma, myelodysplastic syndrome, and CLL[154]. In CLL, lenalidomide was shown to be very efficient in patients with relapsed or refractory disease[155,156]. Lenalidomide has antiangiogenic and antitumor activity as it downregulates cytokines such as VEGF in CLL patients[157]. However, a clinical trial has reported the appearance of venous thrombosis in approximately 18% of the patients receiving lenalidomide, together with the upregulation of soluble vascular endothelial adhesion molecule 1, tumor necrosis factor-α, and C-reactive protein[158]. Other clinical trials have shown that lenalidomide increases the efficiency of fludarabine and rituximab, although this effect appears to be dependent on specific clinical features of the patients[159-161]. Bevacizumab and VEGF receptor inhibitors Bevacizumab and VEGF receptor inhibitors Tested reagents in antiangiogenic therapy for CLL Drug/procedure Target Fludarabine and cyclophosphamide, and rituximab Patients with progressive/advanced CLL Fludarabine Patients with Binet stage B CLL Fludarabine-induction and alemtuzumab Patients with progressive/advanced CLL Fludarabine and thalidomide Patients with progressive/advanced CLL Lenalidomide Patients with relapsed/refractory CLL Lenalidomide Patients with relapsed/refractory CLL Lenalidomide and rituximab Patients with CLL (untreated) and patients with relapsed CLL Lenalidomide, fludarabine and rituximab Patients with CLL (untreated) and patients with relapsed CLL Fludarabine, cyclophosphamide, and lenalidomide Patients with relapsed/refractory CLL Bevacizumab (AVA) AZD2171 and sunitinib malate Patients with untreated CLL Bevacizumab, pentostatin, cyclophosphamide, and rituximab Patients with untreated CLL Vatalanib and pazopanib Primary samples from CLL patients and healthy donors Sorafenib Primary samples from CLL patients Sorafenib Primary samples from CLL patients Sorafenib and rituximab Primary samples from CLL patients and lymphoma/leukemia cell lines PF-03084014 and Fludarabine Primary samples from CLL patients Epigallocatechin gallate (EGCG) Primary samples from CLL patients CLL: Chronic lymphocytic leukemia; ORR: overall response rate; CRR No complete or partial responses. Declined VEGF levels in plasma (AVA) [165] Increased CRR. Increased VEGF levels after treatments. Reduction in CCL3 and CCL4 levels [166] treatment free survival[166]. Vatalanib and pazopanib are potent orally available VEGF receptor tyrosine kinase inhibitors. Both reagents were shown to efficiently induce apoptosis in CLL cells and diminish tumor growth in murine xenograft models[167]. An additional possible avenue to explore VEGF blockage is through epigallocatechin gallate, a green tea extract component that inhibits the VEGFR activation in CLL cells[34,134,168]. Bevacizumab and VEGF receptor inhibitors Bevacizumab (Avastin, AVA) is a monoclonal antibody targeting VEGF with proven antiangiogenic properties in multiple hematologic malignancies, such as acute myeloid leukemia, CLL, and non-Hodgkin’s lymphoma[162-164]. Despite its potential relevance in the clinic, the administration of bevacizumab, as a single agent, to CLL patients has shown no significant improvement in clinical trials[165]. However, when bevacizumab was given to CLL patients in combination with other conventional therapies (pentostatin, cyclophosphamide, and rituximab), clinical trials have shown that it prolongs the progression free and Page 15 of 22 Page 15 of García-Pardo et al. J Cancer Metastasis Treat 2021;7:62 https://dx.doi.org/10.20517/2394-4722.2021.103 22 Table 2. Tested reagents in antiangiogenic therapy for CLL Drug/procedure Target Effect Ref. Fludarabine and cyclophosphamide, and rituximab Patients with progressive/advanced CLL No detectable disease on flow cytometry. Increased ratio of CRR [146] Fludarabine Patients with Binet stage B CLL Diminished microvessel density in BM. Increased ratio of CRR [147] Fludarabine-induction and alemtuzumab Patients with progressive/advanced CLL Diminished microvessel area and CLL and mast cells in BM [148] Fludarabine and thalidomide Patients with progressive/advanced CLL Diminished number of CLL cells. Increased CRR and nodular partial remission [153] Lenalidomide Patients with relapsed/refractory CLL Diminished number of CLL cells. Increased CRR [155] Lenalidomide Patients with relapsed/refractory CLL Diminished CLL survival on HUVEC cells. Diminished microvessel density. Downregulation of VEGF and THBS-1 [157] Lenalidomide and rituximab Patients with CLL (untreated) and patients with relapsed CLL Increased apoptosis of CLL cells. Increased ORR. Absence of mutations in the Notch pathway. Diminished baseline β2- microglobulin [161] Lenalidomide, fludarabine and rituximab Patients with CLL (untreated) and patients with relapsed CLL Increased ORR and MRD negativity. Direct effects on TP53 mutation and Notch [161] Fludarabine, cyclophosphamide and Patients with relapsed/refractory CLL Increased CLL cell death. Increased CRR and ORR [159,160] Table 2. Tested reagents in antiangiogenic therapy for CLL Drug/procedure Target Effect Ref. Fludarabine and cyclophosphamide, and rituximab Patients with progressive/advanced CLL No detectable disease on flow cytometry. Increased ratio of CRR [146] Fludarabine Patients with Binet stage B CLL Diminished microvessel density in BM. Increased ratio of CRR [147] Fludarabine-induction and alemtuzumab Patients with progressive/advanced CLL Diminished microvessel area and CLL and mast cells in BM [148] Fludarabine and thalidomide Patients with progressive/advanced CLL Diminished number of CLL cells. Increased CRR and nodular partial remission [153] Lenalidomide Patients with relapsed/refractory CLL Diminished number of CLL cells. Bevacizumab and VEGF receptor inhibitors Increased CRR [155] Lenalidomide Patients with relapsed/refractory CLL Diminished CLL survival on HUVEC cells. Diminished microvessel density. Downregulation of VEGF and THBS-1 [157] Lenalidomide and rituximab Patients with CLL (untreated) and patients with relapsed CLL Increased apoptosis of CLL cells. Increased ORR. Absence of mutations in the Notch pathway. Diminished baseline β2- microglobulin [161] Lenalidomide, fludarabine and rituximab Patients with CLL (untreated) and patients with relapsed CLL Increased ORR and MRD negativity. Direct effects on TP53 mutation and Notch [161] Fludarabine, cyclophosphamide, and lenalidomide Patients with relapsed/refractory CLL Increased CLL cell death. Increased CRR and ORR [159,160] Bevacizumab (AVA) AZD2171 and sunitinib malate Patients with untreated CLL No complete or partial responses. Declined VEGF levels in plasma (AVA) [165] Bevacizumab, pentostatin, cyclophosphamide, and rituximab Patients with untreated CLL Increased CRR. Increased VEGF levels after treatments. Reduction in CCL3 and CCL4 levels [166] Vatalanib and pazopanib Primary samples from CLL patients and healthy donors Increased CLL cell apoptosis (Caspase dependent). Decreased levels of Mcl-1. Inactivation of VEGFR. Reduction of tumor growth in xenograft models [167] Sorafenib Primary samples from CLL patients Increased CLL cell death. Downregulation of Mcl-1. Destabilization of the mitochondrial membrane potential. Caspase activation [169] Sorafenib Primary samples from CLL patients Increased apoptosis in ZAP70+ CLL cells. Inhibition of ERK pathway. Increased CLL apoptosis in cocultures with nurse- like cells [170] Sorafenib and rituximab Primary samples from CLL patients and lymphoma/leukemia cell lines Increased CLL apoptosis. Downregulation of membrane- bound complement regulatory proteins (mCRPs) [171] PF-03084014 and Fludarabine Primary samples from CLL patients Diminished angiogenesis and CXCL12-induced chemotaxis. Inhibition of Notch pathway. Upregulation of HRK gene and downregulation of MMP-9,IL32, Rac2, and actin polymerization [149] Epigallocatechin gallate (EGCG) Primary samples from CLL patients Apoptosis of CLL cells on stromal cocultures [168] CLL: Chronic lymphocytic leukemia; ORR: overall response rate; CRR: complete remission rate; HUVEC: human umbilical vein endothelial cells. Table 2. Tested reagents in antiangiogenic therapy for CLL Table 2. Kinase inhibitors and other therapies Sorafenib is a well-known multikinase inhibitor with proven effective roles in tumor cell signaling, proliferation, and angiogenesis. Sorafenib has been shown to a potent inducer of apoptosis in CLL cells, by a mechanism which involves downregulation of the anti-apoptotic protein Mcl-1[169]. This study also showed Page 16 of García-Pardo et al. J Cancer Metastasis Treat 2021;7:62 https://dx.doi.org/10.20517/2394-4722.2021.103 22 García-Pardo et al. J Cancer Metastasis Treat 2021;7:62 https://dx.doi.org/10.20517/2394-4722.2021.103 that sorafenib was able to overcome the drug resistance effect of stromal cells on CLL cells[169]. Another report has shown that sorafenib blocked the survival signals induced by CXCL12 engagement to its receptor CXCR4, particularly the activation of ERK and MEK kinases, and induced apoptosis in a caspase dependent manner[170]. In another study, sorafenib improved the response of CLL cells to the cytotoxic effect of rituximab or ofatumumab, and this effect involved a decrease in the expression of complement regulatory proteins[171]. It was therefore suggested that sorafenib could constitute a potential second line therapy for refractory CLL patients. Altogether these studies indicate that treatments addressing angiogenic factors and their receptors may constitute a useful approach to treat CLL, particularly when used in combination therapies. CONCLUSION Increased bone marrow angiogenesis is a common feature found in CLL, and it is widely accepted that it contributes to the pathogenesis of the disease. CLL cells are active contributors to this aberrant angiogenesis, as they produce and secrete many angiogenic factors and express angiogenic receptors. CLL cells in niches establish functional bidirectional interactions with microenvironmental cells, which induce a proangiogenic profile in CLL cells and enhanced the angiogenic capacity of stromal cells. Clinical trials addressing angiogenic factors have proven to be insufficient as single treatments and indicate that these therapies should rather be used in combined treatments. Moreover, CLL cells in tissues receive survival and proliferation signals that contribute to disease progression. Consequently, the CLL microenvironment is now considered a crucial target for treatment, and current therapies are aimed at the signaling pathways induced by CLL cell-microenvironment interactions. DECLARATIONS Authors’ contributions Contributed to the preparation of the manuscript: García-Pardo A, Redondo-Muñoz J DECLARATIONS Authors’ contributions Availability of data and materials Not applicable. Financial support and sponsorship Work for the author’s laboratory was supported by grants SAF2009-07035, SAF2012-31613, SAF2015- 69180-R, PI060400, RD06/0020/0011, RD12/0036/0061 (to García-Pardo A) and SAF2017-86327-R (to Redondo-Muñoz J) from the Ministerio de Ciencia e Innovacion- Fondo Europeo de Desarrollo Regional (FEDER), Madrid; P2010/BMD-2314 from the Comunidad de Madrid/European Union (to García-Pardo A); the Fundación de Investigación Mutua Madrileña (to García-Pardo A); the 2020 Leonardo Grant for Researchers and Cultural Creators (BBVA Foundation) (to Redondo-Muñoz J). Conflicts of interest Conflicts of interest Both authors declared that there are no conflicts of interest. Both authors declared that there are no conflicts of interest. Ethical approval and consent to participate Ethical approval and consent to participate Not applicable. Copyright © The Author(s) 2021. © The Author(s) 2021. Consent for publication Not applicable. Not applicable. 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https://openalex.org/W3110732799	https://zenodo.org/records/4319391/files/jhms0343%20heriberto.pdf	English	null	A Qualitative Study of Patients’ Perceptions of Dental Care in Primary Health Care	Journal of Health and Medical Sciences	2,020	cc-by	7,043	Journal of Health and Medical Sciences Sanchez, Heriberto F., Vargas, Andrea Maria D., Werneck, Marcos Azeredo F., and Ferreira, Efigênia F. (2020), A Qualitative Study of Patients’ Perceptions of Dental Care in Primary Health Care. In: Journal of Health and Medical Sciences, Vol.3, No.4, 535-543. ISSN 2622-7258 DOI: 10.31014/aior.1994.03.04.145 The online version of this article can be found at: https://www.asianinstituteofresearch.org/ Journal of Health and Medical Sciences Sanchez, Heriberto F., Vargas, Andrea Maria D., Werneck, Marcos Azeredo F., and Ferreira, Efigênia F. (2020), A Qualitative Study of Patients’ Perceptions of Dental Care in Primary Health Care. In: Journal of Health and Medical Sciences, Vol.3, No.4, 535-543. ISSN 2622-7258 DOI: 10.31014/aior.1994.03.04.145 The online version of this article can be found at: https://www.asianinstituteofresearch.org/ Journal of Health and Medical Sciences Sanchez, Heriberto F., Vargas, Andrea Maria D., Werneck, Marcos Azeredo F., and Ferreira, Efigênia F. (2020), A Qualitative Study of Patients’ Perceptions of Dental Care in Primary Health Care. In: Journal of Health and Medical Sciences, Vol.3, No.4, 535-543. Sanchez, Heriberto F., Vargas, Andrea Maria D., Werneck, Marcos Azeredo F., and Ferreira, Efigênia F. (2020), A Qualitative Study of Patients’ Perceptions of Dental Care in Primary Health Care. In: Journal of Health and Medical Sciences, Vol.3, No.4, 535-543. ISSN 2622-7258 DOI: 10.31014/aior.1994.03.04.145 DOI: 10.31014/aior.1994.03.04.145 Abstract Knowledge of patients' views can contribute to the strengthening of health services. The aim of this study is to describe the patients' perception of a public oral health service, contributing to evaluations in health services. This is a qualitative study in which a focus group was conducted, with the participation of six patients of the oral health system in the city of Belo Horizonte, MG, Brazil, all with a minimum experience of three years of using the service. A theoretical model with dimensions aimed at assessing integrality and primary care services was used. In conducting the research, a semi-structured script was used. The data were analyzed by content analysis. The most representative categories for evaluating oral health actions in primary care are the health unit; the welcoming and its relation with the creation of the bond; service with a strong emphasis on the humanized relationship between professional and patient and on teamwork and; as a highlight, citizen participation, based on the recognition of a “system” that prevents the proper functioning of services and that must be fought with citizenship. Patients’ perceptions can be used to assess oral health in primary care from the perspective of those who actually use health services, seeking ultimately to constantly improve them. Knowledge of patients' perceptions may enable organizations to know their performance, through assessment methodologies based on the established perceptions. Keywords: Primary Health Care, Health Evaluation, Qualitative Research, Patient Participation 1 Faculty of Dentistry, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil Faculty of Dentistry, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil Correspondence: Heriberto Fiuza Sanchez. Av. Brasil, 1491/406, 30140-002, Belo Horizonte, Minas Gerais, Brazil. E-mail: heribertofsanchez@gmail.com. The online version of this article can be found at: https://www.asianinstituteofresearch.org/ The online version of this article can be found at: https://www.asianinstituteofresearch.org/ Published by: The Asian Institute of Research The Journal of Health and Medical Sciences is an Open Access publication. It may be read, copied, and distributed free of charge according to the conditions of the Creative Commons Attribution 4.0 International license. The Asian Institute of Research Journal of Health and Medical Sciences is a peer-reviewed International Journal. The journal covers scholarly articles in the fields of Medicine and Public Health, including medicine, surgery, ophthalmology, gynecology and obstetrics, psychiatry, anesthesia, pediatrics, orthopedics, microbiology, pathology and laboratory medicine, medical education, research methodology, forensic medicine, medical ethics, community medicine, public health, community health, behavioral health, health policy, health service, health education, health economics, medical ethics, health protection, environmental health, and equity in health. As the journal is Open Access, it ensures high visibility and the increase of citations for all research articles published. The Journal of Health and Medical Sciences aims to facilitate scholarly work on recent theoretical and practical aspects of Health and Medical Sciences. The Asian Institute of Research Journal of Health and Medical Sciences Vol.3, No.4, 2020: 535-543 ISSN 2622-7258 Copyright © The Author(s). All Rights Reserved DOI: 10.31014/aior.1994.03.04.145 The Asian Institute of Research Journal of Health and Medical Sciences Vol.3, No.4, 2020: 535-543 ISSN 2622-7258 Copyright © The Author(s). All Rights Reserved DOI: 10.31014/aior.1994.03.04.145 Heriberto F. Sanchez1, Andrea Maria D. Vargas1, Marcos Azeredo F. Werneck1, Efigênia F. Ferreira1 Heriberto F. Sanchez1, Andrea Maria D. Vargas1, Marcos Azeredo F. Werneck1, Efigênia F. Ferreira1 1. Introduction In 2018, the 40th anniversary of the Alma-Ata World Conference was celebrated, when primary health care (PHC) began to occupy a more central place on the countries' agendas. PHC represents a way to rationalize the increasing costs involved in health care and the possibility of including population contingents that were on the margins of this care. Furthermore, the returns on investing in health, based on methods that include both the benefits of improved economic productivity and the intrinsic value of health, can far exceed the costs (Watkins et al, 2018). The need to improve the oral health conditions of excluded groups motivated the launch of the Oral Health Program of the World Health Organization (WHO, 2002) which includes among its proposals the strengthening of global oral health systems towards PHC (Petersen, 2009). 535 Journal of Health and Medical Sciences Vol.3, No.4, 2020 Asian Institute of Research The Brazilian government decided that oral health should be included in this effort to universalize PHC, through its insertion in the Family Health Strategy (ESF) and based on the guidelines and principles of the Unified Health System (SUS), the Brazilian policy of health. Based on the principle of integrality, public health services must promote health, prevent risk factors and rehabilitate according to the dynamics of the health-disease process (Brasil, 2006). However, there are challenges in the PHC consolidation process in the Brazilian (Mattos et al, 2014) and worldwide (Mosquera et al, 2014; Gurung et al, 2016) context. To overcome this obstacle, the performance and impact of health services on the population's health must be known and the patients’ perception can be a sensitive indicator of the quality of the service provided, related to the greater adequacy in the use of the public health service (Campos, Filho & Castro, 2017). Evaluative research has sought to investigate the expectations and the symbolic universe of these actors, seeking to understand what quality of health services means for them (Amorim et al, 2019). Methodologies that use the patients’ view are seen as an effort in which principles related to individual rights and citizenship are reaffirmed, such as expressed in the concepts of humanization, patient rights, empowerment, assuming a political dimension and a social end (Bosi & Uchimura, 2007). 1. Introduction The perception of patients can originate from factors such as their own experience in the use of services, the experience of other members of the family or the community, their health condition, their view on how the care provided by professionals in the area should be, or their perception of what is disclosed in the media (Vaitsman & Andrade, 2005). Even if the perception of a service is a personal judgment, it is important that the health professional or manager knows the patients' expectations for performance improvements (Fadel & Filho, 2009). The identification of assessment categories for a given group may allow a more consistent assessment, either alone or as a complement to quantitative methodologies (Turato, 2005). The present study aimed to identify categories of evaluation of health services, through the perception of patients about the quality of a public oral health service. 2.1 Qualitative methodology When working with perception, the qualitative methodology was chosen, focused on the meanings and intentionality of actions in the contexts of social structures (Husserl, 1988). Qualitative research is concerned with deepening the understanding of a given issue, much more than the numerical factor. The focus group was the data collection strategy chosen for allowing, through an explicit interaction between the participants, under the guidance and facilitation of the researcher, to explore people's views and experiences on different aspects of daily life. It is possible to have a shared perception on the researched topic, covering the study object more broadly and to understand what people think and why they think about a certain topic, in this case, health services (Kitzinger, 1995). 2.2 Participants The subjects of this research were patients of the public oral health system in Belo Horizonte-MG, a large municipality in Brazil, with approximately 2,500,000 inhabitants, divided into nine health districts and with a tradition in the development of its municipal health system within of PHC. The inclusion criteria aimed to guarantee the heterogeneity and multivocality of the participants, according to the different districts of the municipality. Thus, it was requested that dentists based in different basic units, covering the nine health districts, indicate patients who had a minimum experience of three years of effective use of the oral health service (that is, who sought the service seeking treatments and not just emergency care). They should be adults, of both genders and who show a potential interest in participating in the research. 2.3 Data collection 2.3 Data collection Subsequently, the researcher responsible for data collection contacted the patients indicated by the dentists, when clarifications were made about the research (the objective, the importance of their participation, anonymity), as 536 Journal of Health and Medical Sciences Asian Institute of Research Vol.3, No.4, 2020 well as the invitation to participate in the focus group. After attempts to reconcile the availability of those who were interested in participating, six participants remained, who actually attended the activity. The researcher who conducted the data collection had previous experience in conducting qualitative research and had no connection with the others involved (patients and dentists from the basic units). well as the invitation to participate in the focus group. After attempts to reconcile the availability of those who were interested in participating, six participants remained, who actually attended the activity. The researcher who conducted the data collection had previous experience in conducting qualitative research and had no connection with the others involved (patients and dentists from the basic units). Data collection took place in 2015, in a room specially provided by the Research Institution, which provided the necessary adaptation (comfort, silence, tranquility) for the realization of the focus group. The expenses for locomotion of patients to this location were assumed by the researchers. Initially, the participants were again clarified about the research objectives, oriented about their participation in the group, how to avoid overlapping speech or always talking towards the recorder (in the center of the circle). All 6 subjects remained until the conclusion of the group. There were no interruptions during data collection. For the realization of the focus group, a semi-structured script was used, elaborated from a theoretical model that includes proposals for the evaluation of PHC (Starfield, Shi & Macincko, 2005) and integrality (Silva Jr et al, 2008). After the first conversation with the objective of clarifying and relaxing the group, the motivating question was asked: "How is your arrival, your entry into the dental service at the health unit?" Then the script was explored, in continuity with the established conversation. 2.4 Data analysis The activity was considered finished when all the topics defined in the script were covered and saturation was observed. The recorder was only turned off after everyone had left the site, allowing observations to be recorded after the formal termination of the group. The content obtained was transcribed by the same researcher who conducted the focus group. Data obtained were processed using the content analysis method. The first stage, called pre-analysis, involved the first contacts with the documents to be analyzed and the formal preparation of the material. Subsequently, a fluctuating, exhaustive and repeated reading of the texts was made, which allowed the transformation of raw data into themes and later the obtaining of categories. Next, inferences were made from the data already treated, qualitatively analyzing the themes and categories that constituted the patients' perception of oral health care in PHC. This process was carried out by two researchers, simultaneously and independently and after discussion between the researchers, the categories were agreed (Bardin, 1977). This research was approved by the Research Ethics Committee of the Belo Horizonte City Hall, number 0059.0.410.203-10. All subjects signed an informed consent form. 3.1 My health unit Participants reported the difficulties in gaining access to dental treatment in PHC. Despite the improvement made possible by the insertion of dentistry at PHC, problems persist. Patients speak in few places and at an inappropriate time, in addition to highlighting the need for easy geographic access as a matter of urban planning. "The hours should be longer because there are people who work all day, only at night to look for it (P2)" “... when you are going to create a neighborhood, you have to think about the school, the public health services... the health service unit had to be close to everyone, it is difficult but it had to be. (P4) " Regarding the physical structure of the basic units and the availability of equipment, inputs and human resources, some aspects were pointed out as important in the day-to-day services. “I see the following: ... and if it wasn't so bureaucratic, that it wasn't missing so much material (P5)” “ b d ff l h b f f l (P2)” Regarding the physical structure of the basic units and the availability of equipment, inputs and human resources, some aspects were pointed out as important in the day-to-day services. p p p y y “I see the following: ... and if it wasn't so bureaucratic, that it wasn't missing so much material (P5)” “... our biggest difficulty is the number of professionals (P2)” Regarding the offer of services, patients pointed to PHC as a place where basic clinical procedures are performed, unable to respond to demands. There is dissatisfaction in relation to the functional aspect of access, as there is no provision of enough procedures to the needs of the population. “At the health center, it is really basic, it is the basic of the basic (P6)” “At the health center, it is really basic, it is the basic of the basic (P6)” “Because in the basic [PHC] you did the basics, I dream about orthodontics, I want to make a channel treatment, I want to solve my problem ... (P3)” At the health center, it is really basic, it is the basic of the basic (P6) “Because in the basic [PHC] you did the basics, I dream about orthodontics, I want to make a channel treatment, I want to solve my problem ... (P3)” 3.2 I am welcomed and recognized 3.2 I am welcomed and recognized Although PHC is considered the priority access door to the health system, paradoxically, the main barrier can be located in PHC itself, due to barriers to reception. "Well, it is very complicated to go through the" Can I help? "[first contact at reception], the health agent, the doorman ... (P1)" "Well, it is very complicated to go through the" Can I help? "[first contact at reception], the health agent, the doorman ... (P1)" “The doorman already has to deal with so many things, so many diferente areas of the public health service, .... if there was an entrance straight to the dentist, it would already make it easier. ... (P3) " Welcoming may also be in practice a difficulty for those seeking treatment. ng may also be in practice a difficulty for those seeking treatment. “Many times the professional thinks that the reception hours have passed, but does reception have a schedule? And if I left the service, I was feeling sick there, I resisted as long as I could, won't you be welcomed? .... I can't understand it... (P4) ” “Many times the professional thinks that the reception hours have passed, but does reception have a schedule? And if I left the service, I was feeling sick there, I resisted as long as I could, won't you be welcomed? .... I can't understand it... (P4) ” The participants perceive what it feels like to be welcomed and point out in the service organization itself an impediment for this to occur. They recognize that “welcoming” is not being a welcoming moment. “Welcoming is receiving the person, listening to the person, looking at them, even if I had to go back home I would have been treated with respect and I want that from all the professionals, the cleaning lady, the doorman ... I think because due to the demand, they place the reception with a schedule, but reception is not that ... (P4) ” “In the reception they do not let you talk to any professional, or even ask a question to have an answer (P1)” The participants perceive what it feels like to be welcomed and point out in the service organization itself an impediment for this to occur. They recognize that “welcoming” is not being a welcoming moment. 3.3 The service is very good 3. Results The focus group activity was characterized by intense and spontaneous dynamics, lasting 120 minutes, without fatigue or inattention of the participants. All manifested similarly, without the predominance of the speech of some more than others. Of the six participants, five were women and one was a man. The average age of the participants was 40.1 years (25-61). Only one participant had completed high school, and the other five had incomplete high school or complete elementary school. The statements were grouped into four themes, as shown in Chart 1. The patients will be identified at the end of the speeches through a number, as a way of preserving anonymity. Chart 1: Themes and categories observed in the focus group on dental care in PHC units, Belo Horizonte, Brazi 2015. THEMES CATEGORIES My health unit Access Physical structure and inputs Services/procedures offer Attention network I am welcomed and recognized Welcoming Bond The service is good Humanized relationship s and categories observed in the focus group on dental care in PHC units, Belo Horizonte, Brazil, 537 Journal of Health and Medical Sciences Vol.3, No.4, 2020 Asian Institute of Research 3.2 I am welcomed and recognized “Welcoming is receiving the person, listening to the person, looking at them, even if I had to go back home I would have been treated with respect and I want that from all the professionals, the cleaning lady, the doorman ... I think because due to the demand, they place the reception with a schedule, but reception is not that ... (P4) ” “In the reception they do not let you talk to any professional, or even ask a question to have an answer (P1)” There may be a greater establishment of a bond when the patient and his demands and needs are welcomed. This relationship was perceived and reported. There may be a greater establishment of a bond when the patient and his demands and needs are welcomed. This relationship was perceived and reported. “...I also understand because they [health workers] work a lot, and then it affects the welcoming and the bond... the doctor I deal with calls us by name, knows my husband, knows my son but how much time she spent to achieve this? That's where you have a bond, but it could be longer if you had more professional time ... (P4) ” “...I also understand because they [health workers] work a lot, and then it affects the welcoming and the bond... the doctor I deal with calls us by name, knows my husband, knows my son but how much time she spent to achieve this? That's where you have a bond, but it could be longer if you had more professional time ... (P4) ” 3.3 The service is very good 538 Journal of Health and Medical Sciences Vol.3, No.4, 2020 Asian Institute of Research The quality of care was consistently related to the humanized and compromised relationship that occurs between patients and professionals, capable of overcoming common structural difficulties in the daily lives of countless public health units. “The conditions of the dentist there... it is basic, but the care he does for any human being who enters there are the best possible (...) he does not do it anymore because he has no conditions, but his care for the patient it is the best possible (P3) ” Teamwork was another category perceived as a quality item in care. The statements indicate the perception that, if there is integration between professionals, there is gain. 3.4 My citizen participation The presence of what they called “system” was remarkable, that is, a series of factors, especially related to public policies and management, that hinder or prevent the health care of the population from being offered according to their needs. “No, it is everybody, he alone [the dentist] will not do anything, the system will not allow him to do anything alone (...) I think it is a huge neglect [structural problems] and I think it's our fault and the politicians' fault, ours because we vote for them.. (P1) ” “It is the system that does not allow certain things (...) this is very sad, this is the system that makes it difficult and then they come and offer us a denture; why didn't you offer a decent treatment back there?? (P3) ” However, some patients are aware of their potential citizens and know the strength that their citizenship has in defining different aspects of service management. “that's the issue of the city health council. Patients, health professionals mobilize to demand improvemen 3.2 I am welcomed and recognized “And this fact of helping [team work] is very important because sometimes in the conversations between them [professionals] there is an exchange of information from patients... I have already witnessed this. The dentist also plays this role, they know a little bit of our life, our day-to-day lives they know a little about our history (P6) ” 4. Discussion 4.1 My health unit 4.1 My health unit In the medium or long term, the population's demand could be reduced (Mc Manus, 2015). In the Brazilian case, dental services are organized on levels of complexity or healthcare networks (primary, secondary, and tertiary) and it cannot be forgotten that some procedures are performed by the Dental Specialty Centers (CEO). These health units are aimed to perform some clinical procedures that are not performed in basic units, such as surgerys, endodontics and care for special patients, but it also fails to meet these needs (Chaves et al, 2011), compromising the integrality and generating a feeling of dissatisfaction among patients, since they apparently do not know the network operation of the services, but expect attention beyond the “basic”. An important benefit of the existence of adequate flows between basic and specialized services is the maintenance of the bond between patients and the professional of origin, considered a reference for the individual or family in primary units. Apparently, there is no uniformity of procedures among the professionals involved in basic and specialized health units, with situations in which the user's “walk” through the health system takes place in an appropriate manner and, at other times, in a unsatisfying way, compromising the integrality and user satisfaction. Some statements were linked to the lack of human resources in the units' daily lives. These problems could be solved with management mechanisms that are more committed to better structural work conditions and more attractive salary and career plans, for example. However, in addition to technical capacity, team members need to identify themselves with a work proposal that often requires creativity, initiative and a vocation for community and group work, something that should start in training professional (Ronzani & Silva, 2008). 4.2 I am welcomed and recognized It was observed a relantionship between the welcoming and the bond, with the first acting as a facilitator for the second. The coexistence time, despite having its fundamental base in access/reception, is an indispensable factor in longitudinality and can, consequently, interfere in the patients' satisfaction and in the resolution. The bond created is not limited to different clinical moments, it expands to different situations in the participants' daily lives and is not limited to the figure of the dentist, being a category that arises from the humanized relationship that took place with this professional. 4.1 My health unit The patients' perception points to access difficulties in its multiple dimensions. Although there are ethical issues that define health as a good that should be accessible to every human being, this is not what is observed, because for patients access means having public health services available, in places close to the homes, and with permissive hours for use, especially for those who work daily hours. This perception is consistent with other studies (Viegas, Carmo & Luz, 2015; Al-Jaber & Da’Ar, 2016) that pointed to similar difficulties, related to the number of professionals, the geographical issue and the costs of dental treatments, an impediment for many people. This situation, especially in times of financial or political crises, should necessarily lead to reflection among professionals and managers involved, due to the increase in the number of unemployed or people without health insurance coverage, as they may increase the demand for care. Deficiencies in these aspects compromise the service provided to patients and, consequently, society's view of SUS or any other public health system in the world. The availability of human resources to work in the basic units is, in fact, considered important, as it relates to the possibilities (or not) of providing services that the community needs. The issue of human resources is not limited to just a numerical factor, since the characteristics and attitudes of the professionals involved influence the population's access (Tesser, Neto & Campos, 2010). This situation was highlighted when commenting on the reception at the health unit. Theoretically, the organization of reception of the patients, a moment called welcoming, includes among its purposes, facilitating patients' access (Coelho & Jorge, 2009). However, it was observed that some organizational aspects of health units, aimed at benefiting welcoming, may actually be preventing this from happening. Thus, access and the construction of the SUS itself may be compromised (Souza et al, 2008), as well as other national health policies based on PHC. 539 Journal of Health and Medical Sciences Vol.3, No.4, 2020 Asian Institute of Research It was also possible to verify in the statements that there is dissatisfaction with the list of clinical procedures offered. There is a great demand for oral health among the population, which often requires different procedures. PHC should be able to take care of providing, in addition to curative/restorative procedures, the prevention and promotion of health. 4.1 My health unit Bond allows integrality to be more easily achieved and also contributes to longitudinality and long lasting therapeutic bond between patients and professionals, from the recognition of a regular source of primary care (Cunha & Giovanella, 2011). The bond must be based on the activity of the multidisciplinary team, as the population must believe in the team's ability to solve their health problems and the team must accept these demands. The bond contributes to more accurate diagnoses and treatments, lower costs of care and greater user satisfaction. It can be said that there is a direct relationship, based on access, between welcoming and bonding, the result of which is care, obtained when health professionals feel and experience the reality of patients. Empowered by multiprofessional work, workers must reconcile work and care, which are not opposing categories, but complement each other (Rodrigues, Lima & Roncalli, 2008). Patients also expressed themselves in relation to the lack of bond in secondary care. They stated that, unlike what happens in PHC, at the secondary level there is no bonding. The service organization itself exacerbates this situation, as the specialized professional is placed as a person who fulfills procedures on patients, does what is requested and forwards them to the unit of origin, not even the documentation remains in place. Everything contributes to the distance perceived by patients. In addition, the majority of professionals working in specialized public care have a dual professional activity, as they work in public and private clinics. Although this double action is not conflicting, it can make it difficult to adjust to the field of public health (Chaves et al, 2011). 4.4 My citizen participation 4.4 My citizen participation The perception expressed by patients about the political system was highlighted. For them, this system, which is external to the units' daily life, but dictates rules, commands, has immense responsibility in the daily life of public health services, in what they come from positive or negative for the population. It is important to note that this category "system" was addressed several times among the participants, generating debates that revealed a feeling of rejection of political practices that are not committed to the well-being of the population. One of the reasons for maintaining uncompromised and disconnected management practices from what SUS proposes may be the municipalization of health. This is a principle that guides Brazilian health policy, thought as a possibility to facilitate greater participation of society in health policies, due to a greater proximity between citizens and local managers. However, municipalization does not take into account the demographic reality of most municipalities, which do not have enough population to demand a health system with different levels of complexity, nor the fact that local power in Brazil has traditionally been the basis for representing private interests linked to land ownership and the basis of the oligarchic domination system. There is a natural stimulus for competition between local authorities and power is usually concentrated in a few hands in small municipalities. Power is priority in relation to the broader interests of the population (Rodrigues, 2014). In contrast to the perceived "system", patients continued to demonstrate, affirming their power as citizens. This citizen vision contributes to social control and integral practices, as they can mobilize managers. It will favor the construction of a society more aware of its rights and responsibilities and contribute to changing the health profile of the population. These perceptions are in line with the concept of empowerment and its interface with health promotion, pointing towards a collective utopia/expectation of social justice and inclusion, especially in national contexts characterized by needs and exclusion. Empowerment contributes to the constant need to debate politicization and health practices (Carvalho & Gastaldo, 2008). The improvement of any national health policy based on PHC involves the victory over disenchantment or discrediting over that policy. Its concrete performance depends on overcoming the problems historically present in societies, such as uncompromised politics and the lack of efficient management (Campos, 2007). Citizen participation is essential for the promotion of sustainable health and health care. 4.3 The service is very good The importance of the humanized relationship between professionals and patients is highlighted. A relationship based on respect and humanistic aspects can overcome several difficulties that PHC units face in their daily lives, 540 Journal of Health and Medical Sciences Asian Institute of Research Vol.3, No.4, 2020 helps to qualify assistance, the quality of care and contributes to a process of reorientation in the production of care (Medeiros, Araújo-Souza & Albuquerque-Barbosal, 2010). Quality is not placed sole under the responsibility of the professional. Patients recognize the limit of this dimension. But a professional with a humanizing capacity can control conflicts that will inevitably not bring benefits in the construction of the desired public health service. helps to qualify assistance, the quality of care and contributes to a process of reorientation in the production of care (Medeiros, Araújo-Souza & Albuquerque-Barbosal, 2010). Quality is not placed sole under the responsibility of the professional. Patients recognize the limit of this dimension. But a professional with a humanizing capacity can control conflicts that will inevitably not bring benefits in the construction of the desired public health service. The importance of the humanized relationship in PHC services is pointed out, as humanization is particularly important in this type of service, focused on the most frequent health demands, which are often on the border between “life problems” and “pathology”, as defined by biomedicine (Nora & Junges, 2013). Hence the importance of multi-professional action and intersectoral articulation, since PHC has this vocation as a “gateway” not only for the health service network, but for a multiplicity of other social demands, which end up being translated into health demands or simply present themselves in the absence of other social spaces for expression. Patients expressed themselves in favor of teamwork. Teamwork is something that, in the view of patients, should happen in the daily life of services and that can qualify the assistance and the relationship between the team and the family. For this, it is essential to develop a communicative practice oriented towards mutual knowledge (Peruzzo et al, 2019; Nora & Junges, 2013). This study confirms the understanding that the specific knowledge of each area can and should be used together to understand certain situations that involve the patients' health. 4.4 My citizen participation The citizen posture presented is very positive, as it will make it possible to create potential conditions to improve health public services (Willianson, 2014). 541 Journal of Health and Medical Sciences Vol.3, No.4, 2020 Asian Institute of Research 5. Conclusions In this study, it was observed that, for patients, the health unit is an important topic in the evaluation of a public oral health service; welcoming and creating the bond; care with an emphasis on the humanized relationship between professional and patient and teamwork. The critical citizen view on public management was highlighted, which is incompatible with the development of SUS or any other national health policy based on PHC. The presence of patients, as well as professionals and managers in the effort to build public health systems is essential, The knowledge of the patients' perceptions may enable the elaboration of a characterization of the service provided. The evaluation will allow organizations to know their performance and organize their services not only for the demand, but according to the needs pointed out by the patients, true reasons for the existence of public health services. Although there is a need to caution in generalizing qualitative studies, the results may have applicability in similar contexts. The fact that not all invited patients wanted or were able to participate generated a small universe of participating patients, which may have omitted unreported perceptions and experiences. It is suggested that further studies be carried out using different qualitative approaches and in different contexts. References Al-Jaber, A., Da’Ar, O. (2016). 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https://openalex.org/W2022143936	https://europepmc.org/articles/pmc2968206?pdf=render	English	null	1,2,3,6,7,8-Hexahydrocinnolino[5,4,3-<i>cde</i>]cinnoline	Acta crystallographica. Section E	2,009	cc-by	2,044	organic compounds Monoclinic, P21=c a = 9.698 (5) A˚ b = 5.875 (3) A˚ c = 10.023 (5) A˚ = 117.314 (6) V = 507.4 (4) A˚ 3 Z = 2 Mo K radiation = 0.09 mm1 T = 298 (2) K 0.55 0.41 0.09 mm Data collection Bruker SMART CCD area-detector diffractometer Absorption correction: multi-scan (SADABS; Sheldrick, 1996) Tmin = 0.953, Tmax = 0.992 2508 measured reﬂections 890 independent reﬂections 575 reﬂections with I > 2(I) Rint = 0.028 Reﬁnement R[F 2 > 2(F 2)] = 0.041 wR(F 2) = 0.123 S = 1.01 890 reﬂections 97 parameters All H-atom parameters reﬁned max = 0.16 e A˚ 3 min = 0.15 e A˚ 3 Acta Crystallographica Section E Structure Reports Online ISSN 1600-5368 Acta Crystallographica Section E Structure Reports Online ISSN 1600-5368 Z = 2 Mo K radiation = 0.09 mm1 T = 298 (2) K 0.55 0.41 0.09 mm Zhi-Qiang Gao College of Chemistry and Environmental Engineering, Chongqing University of Arts and Sciences, Chongqing, People’s Republic of China Correspondence e-mail: haowenjuanxz@126.com College of Chemistry and Environmental Engineering, Chongqing University of Arts and Sciences, Chongqing, People’s Republic of China Correspondence e-mail: haowenjuanxz@126.com 97 parameters All H-atom parameters reﬁned max = 0.16 e A˚ 3 min = 0.15 e A˚ 3 Received 23 December 2008; accepted 27 December 2008 Key indicators: single-crystal X-ray study; T = 298 K; mean (C–C) = 0.003 A˚; R factor = 0.041; wR factor = 0.123; data-to-parameter ratio = 9.2. Data collection: SMART (Bruker, 1998); cell reﬁnement: SAINT (Bruker, 1999); data reduction: SAINT; program(s) used to solve structure: SHELXS97 (Sheldrick, 2008); program(s) used to reﬁne structure: SHELXL97 (Sheldrick, 2008); molecular graphics: SHELXTL (Sheldrick, 2008); software used to prepare material for publication: SHELXTL. The title compound, C12H12N4, was synthesized by the reaction of hydrazine hydrate and 9-methyl-3,4,6,7-tetra- hydro-2H-xanthene-1,8(5H,9H)-dione in ethanol. In the crystal, the molecule lies across an inversion centre. The pyridazine rings are coplanar and the C6 rings adopt envelope conformations. Supplementary data and ﬁgures for this paper are available from the IUCr electronic archives (Reference: CI2750). Related literature For the biological properties of cinnoline and its derivatives, see: Abdelrazek et al. (2006); Gomtsyan et al. (2005); Inoue et al. (1993); Lewgowd & Stanczak (2007); Lewgowd et al. (2005); Singh et al. (2003); Stefanska et al. (2003); Tutsumi et al. (1992). 1,2,3,6,7,8-Hexahydrocinnolino[5,4,3- cde]cinnoline Zhi-Qiang Gao References Abdelrazek, F. M., Metz, P., Metwally, N. H. & El-Mahrouky, S. F. (2006). Arch. Pharm. 339, 456–460. Bruker (1998). SMART. Bruker AXS Inc., Madison, Wisconsin, USA. Bruker (1999). SAINT. Bruker AXS Inc., Madison, Wisconsin, USA. ( ) , , Gomtsyan, A. et al. (2005). Med. Chem. 48, 744–752. Gomtsyan, A. et al. (2005). Med. Chem. 48, 744–752. Inoue, S., Yasaki, A., Mochizuki, H., Tutsumi, H., Murata, M. & Sakane, K. (1993). Japanese Patent No. 05213951. Experimental Crystal data C12H12N4 Mr = 212.26 Lewgowd, W. & Stanczak, A. (2007). Arch. Pharm. 340, 65–8 Lewgowd, W., Stanczak, A., Ochocki, Z., Krajewska, U. & Rozalski, M. (2005). Acta Pol. Pharm. 62, 105–110. Sheldrick, G. M. (1996). SADABS. University of Go¨ttingen, Germany Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122. Singh, S. K., Ruchelman, A. L., Zhou, N., Li, T.-K., Liu, An., Liu, L. F. & Lavoie, E. J. (2003). Med. Chem. Res. 12, 1–12. Stefanska, B., Arciemiuk, M., Bontemps-Gracz, M. M., Dzieduszycka, M., Kupiec, A., Martelli, S. & Borowski, E. (2003). Bioorg. Med. Chem. 11, 561– 572. Experimental Tutsumi, H., Terasawa, T., Barret, D., Mutata, M., Sakane, K., Yazaki, A. & Inoue, S. (1992). Japanese Patent No. 9215584; Chem. Abstr. 118, 254944w. Mr = 212.26 Mr = 212.26 Acta Cryst. (2009). E65, o239 Zhi-Qiang Gao o239 doi:10.1107/S1600536808044036 Acta Cryst. (2009). E65, o239 [ doi:10.1107/S1600536808044036 ] Acta Cryst. (2009). E65, o239 [ doi:10.1107/S1600536808044036 ] Comment It is well known that six-membered nitrogen-containing heterocycles are abundant in numerous natural products that exhibit important biological properties. For example, cinnolines and their derivatives exhibit a diverse range of biological properties (Lewgowd & Stanczak, 2007) such as molluscicidal activity (Abdelrazek et al., 2006), cytotoxic activity (Lewgowd et al., 2005), transient receptor potential vanilloid 1 (TRPV1) receptor antagonists (Gomtsyan et al., 2005), and topoisomerase I (TOP1)-targeting activity and cytotoxicity (Singh et al., 2003). They also acted as anticancer agents active on a multidrug resistant cell line (Stefanska et al., 2003). They can also be used as bactericides in pharmaceutical industry (Inoue et al., 1993; Tutsumi et al.,1992). The chemistry of cinnolines has received much attention based on the above facts. The title molecule lies across an inversion centre (Fig. 1). The two pyridazine rings (C1/C2/C2A/C3A/N2/N1 and C1A/ C2A/C2/C3/N2A/N1A) are conjugated and are coplanar. The two cyclohexene rings (C1—C6 and C1A—C6A) adopt en- velope conformations, with atoms C5 and C5A deviate from the C1/C2/C3/C4/C6 and C1A/C2A/C3A/C4A/C6A planes by 0.656 (3) and 0.656 (3) Å, respectively. A view of the crystal packing is shown in Fig.2. Experimental The title compound was prepared by the reaction of 3,4,6,7-tetrahydro -9-methyl-2H-xanthene-1,8(5H,9H)-dione (2 mmol) and hydrazine hydrate 80% (8 mmol) in ethanol (8 ml) stirring at 353 K (yield 84%; m.p. 543–544 K). Single crystals suitable for X-ray diffraction were obtained from an ethanol solution by slow evaporation. 1,2,3,6,7,8-Hexahydrocinnolino[5,4,3-cde]cinnoline Z.-Q. Gao supplementary materials supplementary materials Refinement All H atoms were located in a difference Fourier map and refined freely [C-H = 0.95 (2)-1.04 (2) Å Figures Fig. 1. The molecular structure of the title compound, showing 50% probability displacement ellipsoids and the atom-numbering scheme. Atoms labelled with the suffix A are generated by the symmetry operation (1-x, -y, 1-z). Figures Fig. 1. The molecular structure of the title compound, showing 50% probability displacement ellipsoids and the atom-numbering scheme. Atoms labelled with the suffix A are generated by the symmetry operation (1-x, -y, 1-z). sup-1 supplementary materials Fig. 2. Molecular packing in the title compound, viewed approximately along the a axis. 1,2,3,6,7,8-Hexahydrocinnolino[5,4,3-cde]cinnoline 1,2,3,6,7,8-Hexahydrocinnolino[5,4,3-cde]cinnoline Special details Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes) are estimated using the full covariance mat- rix. The cell e.s.d.'s are taken into account individually in the estimation of e.s.d.'s in distances, angles and torsion angles; correlations between e.s.d.'s in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s. planes. Refinement. Refinement of F2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F2, convention- al R-factors R are based on F, with F set to zero for negative F2. The threshold expression of F2 > σ(F2) is used only for calculating R- factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F2 are statistically about twice as large as those based on F, and R- factors based on ALL data will be even larger. supplementary materials 97 parameters Δρmin = −0.15 e Å−3 Primary atom site location: structure-invariant direct methods Extinction correction: none Δρmin = −0.15 e Å−3 Δρmin = −0.15 e Å−3 Primary atom site location: structure-invariant direct methods Extinction correction: none Primary atom site location: structure-invariant direct methods Extinction correction: none Extinction correction: none 1,2,3,6,7,8-Hexahydrocinnolino[5,4,3-cde]cinnoline 1,2,3,6,7,8-Hexahydrocinnolino[5,4,3-cde]cinnoline Crystal data C12H12N4 F000 = 224 Mr = 212.26 Dx = 1.389 Mg m−3 Monoclinic, P21/c Melting point = 543–544 K Hall symbol: -P 2ybc Mo Kα radiation λ = 0.71073 Å a = 9.698 (5) Å Cell parameters from 734 reflections b = 5.875 (3) Å θ = 2.4–26.3º c = 10.023 (5) Å µ = 0.09 mm−1 β = 117.314 (6)º T = 298 (2) K V = 507.4 (4) Å3 Plate, colourless Z = 2 0.55 × 0.41 × 0.09 mm F000 = 224 Dx = 1.389 Mg m−3 Melting point = 543–544 K Mo Kα radiation λ = 0.71073 Å Cell parameters from 734 reflections θ = 2.4–26.3º µ = 0.09 mm−1 T = 298 (2) K Plate, colourless 0.55 × 0.41 × 0.09 mm Data collection Bruker SMART CCD area-detector diffractometer Radiation source: fine-focus sealed tu Monochromator: graphite T = 298(2) K φ and ω scans Absorption correction: multi-scan (SADABS; Sheldrick, 1996) Tmin = 0.953, Tmax = 0.992 2508 measured reflections Data collection Bruker SMART CCD area-detector diffractometer 890 independent reflections Radiation source: fine-focus sealed tube 575 reflections with I > 2σ(I) Monochromator: graphite Rint = 0.028 T = 298(2) K θmax = 25.0º φ and ω scans θmin = 4.1º Absorption correction: multi-scan (SADABS; Sheldrick, 1996) h = −11→11 Tmin = 0.953, Tmax = 0.992 k = −6→6 2508 measured reflections l = −11→11 uker SMART CCD area-detector fractometer 890 independent reflections diation source: fine-focus sealed tube 575 reflections with I > 2σ( onochromator: graphite Rint = 0.028 = 298(2) K θmax = 25.0º and ω scans θmin = 4.1º bsorption correction: multi-scan ADABS; Sheldrick, 1996) h = −11→11 min = 0.953, Tmax = 0.992 k = −6→6 08 measured reflections l = −11→11 890 independent reflections 575 reflections with I > 2σ(I) Rint = 0.028 θmax = 25.0º θmin = 4.1º h = −11→11 k = −6→6 l = −11→11 Secondary atom site location: difference Fourier map Hydrogen site location: inferred from neighbouring sites All H-atom parameters refined All H-atom parameters refined w = 1/[σ2(Fo 2) + (0.0655P)2 + 0.0552P] where P = (Fo 2 + 2Fc 2)/3 (Δ/σ)max = 0.001 Δρmax = 0.16 e Å−3 w = 1/[σ2(Fo 2) + (0.0655P)2 + 0.0552P] where P = (Fo 2 + 2Fc 2)/3 (Δ/σ)max = 0.001 Δρmax = 0.16 e Å−3 w = 1/[σ2(Fo 2) + (0.0655P)2 + 0.0552P] where P = (Fo 2 + 2Fc 2)/3 (Δ/σ)max = 0.001 Δρmax = 0.16 e Å−3 sup-2 supplementary materials Special details Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) 3 Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) x y z Uiso/Ueq N1 0.7159 (2) −0.2597 (3) 0.65617 (19) 0.0547 (6) N2 0.5861 (2) −0.3398 (3) 0.66438 (18) 0.0555 (6) C1 0.7050 (2) −0.0820 (4) 0.5729 (2) 0.0466 (6) C2 0.56377 (19) 0.0384 (3) 0.49395 (18) 0.0390 (5) C3 0.5474 (2) 0.2352 (3) 0.4062 (2) 0.0450 (5) C4 0.6864 (3) 0.3211 (5) 0.3949 (3) 0.0581 (7) C5 0.8354 (3) 0.2567 (4) 0.5326 (3) 0.0647 (7) C6 0.8445 (3) 0.0004 (4) 0.5597 (3) 0.0621 (7) H1 0.682 (2) 0.250 (4) 0.305 (3) 0.065 (6) H2 0.678 (2) 0.487 (4) 0.382 (2) 0.076 (7)* H3 0.930 (3) 0.308 (4) 0.522 (3) 0.086 (8)* H4 0.839 (3) 0.340 (4) 0.623 (3) 0.079 (7)* H5 0.846 (2) −0.086 (4) 0.470 (2) 0.072 (7)* H6 0.936 (3) −0.039 (4) 0.649 (3) 0.081 (7)* Atomic displacement parameters (Å2) U11 U22 U33 U12 U13 U23 N1 0.0567 (12) 0.0508 (12) 0.0500 (10) 0.0164 (9) 0.0187 (9) 0.0061 (8) N2 0.0652 (12) 0.0458 (11) 0.0474 (11) 0.0115 (9) 0.0189 (9) 0.0086 (8) C1 0.0501 (12) 0.0467 (12) 0.0395 (10) 0.0102 (10) 0.0175 (9) −0.0019 (10) C2 0.0445 (11) 0.0386 (11) 0.0306 (9) 0.0072 (8) 0.0146 (8) −0.0029 (8) C3 0.0574 (13) 0.0386 (12) 0.0351 (10) 0.0051 (10) 0.0178 (9) −0.0010 (9) C4 0.0737 (17) 0.0505 (15) 0.0555 (14) −0.0061 (12) 0.0344 (13) −0.0027 (12) C5 0.0595 (15) 0.0680 (17) 0.0681 (16) −0.0087 (13) 0.0306 (13) −0.0101 (13) C6 0.0484 (14) 0.0707 (18) 0.0650 (16) 0.0111 (11) 0.0242 (13) −0.0033 (12) Geometric parameters (Å, °) N1—C1 1.310 (3) C4—C5 1.518 (3) Atomic displacement parameters (Å2) sup-3 supplementary materials supplementary materials N1—N2 1.381 (2) C4—H1 0.98 (2) N2—C3i 1.309 (3) C4—H2 0.98 (2) C1—C2 1.417 (3) C5—C6 1.526 (3) C1—C6 1.499 (3) C5—H3 1.01 (2) C2—C2i 1.373 (3) C5—H4 1.01 (2) C2—C3 1.417 (3) C6—H5 1.04 (2) C3—N2i 1.309 (3) C6—H6 0.95 (2) C3—C4 1.492 (3) C1—N1—N2 120.01 (17) C5—C4—H2 111.0 (13) C3i—N2—N1 120.67 (18) H1—C4—H2 109.4 (18) N1—C1—C2 121.89 (19) C4—C5—C6 111.2 (2) N1—C1—C6 120.07 (18) C4—C5—H3 111.1 (13) C2—C1—C6 118.0 (2) C6—C5—H3 109.2 (14) C2i—C2—C3 118.3 (2) C4—C5—H4 108.6 (14) C2i—C2—C1 117.8 (2) C6—C5—H4 110.1 (13) C3—C2—C1 123.94 (17) H3—C5—H4 106.5 (19) N2i—C3—C2 121.33 (19) C1—C6—C5 110.70 (19) N2i—C3—C4 120.3 (2) C1—C6—H5 107.1 (12) C2—C3—C4 118.39 (18) C5—C6—H5 110.5 (12) C3—C4—C5 111.3 (2) C1—C6—H6 109.2 (14) C3—C4—H1 105.8 (12) C5—C6—H6 111.1 (15) C5—C4—H1 110.6 (12) H5—C6—H6 108.2 (19) C3—C4—H2 108.6 (13) Symmetry codes: (i) −x+1, −y, −z+1. sup-4 supplementary materials Fig. 1 Fig. 1 sup-5 supplementary materials Fig. 2 sup-6
https://openalex.org/W2961662609	https://europepmc.org/articles/pmc6680637?pdf=render	English	null	In Silico Repositioning of Cannabigerol as a Novel Inhibitor of the Enoyl Acyl Carrier Protein (ACP) Reductase (InhA)	Molecules/Molecules online/Molecules annual	2,019	cc-by	6,317	Received: 25 June 2019; Accepted: 13 July 2019; Published: 15 July 2019 Abstract: Cannabigerol (CBG) and cannabichromene (CBC) are non-psychoactive cannabinoids that have raised increasing interest in recent years. These compounds exhibit good tolerability and low toxicity, representing promising candidates for drug repositioning. To identify novel potential therapeutic targets for CBG and CBC, an integrated ligand-based and structure-based study was performed. The results of the analysis led to the identiﬁcation of CBG as a low micromolar inhibitor of the Enoyl acyl carrier protein (ACP) reductase (InhA) enzyme. Keywords: drug repurposing; molecular modelling; cannabinoids; ligand-based virtual screening; docking; BEAR; natural products In Silico Repositioning of Cannabigerol as a Novel Inhibitor of the Enoyl Acyl Carrier Protein (ACP) Reductase (InhA) Luca Pinzi , Christian Lherbet , Michel Baltas , Federica Pellati and Giulio Rastelli 1 Department of Life Sciences, University of Modena and Reggio Emilia, Via Giuseppe Campi 103, 41125 Modena, Italy 2 LSPCMIB, UMR-CNRS 5068, Université Paul Sabatier-Toulouse III, 118 route de Narbonne, 31062 Toulouse CEDEX 9 France y 2 LSPCMIB, UMR-CNRS 5068, Université Paul Sabatier-Toulouse III, 118 route de Narbonne, 31062 Toulouse CEDEX 9, France 2 LSPCMIB, UMR-CNRS 5068, Université Paul Sabatier-Toulouse III, 118 route de Narbonne, 31062 Toulouse CEDEX 9, France * Correspondence: giulio.rastelli@unimore.it; Tel.: +39-059-2058564 molecules molecules Molecules 2019, 24, 2567; doi:10.3390/molecules24142567 www.mdpi.com/journal/molecules molecules molecules 1. Introduction In recent decades, increasing research eﬀorts have been directed towards identifying novel drugs based on unexplored chemical scaﬀolds. However, the rate of drug approvals has become stable, with only a small number of the developed chemical entities entering in therapeutic use, or even in clinical trials [1–4]. As a consequence, discovery strategies have been adopted to reduce failures, time eﬀorts and expenses; in this context, drug repurposing has become one of the most successful strategies to reduce failures typically associated with drug discovery. Drug repurposing consists of identifying novel therapeutic uses for already approved drugs and/or clinical candidates, as it might allow circumventing preclinical optimization issues, such as adverse toxicology proﬁles. Although most drug repurposing success stories derive from serendipity, current eﬀorts are mainly directed toward rationally predicting repurposing through systematic analysis of bioactivity data with computational approaches. Indeed, in silico methods have been successfully used to help delineating new drug repurposing opportunities [5–7]. Several ligand-based and structure-based virtual screening approaches are currently available to support drug discovery programs. However, each in silico method alone could not be suﬃciently able to model the complex interplay between drugs and targets, because of intrinsic limitations [8]. Therefore, the combination of ligand-based and structure-based methods is expected to: (i) provide more robust results; (ii) help overcoming intrinsic limitations of single approaches, and; (iii) complement each other in a drug discovery workﬂow [8–10]. Interestingly, the combination of ligand-based and structure-based approaches have already been successfully used to identify molecular targets for Mycobacterium tuberculosis phenotypic hits [11]. y g y p yp At present, most drug repurposing studies rely on the analysis of bioactivity data of compounds deriving from chemical synthesis. However, other valuable opportunities might come from natural products [12–14]. Natural products are characterized by a great structural diversity, and can provide 2 of 9 Molecules 2019, 24, 2567 novel chemical entities to be properly optimized in drug discovery campaigns. In this context, cannabinoids, which are terpenophenolics widely present in diﬀerent varieties of Cannabis sativa L., are a very interesting class of bioactive compounds [15]. In particular, the pharmacological proﬁle of non-psychoactive cannabinoids makes them the leading actors of the vast majority of scientiﬁc papers related to the ﬁber-type variety, which is commonly known as industrial hemp or hemp [16]. 1. Introduction Among these compounds, cannabidiol (CBD) represents the best known example from a pharmaceutical point of view, possessing antioxidant, anti-inﬂammatory, antibacterial, anti-proliferative, neuroprotective and anticonvulsant properties [15,16]. Cannabigerol (CBG) and cannabichromene (CBC) are other non-psychoactive cannabinoids (Figure 1a), which can be found in C. sativa inﬂorescences; they are both characterized by antibacterial activity, together with anti-inﬂammatory and anti-proliferative properties [17]. Since the above-mentioned compounds represent good candidates for drug repositioning, the aim of this work was to develop and apply an integrated ligand- and structure-based in silico procedure to unveil possible biological targets of non-psychoactive cannabinoids to be used in future drug discovery campaigns. 2. Results and Discussion Specifically, (a) reports the structures of CBG, 5PP and CBC. The chiral center in CBC is highlighted with a red star. (b) reports the shape-based alignment obtained for CBG (dark teal sticks) Figure 1. Chemical structures and ligand-based alignments of CBG and CBC predicted with ROCS [25]. Speciﬁcally, (a) reports the structures of CBG, 5PP and CBC. The chiral center in CBC is highlighted with a red star. (b) reports the shape-based alignment obtained for CBG (dark teal sticks) and the S stereoisomer of CBC (dark grey sticks) with the 5PP (orange thinner sticks) compound. Figure 1. Chemical structures and ligand-based alignments of CBG and CBC predicted with ROCS [25]. Specifically, (a) reports the structures of CBG, 5PP and CBC. The chiral center in CBC is highlighted with a red star. (b) reports the shape-based alignment obtained for CBG (dark teal sticks) Figure 1. Chemical structures and ligand-based alignments of CBG and CBC predicted with ROCS [25]. Speciﬁcally, (a) reports the structures of CBG, 5PP and CBC. The chiral center in CBC is highlighted with a red star. (b) reports the shape-based alignment obtained for CBG (dark teal sticks) and the S stereoisomer of CBC (dark grey sticks) with the 5PP (orange thinner sticks) compound. Figure 1. Chemical structures and ligand-based alignments of CBG and CBC predicted with ROCS [25]. Specifically, (a) reports the structures of CBG, 5PP and CBC. The chiral center in CBC is highlighted with a red star. (b) reports the shape-based alignment obtained for CBG (dark teal sticks) Figure 1. Chemical structures and ligand-based alignments of CBG and CBC predicted with ROCS [25]. Speciﬁcally, (a) reports the structures of CBG, 5PP and CBC. The chiral center in CBC is highlighted with a red star. (b) reports the shape-based alignment obtained for CBG (dark teal sticks) and the S stereoisomer of CBC (dark grey sticks) with the 5PP (orange thinner sticks) compound. and the S stereoisomer of CBC (dark grey sticks) with the 5PP (orange thinner sticks) compound. Although CBC and CBG showed good shape similarity with the 5PP inhibitor, the two compounds were also docked in the InhA crystal structure (PDB code: 2B36) [20], as described in the “Material and Methods” section. This analysis made it possible to assess whether the investigated compounds also possess good steric and electrostatic complementarity with the InhA binding site. 2. Results and Discussion The 2B36 crystal structure was preferred among others available for InhA, because the enzyme is in complex with 5PP [20]. Indeed, the selection of suitable receptor conformations for docking by means of the similarity between the crystallographic and the screening ligands is among one of the most used methods to improve structure-based virtual screening results [9,26]. Docking analyses were performed with Glide and the Induced Fit Docking protocol available with the Schrödinger suite 2018-3, [27,28]. At this stage of the analysis, different docking protocols, i.e., rigid (Glide) and flexible (Induced Fit), were performed to evaluate whether small structural changes, due, e.g., to receptor flexibility of InhA, might affect ligand binding [29]. To assess the ability of the docking protocol to reproduce the native orientation of the crystallographic ligand, redocking of 5PP into its own 2B36 crystal structure was first performed as a control (see Figure S1 in the Supporting Information), with the evaluated Root Mean Square Deviation (RMSD) being below 2.0 Å. Then, docking of CBG and CBC was performed. A visual inspection of the docking complexes with the best score predicted with Glide showed that CBG could accommodate into the InhA binding site by adopting an orientation similar to that of 5PP (Figure 2a). Interestingly, one of the hydroxyl groups of CBG is involved in a H-bond network of interactions with both Tyr158 and the 2′-hydroxyl group of NADH, similarly to 5PP [20]. These interactions are recognized to be particularly important for the catalytic activity of InhA [20,30]. The phenol ring provides stacking interactions with the nicotinamide ring of NADH. The 3,7-dimethylocta-2,6-dienyl moiety was predicted to be accommodated between the Phe97, Although CBC and CBG showed good shape similarity with the 5PP inhibitor, the two compounds were also docked in the InhA crystal structure (PDB code: 2B36) [20], as described in the “Material and Methods” section. This analysis made it possible to assess whether the investigated compounds also possess good steric and electrostatic complementarity with the InhA binding site. The 2B36 crystal structure was preferred among others available for InhA, because the enzyme is in complex with 5PP [20]. Indeed, the selection of suitable receptor conformations for docking by means of the similarity between the crystallographic and the screening ligands is among one of the most used methods to improve structure-based virtual screening results [9,26]. 2. Results and Discussion To identify potential targets of CBG and CBC that could be of therapeutic interest, a 3D ligand-based virtual screening was ﬁrst performed within the DrugBank database [18]. Indeed, this approach has already demonstrated to provide valuable results for drug repurposing, allowing the identiﬁcation of structurally unrelated compounds with similar bioactivities [19]. Among the available databases, the DrugBank was selected because it provides a comprehensive list of approved and investigational drugs, with trustful bioactivity annotations on relevant therapeutic targets. The DrugBank was ﬁrst prepared for the 3D ligand-based similarity analyses (see “Materials and Methods” section for details). Then, the DrugBank compounds were subjected to a 3D similarity screening against the generated CBG and CBC multi-conformer queries. This analysis allowed prioritizing potential therapeutic targets according to the degree of similarity of CBG and CBC with respect to the DrugBank compounds. Visual inspection of the predicted alignments allowed the identiﬁcation of the Enoyl acyl carrier protein (ACP) reductase (InhA) enzyme as a potential target for both CBG and CBC. In fact, according to the ligand alignments, both cannabinoids resulted to be similar to 5-pentyl-2-phenoxyphenol (5PP, DrugBank ID: DB07178) (Figure 1b), which is a small molecular weight inhibitor of InhA [20]. Ligand similarities are reported in Table S1 of the Supporting Information. In particular, according to the obtained alignments, the 5-pentyl-1,3-dihydroxyphenyl moiety of CBG overlapped well with the 5-pentyl-2-phenoxy group of 5PP, while the 3,7-dimethylocta-2,6-dienyl moiety of CBG provides looser superimposition with the phenyl group of 5PP. Regarding CBC, the n-pentyl-chromene-5-ol group provided a less favorable overlap with the 5-pentyl-2-phenoxy group of 5PP, this moiety occupying signiﬁcant larger volume with respect to the hydroxyphenyl group of 5PP. Good ligand-based alignments of CBC and CBG with other cannabinoids reported in the DrugBank database were also found (see Table S1 in the Supporting Information), such as cannabidiol (DrugBank ID: DB09061) and dronabinol (DrugBank ID: DB00470), which have recently been approved for treating epilepsy in children and nausea associated with cancer chemotherapy, respectively [21,22]. However, we decided to focus our attention to the InhA enzyme, which is a validated target of well-known antitubercular drugs [23,24]. 3 of 9 3 of 9 Molecules 2019, 24, 2567 Molecules 2019 24 x FO ecules 2019, 24, 2567 3 o olecules 2019, 24, x FOR PEER REVIEW 3 Figure 1. Chemical structures and ligand-based alignments of CBG and CBC predicted with ROCS [25]. 2. Results and Discussion Specifically, (a) and (b) report the predicted binding modes of CBG (dark teal sticks) and the S ti f CBC (d k ti k ) i t th I hA t ti l NADH i t d Figure 2. Docking poses of CBG and CBC into the 2B36 crystal structure predicted with Glide. Speciﬁcally, (a,b) report the predicted binding modes of CBG (dark teal sticks) and the S enantiomer of CBC (dark grey sticks) into the InhA receptor, respectively. NADH is reported as raspberry sticks. raspberry sticks. Based on these results, CBG turned out to be the best candidate for inhibition of InhA. In fact, although CBC resulted structurally similar to the 5PP inhibitor according to the ligand-based analyses, it could not be accommodated as good as CBG within the InhA enzyme with docking. To test this prediction, CBG and CBC were purchased from Sigma-Aldrich (Milano, Italy) and then tested for the inhibition of the InhA enzyme activity, as described in the “Material and Methods” section [36]. Notably, the experiments confirmed that CBG inhibits InhA with low micromolar inhibitory activity, the evaluated IC50 being 5.2 ± 0.1 µM (see Table 1, and Figure S4 in the Supporting Information). On the contrary, CBC turned out to be inactive or scarcely active (Table 1), in agreement with the structure-based results. Diﬀerent results were obtained for CBC, for which structure-based predictions did not agree with the ligand-based alignment. In particular, the S stereoisomer of CBC, which provided the best score in the docking calculations, was predicted to bind InhA with a binding mode that is head-to-tail with respect to that of 5PP in the crystal structure (Figure 2b and Figure S2). The diﬀerent binding mode likely originates from steric repulsion of the 2-(4-methylpent-3-enyl) moiety of CBC with the NADH cofactor. In this binding mode, the S stereoisomer of CBC is engaged in H-bonds with both Tyr158 and the 2′-hydroxyl group of NADH, such as 5PP [20]. On the contrary, docking of the R enantiomer of CBC into the 2B36 crystal structure provided a binding pose that did not establish relevant H-bond interactions with the InhA binding site residues or NADH; therefore, this stereoisomer was not further considered. To further reﬁne the results obtained with Glide, a rescoring of the predicted docking poses was performed with BEAR [31]. 2. Results and Discussion Docking analyses were performed with Glide and the Induced Fit Docking protocol available with the Schrödinger suite 2018-3, [27,28]. At this stage of the analysis, diﬀerent docking protocols, i.e., rigid (Glide) and ﬂexible (Induced Fit), were performed to evaluate whether small structural changes, due, e.g., to receptor ﬂexibility of InhA, might aﬀect ligand binding [29]. To assess the ability of the docking protocol to reproduce the native orientation of the crystallographic ligand, redocking of 5PP into its own 2B36 crystal structure was ﬁrst performed as a control (see Figure S1 in the Supporting Information), with the evaluated Root Mean Square Deviation (RMSD) being below 2.0 Å. Then, docking of CBG and CBC was performed. A visual inspection of the docking complexes with the best score predicted with Glide showed that CBG could accommodate into the InhA binding site by adopting an orientation similar to that of 5PP (Figure 2a). Interestingly, one of the hydroxyl groups of CBG is involved in a H-bond network of interactions with both Tyr158 and the 2′-hydroxyl group of NADH, similarly to 5PP [20]. These interactions are recognized to be particularly important for the catalytic activity of InhA [20,30]. The phenol ring provides stacking interactions with the nicotinamide ring of NADH. The 3,7-dimethylocta-2,6-dienyl moiety was predicted to be accommodated between the Phe97, Met103 and the Ala198 residues, while the n-pentyl group binds near to the Phe149, Met155, Tyr158 and Leu218 residues, establishing hydrophobic contacts. 4 of 9 ceptor Molecules 2019, 24, 2567 demonstrating tha fl ibili f I hA ( Figure 2. Docking poses of CBG and CBC into the 2B36 crystal structure predicted with Glide. Specifically, (a) and (b) report the predicted binding modes of CBG (dark teal sticks) and the S enantiomer of CBC (dark grey sticks) into the InhA receptor respectively NADH is reported as Figure 2. Docking poses of CBG and CBC into the 2B36 crystal structure predicted with Glide. Speciﬁcally, (a,b) report the predicted binding modes of CBG (dark teal sticks) and the S enantiomer of CBC (dark grey sticks) into the InhA receptor, respectively. NADH is reported as raspberry sticks. Figure 2. Docking poses of CBG and CBC into the 2B36 crystal structure predicted with Glide. 2. Results and Discussion Indeed, BEAR has already demonstrated to improve docking results in a variety of virtual screening campaigns and retrospective validations, including also the enoyl ACP reductase target [32–35]. Therefore, it represents a valuable approach to reﬁne docking results. Interestingly, BEAR provided MM-PBSA free-energies of binding clearly in favor of cannabigerol, the evaluated free energy scores being −28 Kcal·mol−1 and −20 Kcal·mol−1 for CBG and CBC (S stereoisomer), respectively. Induced Fit docking experiments conﬁrmed Glide results. In fact, docking poses with the best score predicted by the Induced Fit protocol superimposed well with those obtained by Glide, thus demonstrating that the predicted CBC and CBG binding modes were not aﬀected by receptor ﬂexibility of InhA (see Figure S3 of the Supporting Information). Based on these results, CBG turned out to be the best candidate for inhibition of InhA. In fact, although CBC resulted structurally similar to the 5PP inhibitor according to the ligand-based analyses, it could not be accommodated as good as CBG within the InhA enzyme with docking. To test this prediction, CBG and CBC were purchased from Sigma-Aldrich (Milano, Italy) and then tested for the inhibition of the InhA enzyme activity, as described in the “Material and Methods” section [36]. Notably, the experiments conﬁrmed that CBG inhibits InhA with low micromolar inhibitory activity, the evaluated IC50 being 5.2 ± 0.1 µM (see Table 1, and Figure S4 in the Supporting Information). On the contrary, CBC turned out to be inactive or scarcely active (Table 1), in agreement with the structure-based results. 5 of 9 Molecules 2019, 24, 2567 Table 1. Inhibitory activity of CBG and CBC. Triclosan was used as a positive control for the assays Table 1. Inhibitory activity of CBG and CBC. Triclosan was used as a positive control for the assays. Compound % Inhibition at 50 µM IC50 (µM) a CBG 78 5.2 ± 0.1 CBC 31 b nd Triclosan (TCL) 100 (56% at 0.3 µM) a The reported IC50 values are the mean of three experiments ± SD. b Not determined. Finally, docking complexes were used to suggest which structural modiﬁcations of CBG could potentially improve the activity of this cannabinoid. In particular, the substitution of the 3,7-dimethylocta-2,6-dienyl moiety of CBG with aromatic rings able to ﬁt in the pocket lined by Met98, Phe97, Pro99, Gln100, Met103 and Ala198 could improve binding to InhA [20,37,38]. 2. Results and Discussion Likewise, substitutions of the n-pentyl moiety of CBG with cyclic aliphatic or aryl groups would provide additional van der Waals contacts with the Phe149, Met155, Pro193, Ile215 and Leu218 residues, which are expected to provide improved activity for InhA, as already observed for other InhA inhibitors [39]. 3.1. Ligand-Based Virtual Screening on the DrugBank Database Compounds with associated bioactivity annotations were ﬁrst retrieved from the DrugBank database (www.drugbank.ca, accessed on April 20, 2018) [18]. Then, compounds with recognized toxic and reactive functional groups or transition metals, and a molecular weight lower than 150 or higher than 850 Da were removed, yielding a database of 6014 unique compounds. The FILTER software (OpenEye, Santa Fe, Mexico) was used for the ﬁltering calculations [40]. Afterwards, all combinations of stereoisomers, ionization states and tautomers potentially present at neutral pH were generated for both the investigated cannabinoids and the ﬁltered DrugBank ligands by using the Quacpac python toolkits [41]. Pre-deﬁned chiralities were kept unaltered and pre-treated structures were minimized according to the MMFF force ﬁeld [42]. Finally, up to 10 and 600 conformers were generated for the investigated cannabinoids and the ﬁltered DrugBank compounds, respectively. A cutoﬀof 0.5 Å on RMSD and an energy window of 10 kcal/mol were used as parameters to accept conformers during the conformational sampling with the OMEGA software [40,43]. A multi-conformer versus multi-conformer 3D shape-based virtual screening was performed to evaluate the similarity proﬁle of the investigated cannabinoids with respect to the ﬁltered DrugBank compounds. The ROCS software (version 3.2.1.4) was used as the engine for the similarity calculations [25,44]. Finally, ligand alignments were visually inspected, and the activity annotations of the DrugBank compounds were carefully analyzed. 3.2. Structure-Based Virtual Screening on Enoyl Acyl Carrier Protein (ACP) Reductase 3.2. Structure-Based Virtual Screening on Enoyl Acyl Carrier Protein (ACP) Reductase The InhA crystal structure (PDB code: 2B36) was ﬁrst downloaded and pre-processed with the Protein Preparation Wizard module available within the Schrödinger Suite 2018-3 [20,45]. In particular, atom types and bond connectivity issues in the downloaded crystal structure were ﬁxed. Moreover, missing side chains were rebuilt by using Prime (version 5.3) [46]. Then, hydrogen atoms were added to the pre-treated crystal structures and their coordinates were energy-minimized. Finally, ions, solvent and water molecules were removed. Docking calculations were performed by using both Glide (version 8.0.012) and the Induced Fit Docking protocol available within the Schrödinger Suite (New York, NY, USA) 2018-3 [27,28]. The NADH cofactor was included in the docking calculations and considered as a part of the receptor. In the case of Glide, receptor grids were generated around the coordinates of the bound ligands (outer box of 10 Å × 10 Å × 10 Å), with rigid docking calculations performed by using default settings. Both the residues lining the protein-binding site and the NADH cofactor were considered as rigid elements in the Glide calculations, while the “ﬂexible” sampling mode was used for the ligand to 6 of 9 Molecules 2019, 24, 2567 determine the optimal ligand conformation and orientation (default settings). For each ligand, only the pose with the best docking score was retained and visually inspected. In the case of Induced Fit Docking, default settings were used for receptor grid generation, while the calculations for docking the ligands into the 2B36 crystal structure were performed as follows. Firstly, the van der Waals radii of the protein and ligand were scaled by a factor of 0.8, afterwards the compounds were docked into the protein by using the default Glide SP protocol. Then, Prime was used to predict and optimize the protein side chains around the ligand. In this stage of reﬁnement, the residues within 5 Å of each ligand pose were optimized, while the NADH cofactor was kept rigid. Other parameters were set to the default. Finally, the poses were re-docked by using the Glide XP protocol and then scored. Twenty poses with the best scores were retained for each ligand in the ﬁnal step of the docking calculations to be visually inspected. y p The docking procedure was validated by redocking the co-crystallized 5PP ligand into its crystal structure prior to the cannabigerol (CBG) and cannabichromene (CBC) screening. 3.3. In Vitro Testing of the Compounds on the InhA Enzyme CBG, CBC, NADH and the Triclosan standard control were purchased from Sigma-Aldrich. Stock solutions of the investigated compounds were prepared in DMSO to let their ﬁnal concentration be equal to 5% v/v, in a ﬁnal volume of 1 mL for all kinetic reactions. Kinetic assays were performed by using trans-2-dodecenoyl-coenzyme A (DDCoA) and wild type InhA, as previously described [49]. Brieﬂy, reactions were performed at 25 ◦C in an aqueous buﬀer (30 mM PIPES and 150 mM NaCl pH: 6.8), containing additionally 250 µM cofactor (NADH), 50 µM substrate (DDCoA) and the tested compound (at 50 µM or 10 µM). Reactions were initiated by addition of InhA (50 nM ﬁnal) and NADH oxidation was followed at 340 nm. The inhibitory activity of each derivative was expressed as the percentage inhibition of InhA activity (initial velocity of the reaction) with respect to the control reaction without inhibitor. Triclosan was used as the positive control. All activity assays were performed in triplicate. For the most potent compound, IC50 values were determined by using the 4-parameter curve-ﬁtting software XLFit (IDBS), with at least six points. 3.2. Structure-Based Virtual Screening on Enoyl Acyl Carrier Protein (ACP) Reductase Afterwards, CBG and CBC were prepared with LigPrep for the docking calculations [47]. In particular, all combinations of stereoisomers, ionization states and tautomers potentially present at physiological pH in aqueous solution were ﬁrst generated and then minimized according to the OPLS_2005 force ﬁeld [48]. Finally, compounds were docked in the InhA active site. Moreover, the BEAR tool, which integrates molecular dynamics and binding free energy estimations with the aim of reﬁning ligand-protein complexes and estimate binding energetics, was applied to further reﬁne docking results obtained with Glide [31–35]. Default settings were used for rescoring the docking poses with BEAR [31]. A ﬁnal step of visual inspection of the generated poses conﬁrmed the selection of CBG and CBC as potential candidate inhibitors to be experimentally tested on InhA. References 1. 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The potential provided by the proposed approach was tested on cannabigerol (CBG) and cannabichromene (CBC), leading to the identiﬁcation of CBG as a low micromolar inhibitor of the InhA enzyme. Indeed, naturally occurring cannabinoids are known to possess antibacterial activity in various bacterial strains [17,50] but, to the best of our knowledge, their biological target(s) have never been identiﬁed. Our study demonstrates that CBG is an InhA inhibitor. Interestingly, CBG is a small molecular weight compound with a good safety proﬁle. Therefore, it represents a valuable starting point for the design of new synthetic derivatives with improved activity, thus paving the way to novel interesting possibilities for the treatment of infectious diseases. Finally, our study showed that integrating structure-based and ligand-based methods can lead to more accurate predictions 7 of 9 Molecules 2019, 24, 2567 of bioactive compounds [8]. This approach can be applied to eﬃciently repurpose any natural and synthetic ligand towards other therapeutic targets of interest. of bioactive compounds [8]. This approach can be applied to eﬃciently repurpose any natural and synthetic ligand towards other therapeutic targets of interest. Supplementary Materials: The following are available online at http://www.mdpi.com/1420-3049/24/14/2567/s1. Figure S1: superimposition of the 5PP binding modes predicted by Glide and the Induced ﬁt protocols with its crystallographic conformation; Figure S2: superimposition of the predicted ligand-based and structure-based (with Glide) poses of CBC; Figure S3: CBG and CBC binding modes predicted by the Induced Fit Docking protocol; Figure S4: the dose-response curve of CBG on InhA; Table S1: similarity values obtained with ROCS of CBC and CBG with respect to the DrugBank ligands. Author Contributions: The manuscript was written through contributions of all authors. 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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/). © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W2468438354	https://europepmc.org/articles/pmc4937037?pdf=render	English	null	Development of Cerebral Microbleeds in the APP23-Transgenic Mouse Model of Cerebral Amyloid Angiopathy—A 9.4 Tesla MRI Study	Frontiers in aging neuroscience	2,016	cc-by	6,242	ORIGINAL RESEARCH published: 08 July 2016 doi: 10.3389/fnagi.2016.00170 Development of Cerebral Microbleeds in the APP23-Transgenic Mouse Model of Cerebral Amyloid Angiopathy—A 9.4 Tesla MRI Study Björn Reuter 1*, Alexander Venus 2, Patrick Heiler 3, Lothar Schad 3, Anne Ebert 2, Michael G. Hennerici 2, Saskia Grudzenski 2† and Marc Fatar 2† 1 Department of Neurology and Neurophysiology, Freiburg University, Freiburg, Germany, 2 Department of Neurology, Universitätsmedizin Mannheim, Heidelberg University, Mannheim, Germany, 3 Computer Assisted Clinical Medicine, Heidelberg University, Mannheim, Germany Background: Cerebral amyloid angiopathy (CAA) is characterized by extracellular deposition of amyloid β (Aβ) around cerebral arteries and capillaries and leads to an increased risk for vascular dementia, spontaneous lobar hemorrhage, convexal subarachnoid hemorrhage, and transient focal neurological episodes, which might be an indicator of imminent spontaneous intracerebral hemorrhage. In CAA cerebral microbleeds (cMBs) with a cortical/juxtacortical distribution are frequently observed in standard magnetic resonance imaging (MRI). In vivo MRI of transgenic mouse models of CAA may serve as a useful tool to investigate translational aspects of the disease. Edited by: Edited by: Aurel Popa-Wagner, University of Medicine Rostock, Germany Materials and Methods: APP23-transgenic mice demonstrate cerebrovascular Aβ deposition with subsequent neuropathological changes characteristic for CAA. We performed a 9.4 Tesla high ﬁeld MRI study using T2, T2∗and time of ﬂight-magnetic resonance angiograpy (TOF-MRA) sequences in APP23-transgenic mice and wildtype (wt) littermates at the age of 8, 12, 16, 20 and 24 months, respectively. Numbers, size, and location of cMBs are reported. Reviewed by: Johannes Boltze, Fraunhofer-Society, Germany Ana Maria Buga, University of Medicine and Pharmacy Rostock, Germany Results: T2∗imaging demonstrated cMBs (diameter 50–300 µm) located in the neocortex and, to a lesser degree, in the thalamus. cMBs were detected at the earliest at 16 months of age. Numbers increased exponentially with age, with 2.5 ± 2 (median ± interquartilrange) at 16 months, 15 ± 6 at 20 months, and 31.5 ± 17 at 24 months of age, respectively. Received: 21 March 2016 Accepted: 27 June 2016 Published: 08 July 2016 Keywords: amyloid, CAA, cerebral microbleeds, transgenic mice, APP23, MRI Animals APP23-tg mice contain a human amyloid precursor protein (APP751) cDNA with the Swedish double mutation at position 670/671 under the control of the neuron-specific Thy-1 promoter (Calhoun et al., 1999). These animals express APP in sevenfold excess compared with the endogenous murine APP. Histologically visible parenchymal Aβ deposition starts at 6–8 months of age. Mainly affected are the neocortex, hippocampus, and thalamus (Sturchler-Pierrat et al., 1997). Besides to parenchymal deposition the mouse model shows a substantial cerebrovascular accumulation of Aβ, first detectable in histology at the age of 12 months (Calhoun et al., 1999; Sturchler-Pierrat and Staufenbiel, 2000). Leakage of the BBB, cerebral microhemorrhages, and macrohemorrhages are closely connected to the affected vessels and show similarities to human pathologies (Winkler et al., 2001; Beckmann et al., 2003). Magnetic resonance imaging (MRI) as the brain imaging technique of choice demonstrates distinctive features as cerebral microbleeds (cMBs) predominantly located within the cortex and subcortex, white matter changes, superficial siderosis/convexal subarachnoid hemorrhage, silent cortical infarcts, inflammatory disease, and atypical lobar hemorrhage (Chao et al., 2006). Among these neuroimaging findings, cMBs have received the highest attention, being frequently observed in T2∗gradient echo sequences and susceptibility-weighted imaging (SWI; Haacke et al., 2007). They represent focal deposits of perivascular hemosiderin-iron and their distribution pattern in synopsis of further imaging findings may allow for etiologic conclusions, i.e., CAA, severe arterial hypertension, chronic cerebral infarction, head trauma, genetic vessel disease, or endocarditis (Schrag and Greer, 2014). In CAA they are closely connected to Aβ deposits in arterioles and, to a lesser extent, capillaries (Schrag et al., 2010; Park et al., 2013). Focal rupture of the vessel wall is thought to be a consequence of Aβ-induced inflammation and loss of vascular smooth muscle cells (Schrag et al., 2010). Strictly lobar cMBs were positively associated with amyloid burden in Pittsburgh compound B (PiB) positron emission tomography (PET), cognitive function and the risk for spontaneous lobar hemorrhage (Poels et al., 2012; Park et al., 2013; van Etten et al., 2014). p g ) Approval of our experiments was given by the local ethical animal care and use committee (Regierungspräsidium Karlsruhe, 76247 Karlsruhe, Germany, file number 35-9185.81/G-9/10). All procedures were in strict accordance with institutional animal protection guidelines. Animals Heterozygote B6, D2-TgN[Thy- APPSWE]-23-tg mice (APP23-tg) provided by Matthias Staufenbiel (Novartis Institutes for BioMedical Research, Novartis Pharma AG, Basel, Switzerland) were backcrossed twice with C57BL/6 mice (Janvier, Saint Berthevin Cedex, France) and kept under a 12/12 h light/dark cycle with standard food and water ad libitum. Fourty-eight heterozygote mice and 48 wt littermates were bred to reach a final study cohort of 60 mice and compensate for spontaneous death. APP23-tg mice and wt controls (n = 6 each) were measured at the age 8, 12, 16, 20 and 24 months, respectively. No further in- or exclusion criteria were applied and mice of both sexes were used in our study. Wt mice do not develop spontaneous cMBs (Klohs et al., 2011; Hoffmann et al., 2016). With inclusion of wt littermates we aimed to identify possible MRI abnormalities morphologically similar to cMBs, which have to be taken into account for cMB counting in APP23-tg mice. The development of transgenic mice was essential to understand the pathogenesis of Alzheimer’s disease (AD) and CAA and to investigate promising therapeutic strategies. While there are several different transgenic mouse lines which represent the pathological correlate of AD, only few can serve as models for CAA. Those are in particular the APP23, Tg2576, APPDutch, ArcAβ and TgSwDi mouse lines (Hsiao et al., 1996; Calhoun et al., 1999; Davis et al., 2004; Herzig et al., 2004; Elder et al., 2010; Klohs et al., 2011). Histological validation of a therapeutical approach in transgenic mouse models is essential to understand Citation: Conclusion: We report the temporal and spatial development of cMBs in the aging APP23-transgenic mouse model which develops characteristic pathological patterns known from human CAA. We expect this mouse model to serve as a useful tool to non- invasively monitor mid- and longterm translational aspects of CAA and to investigate experimental therapeutic strategies in longitudinal studies. Reuter B, Venus A, Heiler P, Schad L, Ebert A, Hennerici MG, Grudzenski S and Fatar M (2016) Development of Cerebral Microbleeds in the APP23-Transgenic Mouse Model of Cerebral Amyloid Angiopathy—A 9.4 Tesla MRI Study. Front. Aging Neurosci. 8:170. doi: 10.3389/fnagi.2016.00170 July 2016 \| Volume 8 \| Article 170 1 Frontiers in Aging Neuroscience \| www.frontiersin.org cMBs in the Aging APP23 Mouse Model Reuter et al. INTRODUCTION the underlying mechanisms. However, we believe that under translational aspects a non-invasive brain imaging with methods well-established in clinical routine might be of high value. Therefore, we conducted a high-field 9.4 Tesla MRI-study in APP23-tg mice and wildtype (wt) littermates with the objective to characterize the temporal and spatial development of cMBs. Based on the presented data we regard cMBs in the APP23-tg mouse model useful as an outcome marker for preclinical testing of pharmatherapeutical approaches under standardized conditions, which under methodological aspects can also be easily transferred and replicated in human clinical trials. Cerebral amyloid angiopathy (CAA) causes a higher risk for spontaneous lobar hemorrhage and cognitive decline (Yamada, 2015). Although the incidence of CAA increases constantly with age, there is no clear association with the common cerebrovascular risk factors. The prevalence in the elderly population ranges from 28 to 38% in non-demented to 55–59% in demented subjects (Keage et al., 2009). Amyloid beta (Aβ) as the main pathological substrate travels along pericapillary interstitial pathways before it aggregates and deposits more in the arterial than in the capillary system (Smith and Greenberg, 2009). Experimental and histopathological studies suggest that vascular accumulation of Aβ is not only a result of enhanced production of this peptide, but also of its reduced clearance from the brain interstitial fluid into the blood across the blood brain barrier (BBB; Weller et al., 2008). Histological findings in CAA are well known for several decades and comprise of degenerated vascular smooth muscle cells, loss of tight junction proteins, clustered occurrence of activated microglial cells, infiltration of leucocytes, fibrinoid necrosis, double barreling and microaneurysms (Scholz, 1938; Smith and Greenberg, 2009; Carrano et al., 2012). Frontiers in Aging Neuroscience \| www.frontiersin.org RESULTS For imaging a 9.4 T Biospec 94/20 USR small animal system equipped with 740 mT/m gradients and a 1H surface cryogenic probe (Bruker, Ettlingen, Germany) was used as described before (Reuter et al., 2014). T2∗-weighted gradient echo images were used to demonstrate hemosiderin deposits resulting from cMBs. SWI with its higher sensitivity to detect cMBs in humans has been previously described to be impractical for rodent in vivo imaging due to susceptibility interface-related signal loss in the cortex (Chamberlain et al., 2009). We’ve tested for SWI and faced the same problem of artifacts in the air/brain tissue border zones, which interfered with sufficient evaluation of cortical cMBs. Magnetic Resonance Imaging (MRI) Protocol and Analysis In vivo brain imaging was performed in 4-month intervals within an age range of 8–24 months and consisting of six mice each. July 2016 \| Volume 8 \| Article 170 Frontiers in Aging Neuroscience \| www.frontiersin.org 2 cMBs in the Aging APP23 Mouse Model Reuter et al. DISCUSSION Besides slowly progressing CAA-related syndromes, i.e., vascular dementia and gait disturbances, cerebrovascular events as spontaneous lobar intracerebral hemorrhage (ICH), convexal subarachnoid hemorrhage and the very recently discovered CAA-related inflammation require immediate hospitalization. Acute therapy nowadays comprises of admission to organized stroke units or intensive care units and treatment strategies not specifically related to CAA. This includes the consideration of hematoma evacuation in lobar ICH, immunosuppressive therapy Besides slowly progressing CAA-related syndromes, i.e., vascular dementia and gait disturbances, cerebrovascular events as spontaneous lobar intracerebral hemorrhage (ICH), convexal subarachnoid hemorrhage and the very recently discovered CAA-related inflammation require immediate hospitalization. Acute therapy nowadays comprises of admission to organized stroke units or intensive care units and treatment strategies not specifically related to CAA. This includes the consideration of hematoma evacuation in lobar ICH, immunosuppressive therapy istological Matching of MRI Findings Histological Matching of MRI Findings Following brain imaging a neuropathologic examination was performed in a 24 month old APP23-tg mouse for spatial colocalization of cMBs (Figure 5). Using T2∗imaging sequences as reference a matched histological section with PB staining is demonstrated. Bright field analysis was done using a Leica DM 4500 B fluorescence microscope (Leica, Wetzlar, Germany). Pictures were taken with Leica IM50 Image Manager Software (Leica, Cambridge, UK). Spatial Distribution of cMBs Depending on Age g Total cMB numbers as well as the amount of cMBs located in cortex and thalamus were obtained for each age-group (Figure 1A, Supplementary Table S1). Results showed that up to 12 months APP23-tg mice displayed no cMBs. Starting at 16 months total cMB numbers increased exponentially with age (p < 0.001). Approximately two thirds of the cMBs were located in the cortex and one third in the thalamus. Representative images on cMBs in cortical and thalamic location at the age of 16, 20, and 24 months are shown in Figure 2. Rarely an adjacent arterial vessel was observed in TOF-MRA images and one mouse 20 months of age displayed a spontaneous lobar hemorrhage in the left hemispheric frontal cortex (Figures 3, 4, respectively). Wt littermates displayed a low background level of hypointense focal lesions of unclassified origin first observed at the age of 16 months, where the presence of cMBs could not be fully excluded by neuroimaging. Due to their insignificant frequency no adjustment of cMB counts in APP23-tg mice is regarded necessary. Twelve coronary sections covering the whole brain were analyzed. Hypointense regions in T2∗-weighted images considered to be cMBs were verified by comparison to time of flight-magnetic resonance angiograpy (TOF-MRA) raw data to distinguish vessel related flow void. cMBs were quantified and graded in vivo in APP23-tg and wt littermates depending on size (cMBs with size ≤100 µm, 150–200 µm or >200 µm) and spatial distribution (cortex and thalamus). Age-matched wt mice served as controls to assess the frequency of artifacts susceptive for cMBs. The quantification and size-grading of cMBs was performed by an investigator blinded for age and genetic status. Histology l d g Numbers of cMBs with size of ≤100 µm, 150–200 µm or >200 µm were obtained for each age-group (Figure 1B, Supplementary Table S2). At the age of 16 months cMBs were at maximum 200 µm in diameter. With increasing age small cMBs (cMBs ≤100 µm) were observed in highest frequency (median 22, IQR 13 at the age of 24 months, p < 0.001) followed by cMBs 150–200 µm in diameter (median 13, IQR 8 at 24 months, p < 0.001), whereas the frequency of large cMBs >200 µm was generally low and no association with age was observed (median 1, IQR 0 at 24 months, p = 0.1). Hypointense focal lesions in wt mice were generally small ≤100 µm in diameter and their frequency was independent from age (p = 0.2). Histology was done as described previously (Reuter et al., 2016). In short, animals were sacrificed within 3 days after MRI under deep Isoflurane anesthesia by transcardial perfusion with 4% acid free formalin (Roth, Karlsruhe, Germany). The harvested brains were incubated over night in 4% acid free formalin at 4◦C, cut into blocs with 2 mm thickness, dehydrated with ethanol and xylol and embedded in paraffin. For histochemical analysis 4 µm sections were dewaxed in xylene and rehydrated in alcohol and distilled water. For detection of cMBs Prussian blue (PB) staining was performed using the Accustain® Iron Stain Kit as described in the manufacturer’s protocol (Sigma-Aldrich, St. Louis, MO, USA). Nuclei were counterstained using nuclear fast red 0.1% (Merck, Darmstadt, Germany) for 10 min. Following dehydration steps in alcohol and xylol the sections were preserved in mounting medium (Eukitt, O. Kindler, Freiburg, Germany). Frontiers in Aging Neuroscience \| www.frontiersin.org Statistical Analysis Statistical analysis was performed with a standard software package (SPSS 22, ‘‘SPPS Inc.’’, Chicago, IL, USA). The statistical evaluation was performed using univariate analysis of variance. After significant analyses of variance, multiple post hoc comparisons were carried out using the Scheffé test. Data were visualized with boxplots and expressed as median and interquartile range. A p < 0.05 was considered significant. July 2016 \| Volume 8 \| Article 170 Frontiers in Aging Neuroscience \| www.frontiersin.org 3 cMBs in the Aging APP23 Mouse Model Reuter et al. FIGURE 1 \| Spatial and size distribution of cerebral microbleeds (cMBs) depending on age. (A) Total cMB numbers and the amount of cMBs located in cortex and thalamus were obtained for APP23-tg mice aged 8, 12, 16, 20 and 24 months. Since APP-tg mice aged 8 and 12 months did not display cMBs only values for APP-tg mice aged 16, 20 and 24 months (each group n = 6) are shown. (B) cMBs were graded depending on size (≤100 µm, 100–150 µm and >200 µm) for APP-tg mice aged 16, 20 and 24 months (each group n = 6). FIGURE 1 \| Spatial and size distribution of cerebral microbleeds (cMBs) depending on age. (A) Total cMB numbers and the amount of cMBs located in cortex and thalamus were obtained for APP23-tg mice aged 8, 12, 16, 20 and 24 months. Since APP-tg mice aged 8 and 12 months did not display cMBs only values for APP-tg mice aged 16, 20 and 24 months (each group n = 6) are shown. (B) cMBs were graded depending on size (≤100 µm, 100–150 µm and >200 µm) for APP-tg mice aged 16, 20 and 24 months (each group n = 6). are safety concerns regarding CAA patients in need for either acute ischemic stroke treatment with intravenous recombinant tissue plasminogen activator (rtPA) or long-term treatment with platelet aggregation inhibitors and/or oral anticoagulants (Reuter et al., 2014; Charidimou et al., 2015a; Wilson et al., 2015). cMBs are one of the most important clinicoradiological findings for risk stratification prior to treatment with either rtPA or antithrombotics. in CAA-related inflammation and avoidance of immobility- related complications with early mobilization and rehabilitation strategies (Steiner et al., 2014; AVERT Trial Collaboration Group, 2015; Raghavan et al., 2016). Since the incidence of CAA and subsequent vascular disease is expected to increase in aging societies, strong efforts are undertaken to develop new treatment options. Statistical Analysis July 2016 \| Volume 8 \| Article 170 Frontiers in Aging Neuroscience \| www.frontiersin.org 5 cMBs in the Aging APP23 Mouse Model Reuter et al. the technical improvement of current non-contrast enhanced rodent MRI (Beckmann et al., 2011). Compared to the gold standard histology T2∗high field MRI now offers a valid option to rapidly and serially determine the whole brain cMB load in vivo. Using the same MRI protocol, we’ve recently demonstrated a good correlation between MRI and histology-derived numbers of cMBs in the APP23-tg mouse model and thus the validity of MRI derived data (Reuter et al., 2016). FIGURE 4 \| Spontaneous lobar intracerebral hemorrhage in the left hemispheric frontal lobe of an APP23-tg mouse 20 months of age. Prior to clinical trials novel agents need to prove their efficacy and safety in animal models of CAA and transgenic mouse models are regarded invaluable for this undertaking. Moreover, in a retrograde approach transgenic mouse models may pathophysiologically explain findings made from observational or clinical studies. There are several possibilities to invasively and non-invasively monitor the progression of CAA, i.e., histological analysis with Congo Red, Thioflavin S or immunostaining, brain imaging with MRI, PiB PET, and light-based microscopy methods, i.e., two-photon microscopy or optical coherence tomography (for detailed information, see Klohs et al., 2014). Compared to other imaging parameters, cMBs in cortical distribution are frequently observed in patients suffering CAA. Although cMBs are not a clinical outcome marker per se, their relationship to morphological and clinical CAA severity and progression and their high incidence in standard non-invasive brain imaging make them sufficiently sensitive and specific as an outcome parameter for clinical trials (Greenberg et al., 2014; Charidimou et al., 2015b). Therefore, the longitudinal assessment of cMB numbers is regarded to be a useful tool to non-invasively monitor CAA progression, which in the future could be further improved by 3D techniques at very high spatial resolution (Greenberg et al., 2014; Chacon-Caldera et al., 2016). Moreover, in many healthcare systems cranial MRI scanners including gradient echo sequences or susceptibility weighted FIGURE 4 \| Spontaneous lobar intracerebral hemorrhage in the left hemispheric frontal lobe of an APP23-tg mouse 20 months of age. 2 at 16 months and 17 at 24 months of age. Previous work has reported an age-dependent exponential increase of cMBs in this mouse model using histology or T2∗-weighted MRI at 4.7 T field intensity, respectively. Statistical Analysis These strategies are closely related to therapy approaches in AD, which have received a much higher public and financial support for many decades. However, in CAA they try to target the vascular accumulation of Aβ itself and/or aim to stabilize vascular function (Saito and Ihara, 2014; Reuter et al., 2016). A second aspect of growing interest We present a transgenic mouse model of CAA which develops cMBs at the earliest between 12–16 months of age. The majority of cMBs detected in T2∗-weighted imaging are 50–200 µm in size, while cMBs larger than 200 µm were less frequently July 2016 \| Volume 8 \| Article 170 Frontiers in Aging Neuroscience \| www.frontiersin.org 4 Reuter et al. cMBs in the Aging APP23 Mouse Model FIGURE 2 \| Representative T2∗magnetic resonance images of cortical and thalamic cMBs in the APP23-tg mouse model at 16, 20, and 24 months of age. FIGURE 2 \| Representative T2∗magnetic resonance images of cortical and thalamic cMBs in the APP23-tg mouse model at 16, 20, and 24 months of age. FIGURE 2 \| Representative T2∗magnetic resonance images of cortical and thalamic cMBs in the APP23-tg mouse model at 16, 20, and 24 months of age observed. About two thirds of the cMBs are located in the neocortex and, contrary to human disease, about one third in the thalami. APP23-tg mice are known to develop severe CAA in thalamic vessels (Thal et al., 2009). Since they do not connect to human CAA it thus might be suitable in experimental studies to focus solely on cortical cMBs. The overall burden of cMBs showed a significant and exponential increase over time with median numbers ranging from 2.5 in 16 months old mice to 31.5 in 24 months old mice. However, in aged mice an increasing variability in numbers was observed, with an IQR of FIGURE 3 \| Colocalization of a thalamic cMB and an adjacent arterial vessel using T2∗imaging and time of ﬂight-magnetic resonance angiograpy (TOF-MRA). FIGURE 3 \| Colocalization of a thalamic cMB and an adjacent arterial vessel using T2∗imaging and time of ﬂight-magnetic resonance angiograpy (TOF-MRA). a thalamic cMB and an adjacent arterial vessel using T2∗imaging and time of ﬂight-magnetic resonance angiograpy FIGURE 3 \| Colocalization of a thalamic cMB and an adjacent arterial vessel using T2∗imaging and time of ﬂight-ma TOF-MRA). FUNDING The study was funded by an internal grant of the Medical Faculty of Mannheim, University of Heidelberg. The study was funded by an internal grant of the Medical Faculty of Mannheim, University of Heidelberg. Statistical Analysis In histology the mean number of cMBs at 27 months of age was reported to be 14 (right hemisphere only, every 10th section, total estimated cMB load >100), and a positive correlation with CAA-severity was observed (Winkler et al., 2001). In MRI at 4.7 T the mean number of discovered cMBs at the age of 24 months was reported 5 compared to 31.5 in our study at 9.7 T field intensity, thereby demonstrating FIGURE 5 \| Comparison of cMBs in the APP23-tg mouse model using T2∗magnetic resonance imaging (MRI) and Prussian blue (PB) staining. In MRI the detection of the right hemispheric cMB was hindered by an artifact but approved in the neighboring T2∗sequence and TOF-MRA raw data. FIGURE 5 \| Comparison of cMBs in the APP23-tg mouse model using T2∗magnetic resonance imaging (MRI) and Prussian blue (PB) staining. In MRI the detection of the right hemispheric cMB was hindered by an artifact but approved in the neighboring T2∗sequence and TOF-MRA raw data. July 2016 \| Volume 8 \| Article 170 6 Frontiers in Aging Neuroscience \| www.frontiersin.org cMBs in the Aging APP23 Mouse Model Reuter et al. imaging are widely available and offer a good opportunity for safety monitoring in phase IV clinical trials, when a therapeutic agent has received approval for CAA. With our study we demonstrate that cMBs in the APP23-tg mouse model are easily and reliably detectable using whole brain high field 9.4 Tesla MRI. cMBs are not uniquely observed in APP23-tg mice but have also been described in Tg2576, TgSwDI and arcAβ mice (Fisher et al., 2011; Klohs et al., 2011; Yang et al., 2011). Yet to the best of our knowledge, we provide the most comprehensive description of cMBs regarding their incidence in ageing, their distribution, and their size. Based on the numbers of cMBs observed at 24 months of age the following sample sizes were calculated to detect a significant reduction of cMBs in experimental trials with two groups. Power analysis was performed for a single- tailed two-group independent sample t-test (α 0.05, β 0.80). Regarding a therapeutical approach with an assumed large effect size (d = 0.80) N = 21 mice per group are required and for a medium effect size (d = 0.50) N = 51 per group. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fnagi. 2016.00170/abstract CONCLUSION In a longitudinal MRI study we demonstrate that APP23 transgenic mice develop cMBs at the earliest between 12 and 16 months of age. As in human CAA cMBs are located within the neocortex but contrary to human disease to a lesser degree also in the thalami. The overall burden of cMBs exponentially increases with age but also shows an increasing inter-individual variability in numbers. This needs to be taken into account for sample size calculation in experimental studies. Under translational aspects we expect this mouse model and method to serve as a useful tool to non-invasively monitor the development of CAA and to investigate experimental therapeutic strategies in longitudinal studies. Possible pitfalls comprise of the great variability of cMB prevalence in aged mice, which require careful consideration of the optimal timing of brain imaging and also a sufficient size of the study cohort. AUTHOR CONTRIBUTIONS Current therapeutical approaches are closely connected to AD research. Possible targets to reduce the vascular burden of Aβ are proteolytic pathways, uptake and degradation by microglial cells and astrocytes, the modulation of Aβ efflux and influx over the BBB, and finally the stimulation of perivascular drainage alongside of small arteries and arterioles within the brain interstitial fluid (Charidimou et al., 2012; Saito and Ihara, 2014). In future studies we intent to investigate the effect of ligand stimulated nuclear receptors (NR) on Aβ secretion and APP processing in APP23-tg mice. NRs are ligand-activated transcription factors providing a critical role between the genome and the environment (Mandrekar-Colucci and Landreth, 2011). It was shown that drugs targeting NRs may strongly influence the regulation of cholesterol efflux and generation of high density lipoproteins (HDL), thus leading to diminished Aβ secretion (Riddell et al., 2007; Fitz et al., 2010; Sandoval-Hernández et al., 2015). Furthermore it has been shown that different mouse strains of AD including APP23-tg BR, MGH, SG, and MF conceived and designed the experiments; BR, AV, and SG performed the experiments; BR, SG, and MF analyzed the data; PH and LS contributed analysis tools; BR wrote and submitted the article. All authors agree to be accountable for all aspects of the work and gave final approval for this version to be published. Statistical Analysis In case research groups aim to investigate other age cohorts or cMBs in cortical or thalamic localization only the raw data is provided as a supplementary file (Supplementary Table S3) to perform the respective power analyses. We hope that this data facilitates the conduction of future experimental studies with the APP23-tg mouse model. mice being deficient for NR targeted genes present altered Aβ levels (Hirsch-Reinshagen et al., 2005; Koldamova et al., 2005). REFERENCES Int. J. Mol. Sci. 17:126. doi: 10.3390/ijms17 010126 Fisher, M., Vasilevko, V., Passos, G. F., Ventura, C., Quiring, D., and Cribbs, D. H. (2011). Therapeutic modulation of cerebral microhemorrhage in a mouse model of cerebral amyloid angiopathy. Stroke 42, 3300–3303. doi: 10. 1161/strokeaha.111.626655 Riddell, D. R., Zhou, H., Comery, T. A., Kouranova, E., Lo, C. F., Warwick, H. K., et al. (2007). 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https://openalex.org/W2172900601	https://europepmc.org/articles/pmc4646466?pdf=render	English	null	The Season for Peace: Reconciliation in a Despotic Species (Lemur catta)	PloS one	2,015	cc-by	11,247	RESEARCH ARTICLE The Season for Peace: Reconciliation in a Despotic Species (Lemur catta) Elisabetta Palagi1,2, Ivan Norscia1 1 Natural History Museum, University of Pisa, Calci, Pisa, Italy, 2 Institute of Cognitive Sciences and Technologies, Unit of Cognitive Primatology & Primate Center, CNR, Rome, Italy Elisabetta Palagi1,2, Ivan Norscia1 Elisabetta Palagi1,2, Ivan Norscia1 1 Natural History Museum, University of Pisa, Calci, Pisa, Italy, 2 Institute of Cognitive Sciences and Technologies, Unit of Cognitive Primatology & Primate Center, CNR, Rome, Italy 1 Natural History Museum, University of Pisa, Calci, Pisa, Italy, 2 Institute of Cognitive Sciences and Technologies, Unit of Cognitive Primatology & Primate Center, CNR, Rome, Italy elisabetta.palagi@unipi.it * elisabetta.palagi@unipi.it Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: The authors only received reimbursement for travel and accommodation fees from Giardino Zoologico di Pistoia, Parco Zoo Falconara (Falconara, Italy), and Parco Zoo Punta Verde (Lignano Sabbiadoro, Italy). No official grant or funding (with grant numbers) was obtained and no salary was received for the investigation, which was carried out with the authors' own funds. Abstract 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. OPEN ACCESS Citation: Palagi E, Norscia I (2015) The Season for Peace: Reconciliation in a Despotic Species (Lemur catta). PLoS ONE 10(11): e0142150. doi:10.1371/ journal.pone.0142150 Editor: Karen E. Samonds, Northern Illinois University, UNITED STATES Received: February 9, 2015 Accepted: October 19, 2015 Published: November 16, 2015 Editor: Karen E. Samonds, Northern Illinois University, UNITED STATES Received: February 9, 2015 Accepted: October 19, 2015 Published: November 16, 2015 Copyright: © 2015 Palagi, Norscia. 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 However despotic a social group may be, managing conflicts of interest is crucial to pre- serve group living benefits, mainly based on cooperation. In despotic groups, post-conflict management via reconciliation (the first post-conflict reunion between former opponents) can occur, even if conciliatory rates are considerably different. Lemur catta is defined as a despotic species because groups are characterized by a strict linear hierarchy maintained by the adult females (the dominant sex) mainly via aggression. Reconciliation was reported in one out of four captive groups of L. catta. Here we investigate which variables influence the occurrence of reconciliation in these despotic groups. We analyzed 2339 Post Conflict (PC)-Matched Control (MC) observation pairs, collected on eight groups (five in the Berenty forest, Madagascar; three hosted at the Pistoia Zoo, Italy). Since L. catta is characterized by steep female dominance but shows female-female coalitionary support, we expected to confirm the presence of reconciliation in the study species. Consistently, we found reconcili- ation in one captive group and two wild groups, thus providing the first evidence of the pres- ence of this phenomenon in wild L. catta. Moreover, because this species is a seasonal breeder (with mating occurring once a year), we expected seasonal fluctuations in reconcili- ation levels. Via a GLMM analysis using data from all wild groups and on a captive group fol- lowed for more than one year, we found that season (but not rank; individuals’ identity, sex, and age; or group identity) significantly affected individual reconciliation rates, and such rates were lowest during the mating period. Thus, reconciliation can be present in groups in which dominants strongly influence and limit social relationships (steep dominance hierar- chy) except when the advantages of intra-group cooperation are overcome by competition, as occurs in seasonal breeders when reproduction is at stake. We conclude that in despotic social groups in which coalitions are observed, the right question is not if but when reconcili- ation can be present. OPEN ACCESS Citation: Palagi E, Norscia I (2015) The Season for Peace: Reconciliation in a Despotic Species (Lemur catta). PLoS ONE 10(11): e0142150. doi:10.1371/ journal.pone.0142150 Editor: Karen E. Samonds, Northern Illinois University, UNITED STATES Received: February 9, 2015 Accepted: October 19, 2015 Published: November 16, 2015 Copyright: © 2015 Palagi, Norscia. Introduction The management of conflicts of interest is crucial to preserve group living benefits, even in des- potic societies. In these kinds of societies, to preserve social integrity, violence is minimized via Competing Interests: The authors have declared that no competing interests exist. 1 / 21 PLOS ONE \| DOI:10.1371/journal.pone.0142150 November 16, 2015 Season Dependent Reconciliation in Lemur catta the strict control exerted by dominants over other individuals (“negative peace”, sensu Galtung [1]). Yet, in humans and other social mammals, dominant individuals or subgroups may need the support of others to obtain resources and maintain the status quo [2–7]. Consequently, strategies of mutual help other than competition for dominance and resources must be enabled, such as cooperative breeding, hunting, and coalitionary support during between- group conflicts [8–11]. Reconciliation or peace-making, defined as the first affinitive contact between former oppo- nents occurring within few minutes after the conflict, is one of the main mechanisms to man- age conflicts [12]. The phenomenon is present in social animals, including a bird species (e.g. ravens, Corvus corax [13]), various non primate mammals (e.g. domestic goats, Capra hircus [14]; dolphins, Tursiups troncatus [15]; domestic dogs, Canis lupus familiaris [16]; horses, Equus caballus [17]; red-necked wallabies, Macropus rufogriseus [18]), and human and non human primates (Homo sapiens [19]; chimpanzees, Pan troglodytes [20], [21]; bonobos, Pan paniscus [22]; Gorilla beringei and Gorilla gorilla [23–25]; wild macaques, Macaca spp. [26, 27]; captive guereza, Colobus guereza [28]; captive patas monkeys, Erythrocebus patas [29]; captive squirrel monkeys, Saimiri sciureus [30]; captive white-faced capuchins, Cebus capuci- nus [31]). By restoring the relationship between former opponents [32–39], reducing the probability of further fights [23], [33], [34], [40–45] and/or reducing anxiety in the victim [21], [46–50], reconciliation is crucial to preserving social unity from the disruption caused by uncontrolled conflict spreading in the group. Therefore, reconciliation is expected to be present any time that it is valuable for the group members (including dominants) to preserve the alliances that facilitate group survival, thus preserving the benefits of group living [51]. Consistently, reconciliation has been found also in species with a despotic dominance style [5; 52–55]. According to the definition of Flack and de Waal [52], in despotic groups domi- nance dyadic asymmetries remains quite stable over time because they reinforced through severe aggression. Instead, in tolerant groups dyadic asymmetries can exist but many relation- ships are unresolved. PLOS ONE \| DOI:10.1371/journal.pone.0142150 November 16, 2015 Prediction 2 In the animals breeding once or twice in the year, seasonality strongly affects social behaviour and competition levels [83]. Majolo & Koyama [84] found that in the population of despotic Macaca fuscata from Yakushima Island reconciliation levels changed seasonally. As most lemur species, L. catta lives and has evolved in a highly seasonal environment [61], [85,86] and is a seasonal breeder [58]. In fact, females are receptive once a year [87–89] and the mating period (from three weeks to two months depending on the site and the definition; see also: [57], [58], [90], [91]) is characterised by high competition and low affiliation levels. During the mating period, competition within and between sexes is extremely high and affiliation levels are low [58], [77], [92], [91]. Therefore, we expected that in L. catta seasonality would particu- larly affect reconciliation levels. Introduction Examples of animals living in despotic groups and that are able to recon- cile include spotted hyenas (Crocuta crocuta [53]), wolves (Canis lupus lupus [5]), Japanese macaques (Macaca fuscata [54]), and wild chacma baboons (Papio ursinus: Cheney, Seyfarth & Silk [55]). Similar to these species, Lemur catta can be defined as despotic because groups are characterised by a linear and steep hierarchy with clear-cut dominance relationships [56]. Females are dominant and their dominance is maintained also through severe aggression by dominants over subordinates [56–63]. In this species, the presence of reconciliation was found in one out of four captive troops in which post-conflict management was studied [64],[65]. The linkage between reconciliation and the level of authoritativeness (or despotism) has been qualitatively examined in humans, with friendly peacemaking being favored by minimal authority (power exercised over others; [66]). The linkage between reconciliation and domi- nance style has been also quantitatively assessed in tolerant to despotic macaque species ([52], [67]), with tolerant species (e.g. Tonkean macaques, Macaca tonkeana [37],[68], [69–71]) showing higher reconciliation levels than despotic species (Japanese macaques, Macaca fuscata [54]). The same linkage has been hypothesized in strepsirrhine primates [64], which can also show more or less mild and flexible dominance hierarchies [56]. In this primate taxon, recon- ciliation was indeed found in species with more relaxed (i.e. less steep or transitive) dominance relationships (captive Eulemur wild Eulemur rufusxcollaris [45]) rufus [64], [72]; wild Propithe- cus verreauxi [73]) but not in captive Eulemur macaco showing strong female dominance [72]. In the present study, we investigate the factors that can explain the occurrence of reconcilia- tion (or lack thereof) in different captive and wild groups of L. catta and make inferences about the conditions that favor the presence of reconciliation in despotic groups. As a primate species PLOS ONE \| DOI:10.1371/journal.pone.0142150 November 16, 2015 2 / 21 Season Dependent Reconciliation in Lemur catta belonging to the group (strepsirrhines) that diverged from the common ancestor some 60 mil- lion years ago [74], L. catta also offers the possibility to make inferences about the biological roots of peace-making dynamics found in humans and all other primates. For this investiga- tion, we analyzed the data collected on the focal species both in the wild and in captivity across more than a decade to verify the following predictions: Prediction 1 Similar to wolves and hyenas [5], [75], [76] L. catta is characterized by rigid hierarchy and high competition levels [57–63], [77–79]. Analogous to ring-tailed lemur troops, packs (in the case of wolves; [80]) and clans (in the case of hyenas [81], [82]) strictly defend their territories by directing severe aggression towards potential immigrants. Finally, although in a more limited form compared to canids and hyenids, L. catta females (the dominant sex in this species) are able to form coalitions, especially against other females, to preserve their dominance status or to gain the possibility to use a territory [10]. These traits led us to predict that, as in other des- potic but cooperative species [58], reconciliation may be present in L. catta not only in captivity but also in the wild. Ethics statement Since the study was purely observational the Animal Care and Use board (University of Pisa) waived the need for a permit. The study was conducted with no manipulation of animals. The study was carried out in the private Reserve of Berenty (South Madagascar) and at the Pistoia Zoo (Pistoia, Italy). De Heaulme and family, owners of Berenty and Mr Cavicchio, owner and director of the Pistoia Zoo, permitted us to observe animals. PLOS ONE \| DOI:10.1371/journal.pone.0142150 November 16, 2015 Study location and subjects Berenty (Madagascar). We conducted this research on wild lemurs in the gallery forest of Berenty, a reserve on the Mandrare River in Southern Madagascar (for an extensive description of the forest, see [105]). Data collection was conducted in the northern part of the forest called Ankoba (S 24.998; E 46.298), a 40-ha secondary forest 50- to 60-years-old, with canopy at 10– 15 m (except for few emergent acacias to more than 20 m) and high lemur density [105]. Observations were carried out in the periods November 2006-February 2007, April-July 2008, and March-April 2011 on five troops of L. catta. Details on group composition and observation periods are reported in Table 1. Kin relationships among group members were unknown but groups at Berenty (and other sites) are largely female matrilines (including sibling and off- spring of the alpha female [10],[59], [106], [107]. The individuals were well habituated to the presence of humans. As in previous studies, individual identification was based on sex and dis- tinctive external features [56–58]. Pistoia Zoo (Italy). We studied three captive troops (here named A, B, and C) at the Pistoia Zoo (Italy) in the periods February-May 1999, November 2003-February 2005. Details on group composition and observation periods are reported in Table 1. The captive groups were largely composed by the alpha female and kin (siblings and offspring of the alpha female). The lemurs were housed in an outside grassy enclosure (98 m2). In 1999, groups A and B were kept in two separated indoor halls on the coldest days of the year (A: 10 m2 indoor facility; B: 20 m2 indoor facility). Large glass windows in the two indoor facilities allowed the lemurs to follow the natural day-light 24-h cycle. Each group utilized the outside enclosure for 4–6 h per day, separately. In 2003–2005, another group (Cc) was hosted at the zoo and could use the indoor facility previously used by the other groups (not present anymore). The observations took place outdoors and lasted from the end of October 2003 to February 2015. As in the wild and in previous studies at Pistoia Zoo, individual identification in captivity was based on sex and distinctive external features [57] [65], [73], [74]. Table 1. Composition of wild and captive groups, observation n periods and study sites. Study species Lemur catta (ring-tailed lemur) is a cathemeral species characterized by seasonal fluctuations in olfactory behavior, group dispersal, tolerance level, and reproduction [58], [78], [79], [93– 97]. Lemur catta has a steep, consistent, highly transitive and cohesive hierarchy (sensu Norscia and Palagi [56]), with females dominant over males [58], [59], [78], [98–99]). Male hierarchy is unstable, and at times, non-transitive, and both female-female and male-male dominance hier- archies are fluid and can change over time [100–102]. The mating season overlaps among the different groups of a population and can last from three weeks to two months (depending on the site, the year, the definition of mating period; [57], [58], [90], [91], [103]. However, the onset of the mating period varies between groups, PLOS ONE \| DOI:10.1371/journal.pone.0142150 November 16, 2015 3 / 21 Season Dependent Reconciliation in Lemur catta and the whole mating season for the lemur population spans up to four months [57],[103]. Females experience an annual estrus of a few hours to days, and receptivity lasts 10–24 h after which the estrus period ends [58], [87]-[89], [59]. A second or third belated estrus is possible [58], [78], [79]). Lemur catta females have a visible estrus, which may be asynchronous with other females in their group [104]. The mating period starts about one month before copula- tions, when female perineal area starts to enlargen and the center of the genitalia becomes larger and pinker: this period of swelling anticipates estrus [58], [87]. Generally, receptivity coincides with the last day of maximal pink coloration of vaginal labia ([87], [103]. Data collection Systematic data collection was preceded by a training period that lasted until the data collected by the two observers (on aggression and affiliation behavioral patterns) matched in 95% of cases [108]. The excellent visibility condition of the Berenty forest allowed us to apply the same protocol to the wild as was used in captivity. For each agonistic encounter we recorded: (1) identity of the two opponents; (2) aggressive behavioral patterns (mainly chase, bite, grab, jump); and (3) submissive/frightened patterns (flee and vocalization). The agonistic interaction was labeled as “decided” when one of the two opponents gave up the fight (by retreating, flee- ing or running away) and the winner could be therefore determined with certainty. For a com- prehensive ethogram see [109]. After the last aggressive pattern of any given agonistic event, we followed the loser of the interaction (as the focal individual) for a 10 min post-conflict period (PC). Matched control observations (10 minute long MCs) took place during the next possible day at the same time, context (feeding, resting or travelling) and physiological season (lactation, pre-mating, mating, and pregnancy; see details below) as the original PC. MC data were collected only if all these conditions were met. The MC was conducted on the same focal animal, in the absence of ago- nistic interactions during the 10 min before the beginning of the MC and when the opponents had the opportunity to interact, within a distance of 10 m maximum [110], [111]). We considered four groups of affinitive behaviors to identify the first conciliatory contact: body contact (body-to-body contact excluding tails, huddle); greeting (naso-nasal, face groom- ing); grooming (unidirectional, reciprocal or mutual); olfactory contact (sniffing body, sniffing genitals, and skin licking) [109]). Proximity was not considered because it does not necessarily indicate affiliation. We collected a total of 2339 PC-MC (1461 in captivity and 878 in the wild). For both PCs and MCs we recorded: (1) starting time; (2) type of first affinitive interaction; (3) time of first affinitive contact; (4) partner identity. Study location and subjects Group Observation months Period Malesadult Femalesadult Malesjuvenile Femalesjuvenile Study site WILD Aw Nov2006-Feb2007 Lactation 4 4 1 0 Berenty Bw Nov2006-Feb2007 Lactation 4 6 2 1 Berenty Cw May-Jul2008 Pregnancy 3 6 1 2 Berenty Dw Apr-Jun2008 Mating 6 8 1 3 Berenty Ew Mar-Apr2011 Premating 5 5 5 2 Berenty CAPTIVITY Ac Feb-Mar1999 Pregnancy 2 3 0 0 Pistoia Bc Apr-May1999 Lactation 2 4 2 0 Pistoia Cc Nov2003-Feb2005 Premating, Mating, Lactation, Pregnancy 4 4 0 2 Pistoia doi:10.1371/journal.pone.0142150.t001 PLOS ONE \| DOI:10.1371/journal.pone.0142150 November 16, 2015 4 / 21 . Composition of wild and captive groups, observation n periods and study sites. 4 / 21 Season Dependent Reconciliation in Lemur catta PLOS ONE \| DOI:10.1371/journal.pone.0142150 November 16, 2015 Operational definitions and data analysis We considered the same fixed factors included in the first GLMM except for group ID. Since CCT distribution was normal in both cases (Kolmogorov-Smirnov, p = n.s.), an iden- tity link function was used. We tested models for each combination involving the variables of interest, spanning from the null model (only intercept) to the model including all the fixed fac- tors (full model). To select the best model, we used the Akaike’s Corrected Information Crite- rion (AICc), a measure for comparing mixed models based on the −2 (Restricted) log likelihood. The AICc corrects the Akaike’s Information Criterion (AIC) for small sample sizes. As the sample size increases, the AICc converges to AIC. The model with a lower value of AICc was considered to be the best model. To avoid the increase of type II errors, factors were excluded from a model only if this improved the model fit by >2 AICc units [115]. The value of degrees of freedom is given by the effective sample size (N) minus the rank design matrix of fixed effects (X). The denominator degree of freedom is estimated by SPSS via Satterthwaite’s approximation. We used all dyadic decided agonistic interactions to prepare a winner/loser socio-matrix and carry out hierarchical rank order analysis, by using MatMan 1.0 based on I&SI rankings (Noldus Information Technology, Wageningen, Netherlands; [116]). To assign the age class to each animal, the individuals were distinguished between adults (regularly performing genital marking, informing an age >18 months) and juveniles (not performing genital marking) [117]. Four seasons were recognized: lactation (1), pre-mating (2), mating (3), pregnancy (4) (The numbers correspond to how the seasons have been entered in the GLMM model). For the cap- tive groups (in the northern hemisphere) the different seasons were: lactating season (group Bc: April-May 1999; group Cc: April-August 2004); pre-mating (group Cc: September-October 2004), mating (group Cc: November-December 2003; November-December 2004), pregnancy (group Ac: February-March 1999; group Cc: January-March 2004; January-February 2005). Individual CCTs for group Cc (observed for more than one season) were calculated using the PC-MC collected for each season. In the wild the mating period varied depending on the group (refer to Table 1 for the groups): pre-mating (group Ew: March-April: 2011), mating (group Dw: April-May-beginning of June 2008), pregnancy (group Cw: May-July 2008), and lactating season (groups Aw and Bw: November-February 2006). Operational definitions and data analysis Reconciliation analysis was carried out at the individual level, taking the recipient of the aggres- sion as the individual of reference. For each animal we determined the number of attracted, dis- persed and neutral pairs over all PC-MC pairs. In attracted pairs, affinitive contacts occurred earlier in the PC than in the MC (or they did not occur at all in the MC), whereas in dispersed pairs the affinitive contacts occurred earlier in the MC than in the PC (or they did not occur at all in the PC). In neutral pairs, affinitive contacts occurred during the same minute in the PC and the MC, or no contact occurred in either the PC or the MC [110]. Due to the small sample size and/or deviation from normality (Exact Kolmogorov-Smirnov, p<0.05) we used the Exact Wilcoxon signed-ranks test [112], [113] to compare attracted versus dispersed pairs. Attracted and dispersed pairs were measured at the individual level, thus ensuring the independency of data points. The pair-wise comparison between attracted and dispersed pairs allows checking whether reconciliation is present (if the number of attracted pairs is significantly higher than the number of dispersed pairs) or not. In addition to determining whether reconciliation was present or not, we assessed the indi- vidual rates of conciliatory tendencies of individuals. The measure of corrected conciliatory tendency (CCT; [114]) allows evaluating the level of individual reconciliation by considering the attracted minus dispersed pairs divided by the total number of PC-MC pairs. Individual CCTs were used to determine the mean CCT in wild and captive conditions. To assess the effect of the different factors on individual CCTs (scalar, dependent variable), we ran two sets of General Linear Mixed Model (GLMM). The first GLMM was performed on all the study groups (Table 1). As fixed factors, we considered sex (binomial: male/female), age 5 / 21 Season Dependent Reconciliation in Lemur catta (binomial: juvenile/adult), rank position (scalar), season (multinomial: 1–4), individuals (nom- inal), and groups (nominal). Due to the inter-independence of sex and age, and sex and rank (because females outrank males and adults outrank subadults), these three factors were entered as two combined variables (sexrank and agerank). In order to attempt to remove possible confounding variables, the second GLMM was performed only on groups Cc for which data collection had covered all seasons (Table 1). Operational definitions and data analysis The mating period began when at least one female of the group started showing genital swelling from about 1.5–3 cm in length and developing a pink center [57], [58]. In a group, the pregnancy was considered as starting after the last copulation day (confirmed ex-post by births) whereas lactation started when a female in the group gave birth. Overall two mating periods were available in captivity and one in the wild. Results A previous study [65] showed that reconciliation was present in captive group Ac but not in group Bc (Table 1) so those analyses are not reported here. The overall CCT calculated here for the first time for all groups was 10.25% ±2.24 (Mean ±SE). In the wild the CCT was 10.99% ±2.44 and in captivity 9.62% ±3.60 (Mean ±SE). Mean CCT% (±SE) for each group are reported in Table 2. 6 / 21 PLOS ONE \| DOI:10.1371/journal.pone.0142150 November 16, 2015 Season Dependent Reconciliation in Lemur catta Table 2. Mean Corrected Conciliatory Tendency (CCT %) ± Standard Error (SE) for each study group. Group CCT%: Mean±SE Aw 19.55±7.52 Bw 18.62±8.51 Cw 14.63±6.96 Dw 5.74±2.72 Ew 3.69±2.20 Ac 43.17±19.24 Bc -14.83±4.23 Cc 9.47±6.73 doi:10.1371/journal.pone.0142150.t002 Table 2. Mean Corrected Conciliatory Tendency (CCT %) ± Standard Error (SE) for each study group. Group CCT%: Mean±SE Aw 19.55±7.52 Bw 18.62±8.51 Cw 14.63±6.96 Dw 5.74±2.72 Ew 3.69±2.20 Ac 43.17±19.24 Bc -14.83±4.23 Cc 9.47±6.73 doi:10.1371/journal.pone.0142150.t002 For captive group C (Table 1) we found a significant difference between the number of attracted pairs (in which affinitive contacts occurred earlier in the PC than in the MC or they did not occur at all in the MC) and the number of dispersed pairs (in which affinitive contacts occurred earlier in the MC than in the PC or they did not occur at all in the PC; attrac- ted>dispersed pairs: T = 5, N = 10, ties = 1, p = 0.004; Fig 1). In the wild, reconciliation was present in two groups out of five (groups Cw and Ew). In fact, we found a significant difference between attracted and dispersed pairs (attracted>dispersed) for group Cw (T = 0, N = 12, ties = 6, p = 0.031; Fig 2A) and group Ew (T = 2.50, N = 15, ties = 6, p = 0.020; Fig 2B). No sig- nificant difference between attracted and dispersed pairs was found for group Aw (T = 0, N = 8, ties = 4, p = 0.125), group Bw (T = 12, N = 11, ties = 2, p = 0.254) and group Dw (T = 19.50, N = 18, ties = 7, p = 0.254). Results For both captive and wild settings, the aggression distribution according to the different sex class combination is reported in Table 3 and shows that aggression levels of females toward males and between males were maximum during the mating season. During pregnancy and lactation the majority of conflicts involved females. Of all the GLMM models tested on all groups (AICc range = 393.675–1107.725) the best one was the full model (Intercept: F = 1.104, df1 = 77, df2 = 38, p = 0.376), including the combi- nation of individual features (sexrank: F = 1.448, df1 = 1, df2 = 38, p = 0.236; agerank: F = 0.849, df1 = 1, df2 = 38, p = 0.363), the group identification (F = 1.779, df1 = 1, df2 = 38, p = 0.190), individual identity (F = 0.698, df1 = 64, df2 = 38, p = 0.899), and the season (lacta- tion, pre-mating, mating, and pregnancy; F = 5.282, df1 = 3, df2 = 40, p = 0.004). Fig 3 shows the model output for the best model. Even if part of variability is influenced by individual CCT levels, only the season had a significant effect on the distribution of CCTs, lowest during the mating season (Figs 3 and 4). Of all the GLMM models tested for group Cc (AICc range = 393.675–534.649), the best one was the full model (Intercept: F = 3.103, df1 = 15, df2 = 38, p = 0.002), including the combina- tion of individual features (sexrank: F = 1.448, df1 = 1, df2 = 38, p = 0.236; agerank: F = 0.849, df1 = 1, df2 = 38, p = 0.363), individual identity (F = 1.805, df1 = 9, df2 = 38, p = 0.099), and the season (lactation, pre-mating, mating, and pregnancy; F = 3.844, df1 = 3, df2 = 38, p = 0.017). Fig 5 shows the output for the best model. Again, two individuals accounted for part of the CCT variation but only the season had a significant effect on the dis- tribution of CCTs throughout the year, with CCT values being minimum during the mating season (Figs 5 and 6). Both in captivity and in the wild, males (Min) and females (Fin) initiated the first affinitive contact with comparable frequencies in all seasons (captivity, range: Min = 47,22–51.72%; Fin = 48.28–52.77%; wild, range: Min = 46,88–50.00%; Fin = 50,00–60,00%). PLOS ONE \| DOI:10.1371/journal.pone.0142150 November 16, 2015 Results 7 / 21 PLOS ONE \| DOI:10.1371/journal.pone.0142150 November 16, 2015 Season Dependent Reconciliation in Lemur catta Fig 1. Box plot showing the significant difference (Exact Wilcoxon’s test, p<0.05) between the number of attracted versus dispersed pairs in the Lemur catta troop Cc (November 2003-February 2005), observed at the Pistoia Zoo (Italy). Solid horizontal lines indicate medians; the length of the boxes corresponds to inter-quartile range; thin horizontal lines indicate range of observed values. Fig 1. Box plot showing the significant difference (Exact Wilcoxon’s test, p<0.05) between the number of attracted versus dispersed pairs in the Lemur catta troop Cc (November 2003-February 2005), observed at the Pistoia Zoo (Italy). Solid horizontal lines indicate medians; the length of the boxes corresponds to inter-quartile range; thin horizontal lines indicate range of observed values. doi:10.1371/journal.pone.0142150.g001 PLOS ONE \| DOI:10.1371/journal.pone.0142150 November 16, 2015 Discussion Reconciliation was present both in the wild and in captivity (prediction 1 supported), and spe- cifically in two out of five wild troops of L. catta (Fig 2) and in two captive troops (group Cc, present study; group Ac, [65]) (Fig 1). When considering either all the study groups or group Cc only (for which longitudinal data were available), season was the only effect that signifi- cantly influenced the fluctuation in the frequency of reconciliation events (Figs 3 and 5). In particular, the conciliatory tendency was lowest during the mating season (prediction 2 sup- ported; Figs 4 and 6). Reconciliation was found in another despotic species with linear hierarchy, the wolf (Canis lupus; mean conciliatory tendency, 44.1% in the wild [11]; 53.3% in captivity [5]). In wolves, each group defends its own territory as a unit [118]. Yet, even if the alpha male normally PLOS ONE \| DOI:10.1371/journal.pone.0142150 November 16, 2015 8 / 21 Season Dependent Reconciliation in Lemur catta Fig 2. Box plot showing the significant difference (Exact Wilcoxon’s test, p<0.05) between the number of attracted versus dispersed pairs in two wild Lemur catta troops (Cw: May-July 2008, Fig 2a on the left; Ew: March-April 2011, Fig 2b on the right) observed in the Berenty Forest (Madagascar). Solid horizontal lines indicate medians; the length of the boxes corresponds to inter-quartile range; thin horizontal lines indicate range of observed values. Fig 2. Box plot showing the significant difference (Exact Wilcoxon’s test, p<0.05) between the number of attracted versus dispersed pairs in two wild Lemur catta troops (Cw: May-July 2008, Fig 2a on the left; Ew: March-April 2011, Fig 2b on the right) observed in the Berenty Forest (Madagascar). Solid horizontal lines indicate medians; the length of the boxes corresponds to inter-quartile range; thin horizontal lines indicate range of observed values. doi:10.1371/journal.pone.0142150.g002 guides the movements of the wolf pack and initiates aggressions against intruders [118], the subordinate members can sometimes oppose their leader’s actions. According to Zimen [119], no subject decides alone the carrying out of activities that are vital to the group cohesion. In short, wolves are highly despotic but also extremely cooperative. The existence of an extremely cooperative pack has presumably to do not only with hunting but also with the collective rear- ing of offspring and, consequently, with reproductive success [120]. PLOS ONE \| DOI:10.1371/journal.pone.0142150 November 16, 2015 Discussion Probably, in wolves the benefit of preserving the social bonds through reconciliation outweighs the cost of pack disrup- tion, which would be detrimental for both dominants and subordinates. Thus, reconciliation can be found in despotic groups provided that they show some form of cooperation [51]. Fur- ther evidence of this assumption is the presence of reconciliation in spotted hyenas (Crocuta crocuta [53]). Hyenas are despotic but often depend on the help from other group members Table 3. Aggression distribution (%) according to the different sex class combinations for all sea- sons, in the wild (W) and in captivity (C). Sex class combinations are: ff (females attacking female), fm (female attacking male), mf (male attacking female), mm (male attacking male). Table 3. Aggression distribution (%) according to the different sex class combinations for all sea- sons, in the wild (W) and in captivity (C). Sex class combinations are: ff (females attacking female), fm (female attacking male), mf (male attacking female), mm (male attacking male). ff% fm% mf% mm% matingC 11,76 56,62 3,68 27,94 prematingC 50 25 12,5 12,5 pregnancyC 43,67 38,61 5,7 12,03 lactationC 51,78 27,74 3,12 17,6 matingW 8,93 65,57 1,1 24,41 prematingW 28,55 56,62 0,12 14,7 pregnancyW 35,21 40,37 0 23,83 lactationW 45,64 41,9 8,23 4,24 doi:10.1371/journal.pone.0142150.t003 PLOS ONE \| DOI:10.1371/journal.pone.0142150 November 16, 2015 9 / 21 doi:10.1371/journal.pone.0142150.g003 Season Dependent Reconciliation in Lemur catta Fig 3. Output of the best model explaining the distribution of Corrected Conciliatory Tendencies (CCT %, scalar target var groups. AICc = 430, 295. Season: 1 = lactation; 2 = pre-mating; 3 = mating; 4 = pregnancy. Sex: 0 = male; 1 = female. Age class: 0 ange: 1–16 (rank position is relative to each group) aRedundant coefficients Please refer to S1 Fig in the Supporting Information f PLOS ONE \| DOI:10.1371/journal.pone.0142150 November 16, 2015 10 / 21 Season Dependent Reconciliation in Lemur catta Fig 4. SPSS 20.0 output bar graph showing estimated means of Corrected Conciliatory Tendency (CCT, %) for the significant effect (season: 1 = lactation; 2: pre-mating; 3 = mating; 4 = pregnancy), for all the study groups. Season is the only factor that significantly influences the CCT distribution in the study groups (GLMM; F = 0.718, df1 = 73, df2 = 40, p = 0.890). The conciliatory tendency % is lowest during mating. d i 10 1371/j l 0142150 004 Fig 4. SPSS 20.0 output bar graph showing estimated means of Corrected Conciliatory Tendency (CCT, %) for the significant effect (season: 1 = lactation; 2: pre-mating; 3 = mating; 4 = pregnancy), for all the study groups. Season is the only factor that significantly influences the CCT distribution in the study groups (GLMM; F = 0.718, df1 = 73, df2 = 40, p = 0.890). The conciliatory tendency % is lowest during mating. doi:10.1371/journal.pone.0142150.g004 doi:10.1371/journal.pone.0142150.g004 during hunts, defence of ungulate carcasses against competitors, and coalition formation that is important in both the acquisition and maintenance of social rank [53]. Cooperation and des- potism are two opposite forces that contribute in shaping reconciliation patterns, as it becomes especially clear when comparing species differing only in some aspects of the social system. In hyenas, as in wolves, the necessity to cooperate overcomes the competition between domi- nants and subordinates, which explains the presence of reconciliation. The lower levels of rec- onciliation observed in hyenas (mean conciliatory tendency: 11.3% [53]) may be due to the fact that, contrary to wolves, spotted hyenas live in a fission fusion society allowing dispersal (other than reconciliation) as an exit strategy. The influence of the cooperation pressure over the suit- ability of engaging in reconciliation is even more evident when comparing spotted hyenas with ring-tailed lemurs. PLOS ONE \| DOI:10.1371/journal.pone.0142150 November 16, 2015 Although both species possess steep female dominance, they strongly differ in the level of cooperation. Unlike hyenas, cooperation in L. catta is limited to the coalitionary support provided to the dominant female by other females during targeted aggression toward during hunts, defence of ungulate carcasses against competitors, and coalition formation that is important in both the acquisition and maintenance of social rank [53]. Cooperation and des- potism are two opposite forces that contribute in shaping reconciliation patterns, as it becomes especially clear when comparing species differing only in some aspects of the social system. In hyenas, as in wolves, the necessity to cooperate overcomes the competition between domi- nants and subordinates, which explains the presence of reconciliation. The lower levels of rec- onciliation observed in hyenas (mean conciliatory tendency: 11.3% [53]) may be due to the fact that, contrary to wolves, spotted hyenas live in a fission fusion society allowing dispersal (other than reconciliation) as an exit strategy. The influence of the cooperation pressure over the suit- during hunts, defence of ungulate carcasses against competitors, and coalition formation that is important in both the acquisition and maintenance of social rank [53]. Cooperation and des- potism are two opposite forces that contribute in shaping reconciliation patterns, as it becomes especially clear when comparing species differing only in some aspects of the social system. PLOS ONE \| DOI:10.1371/journal.pone.0142150 November 16, 2015 11 / 21 Season Dependent Reconciliation in Lemur catta Fig 5. Output of the best model explaining the distribution of Corrected Conciliatory Tendencies (CCT %, scal AICc = 398.767. Season: 1 = lactation; 2 = pre-mating; 3 = mating; 4 = pregnancy. Sex: 0 = male; 1 = female. Age clas coefficients. Fig 5. Output of the best model explaining the distribution of Corrected Conciliatory Tendencies (CCT %, scalar target variable) for group Cc. AICc = 398.767. Season: 1 = lactation; 2 = pre-mating; 3 = mating; 4 = pregnancy. Sex: 0 = male; 1 = female. Age class: 0 = subadult; 1 = adult. aRedundant coefficients. Fig 5. Output of the best model explaining the distribution of Corrected Conciliatory Tendencies (CCT %, scalar target variable) for group Cc. AICc = 398.767. Season: 1 = lactation; 2 = pre-mating; 3 = mating; 4 = pregnancy. Sex: 0 = male; 1 = female. Age class: 0 = subadult; 1 = adult. aRedundant coefficients. doi:10.1371/journal.pone.0142150.g005 PLOS ONE \| DOI:10.1371/journal.pone.0142150 November 16, 2015 12 / 21 Season Dependent Reconciliation in Lemur catta Fig 6. SPSS 20.0 output bar graph showing estimated means of Corrected Conciliatory Tendency (CCT, %) for the significant effect (season: 1 = lactation; 2: pre-mating; 3 = mating; 4 = pregnancy), for group Cc. Season is the only factor that significantly influences the CCT distribution in the study groups (GLMM; F = 1.674, df1 = 15, df2 = 36, p = 0.102). The conciliatory tendency % is lowest during mating. Fig 6. SPSS 20.0 output bar graph showing estimated means of Corrected Conciliatory Tendency (CCT, %) for the significant effect (season: 1 = lactation; 2: pre-mating; 3 = mating; 4 = pregnancy), for group Cc. Season is the only factor that significantly influences the CCT distribution in the study groups (GLMM; F = 1.674, df1 = 15, df2 = 36, p = 0.102). The conciliatory tendency % is lowest during mating. Fig 6. SPSS 20.0 output bar graph showing estimated means of Corrected Conciliatory Tendency (CCT, %) for the significant effect (season: 1 = lactation; 2: pre-mating; 3 = mating; 4 = pregnancy), for group Cc. Season is the only factor that significantly influences the CCT distribution in the study groups (GLMM; F = 1.674, df1 = 15, df2 = 36, p = 0.102). The conciliatory tendency % is lowest during mating. PLOS ONE \| DOI:10.1371/journal.pone.0142150 November 16, 2015 conspecifics (to defend territory boundaries or to evict them from the group or the core area of the home range; [10], [58], [121]). This limited cooperation can explain why L. catta show the lowest conciliatory tendencies (9–10%). In some macaque species, it has been observed that the higher the cooperation and tolerance levels, the higher the reconciliation rates [52]. This prin- ciple can be extended to include other primates. For example, conciliatory tendencies can reach more than 40% in bonobos (Pan paniscus) and crested macaques (Macaca nigra) [22], [122] and plummet to less than 15% in more despotic and less cooperative species such as Assamese macaques (Macaca assamensis) and western gorillas (Gorilla beringei) [25], [123]. Of course, the distinction between more and less cooperative species is not always clear cut because primates can form rather complex societies and the individuals of certain subgroups can be more cooperative than the group as a whole, as occurs when cooperative breeding, matriline support, or brotherhood coalitions are in place [124]. 13 / 21 PLOS ONE \| DOI:10.1371/journal.pone.0142150 November 16, 2015 Season Dependent Reconciliation in Lemur catta Although at low levels, reconciliation seems to be possible in despotic species like L. catta when the cooperation-competition balance tilts in favor of cooperation because the benefits of peace making overcome the costs of leaving conflicts unmanaged. But when reproduction is at stake, as it is in lemurs during the once-a-year mating period, both male-female and male-male competition is too high [58],[125] for conflicts to be peacefully resolved. In our study we found that aggression in the mating period was particularly high between males and between females and males (with females initiating the aggression). Consistently, conciliatory rates of both males and females were minimal in the mating season (Figs 4 and 6) likely because in this period the behaviors of individuals are oriented toward reproduction more than maintenance of social stability. Based on these results, it is possible to assert that reconciliation is season- dependent in L. catta. Sex was not the explaining variable for the observed fluctuations in con- ciliatory tendencies. Consistently, both males and females initiated the post-conflict reunion with comparable frequencies throughout the year. The only study to date that has investigated the seasonal fluctuations of reconciliation in another despotic primate species [84] reported that in female Japanese macaques (Macaca fuscata) mating—and not other factors such as changes in activity budgets and dietary composition—had profound effects on peace-making. In fact, the conciliatory tendency–informing reconciliation rates—was significantly lower during the mating season than the non mating season [84]. The authors commented that the negative effects of the mating season on reconciliation within female Japanese macaques may be due to the relevance of female competition for access to male partners in multimale, multifemale societies characterized by adult male dominance. In L. catta the situa- tion is reversed: adult females are dominant over males [57–59] and the competition and stress lev- els during the mating period are highest among males trying to gain access to female partners [125]. Despite the difference in the dominant sex between L. catta and M. fuscata, the result is sim- ilar: reconciliation is lowest during the extremely competitive mating period. A possible explanation for the seasonal distribution of reconciliation can lie in how hor- mones modulate the propensity to affiliate with others, and consequently to reconcile. It is worth remembering that the very definition of reconciliation implies the use of affinitive con- tacts for the purpose of peace making [20]. As well as in other animals in which the sexual con- text is associated with aggression and competition [126], [127], L. catta males experience the highest levels of testosterone during the extremely high competitive mating period [128], which also coincides with the lowest levels of inter-male affiliation [91]. The stress hormones may also increase as a result of aggression, eliciting the fight or flight response [129] and there- fore leaving little space for post-conflict affiliation among males. However, literature reports contrasting results on the level of stress hormones (fecal glucocorticoid) in L. catta males dur- ing the mating period [125]. Besides male affiliation, the high levels of estradiol associated with the mating period can reduce affiliation between primate females, for example in rhesus monkeys (Macaca mulatta [130]). Additionally, in human and non human primates, other hormones such as oxytocin and prolactin may influence female affiliation levels throughout the year because they can enhance individual propensity to affiliate and are higher in non-mating periods [131–138]. Consistently, L. catta females (aggressors) mainly initiated conciliatory affiliation in group Ac [65]. PLOS ONE \| DOI:10.1371/journal.pone.0142150 November 16, 2015 Therefore, hormonal influence may partly explain the variation in post-conflict concilia- tory affiliation across the year. The seasonality of the conciliatory tendency in L. catta documented in the present study is also consistent with the variation of inter-male affiliation rates recorded by Gabriel, Gould & Kelley [91] in the same species, at four sites of Madagascar. These authors observed that inter- male affiliation levels varied across reproductive periods, with the lowest frequencies occurring 14 / 21 Season Dependent Reconciliation in Lemur catta during the mating period. Overall, the seasonal fluctuations of the reconciliation tendency observed in L. catta appear to be sustained by both physiological and socio-ecological data. Access to females is not the only item worth competing for. Another valuable resource con- nected to reproductive success is offspring. We observed that in both the wild and captivity female-female aggression was highest during pregnancy and during the lactation period (Table 3), when the newborn is still carried out by the mother. It has been hypothesised that dominant females may target subordinate ones to prevent them from conceiving or to cause them to lose their infants because subordinate females with vital offspring can potentially acquire a higher ranking status in the social group and subtract resources [58], [121]. Food also represents a valuable commodity for the members of social groups, eliciting competition more than cooperation. Consistently, in the wild, reconciliation was found in a group of Eulemur rufus x collaris and in two groups of Propithecus verreauxi but never in the feeding context [45], [73]. This situation reinforces the idea that when a valuable resource is concerned and cooperation is low (e.g. mate for reproduction, high energy food), gaining access to that resource can be more rewarding than repairing the relationship with a former opponent in the short term, via post-conflict reunions. As a future direction, it would be interesting to assess if and how conciliatory tendencies of L. catta are influenced by the context and the individuals involved in the conflicts within each season. We expect that post conflict reunions are lowest in competive contexts (e.g. feeding) and between competing individuals (e.g. females during preg- nancy and lactation; males during mating, etc.). In conclusion, we posit that the ability to reconcile is expressed whenever the benefits of intra-group cooperation overcome the costs of competition, as occurs when a limited, wanted resource is at stake. Acknowledgments The authors thank the De Heaulme family and Alison Jolly for the possibility of doing this research at Berenty (Madagascar). They express gratitude to the Director of the Giardino Zool- ogico di Pistoia (Pistoia, Italy), Paolo Cavicchio and the lemur keepers for allowing and facili- tating the work in captivity. The authors wish to thank also Elena Bastianelli, Elisa Rigosi, Manrica Cresci, Daniela Antonacci, Stefano Kaburu, Chandra Brondi, Stefania Dall'Olio, Valentina Sclafani, Viola Caltabiano, Giulia Spada, Alessandra Zannella for helping with data collection over the years, Stacey Schmidt Zander for language revision, and Beronia Rioja for enhancing the discussion over results. Finally the authors are grateful to the parks Giardino Zoologico di Pistoia, Parco Zoo Falconara (Falconara, Italy), and Parco Zoo Punta Verde (Lig- nano Sabbiadoro, Italy) for supporing the field data collection in the wild. S1 Dataset. Dataset used to investigate the occurrence and seasonality reconciliation in Lemur catta. (XLSX) S1 Dataset. Dataset used to investigate the occurrence and seasonality reconciliation in Lemur catta. ( S ) S1 Fig. Full size version of Fig 3. (TIF) S1 Fig. Full size version of Fig 3. (TIF) In summary, this study shows that in despotic social groups in which coali- tions are observed, the right question is not if but when reconciliation can be present. References 1. Galtung J (1969) Violence, peace, and peace research. J Peace Research 6: 167–191. 2. Bygott J, Bertram B, Hanby J (1979) Male lions in large coalitions gain reproductive advantages. Nature 282: 839–841. 3. Clutton-Brock T (1998) Reproductive skew, concessions and limited control. Trends Ecol Evol 13: 288–292. PMID: 21238306 4. Duffy KG, Wrangham RW, Silk JB (2007) Male chimpanzees exchange political support for mating opportunities. Curr Biol 17: R586– R587. PMID: 17686425 5. Cordoni G, Palagi E (2008). 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https://openalex.org/W4327914592	https://www.nature.com/articles/s41534-023-00745-1.pdf	English	null	Spectral kissing and its dynamical consequences in the squeezed Kerr-nonlinear oscillator	Research Square (Research Square)	2,023	cc-by	7,878	INTRODUCTION features at long times33–35, isomerization reactions36, and the creation of Schrödinger cat states20. Recent developments in superconducting circuits have opened the pathway to explore long standing predictions of quantum physics. They have been used to study dynamical bifurcation1,2, to squeeze quantum ﬂuctuations3, to prepare exotic quantum states, and to process and stabilize quantum information4,5. Here, we propose to use this platform as a quantum simulator of excited state quantum phase transitions (ESQPTs), a phenomenon that occurs in various nuclear, atomic, molecular, and condensed matter systems. The superconducting circuit considered is a driven system, whose static effective Hamiltonian describes a double-well system and thus exhibits an ESQPT. This perspective adds another layer of interest to the long history of studies on driven nonlinear oscillators6–14, where the emergence of a double well, reached by driving the oscillator at twice its original frequency6, has been explored in studies of quantum activation6,7, quantum tunneling8,9, and the preparation of selected superpositions of quasienergy states10 with applications to quantum information science, such as the generation of Schrödinger cat states. The main signature of an ESQPT is a singularity in the density of states (DOS) that moves to higher excitation energies as the control parameter increases, and may be accompanied by the closing of energy gaps between excited states. The energy where the divergence of the DOS takes place is the ESQPT critical energy. These and related features have been theoretically identiﬁed in various quantum systems with few degrees of freedom15–49, and a proposal to detect the ESQPT with spinor Bose–Einstein con- densates also exists50. Even though spectroscopic signatures of the ESQPT have been experimentally observed51–55 and its presence suggested from the bifurcation phenomenon detected in refs. 56–58, presently none of these systems provides the means to analyze the spectrum as a function of the control parameter and to simultaneously observe the dynamical consequences of an ESQPT in a controllable way. Superconducting circuits close this gap by offering a platform that has an experimental realizable classical limit and provides both frequency- and time-resolved high quantum non-demolition measurements ﬁdelity59. A quantum phase transition (QPT) corresponds to an abrupt change in the ground state of a physical system when a control parameter reaches a critical point. It occurs in the thermodynamic limit, but scaling analyses of ﬁnite systems can signal its presence. 1Department of Physics, University of Connecticut, Storrs, CT 06269, USA. 2Department of Physics, Yeshiva University, New York, NY 10016, USA. 3Department of Applied Physics and Physics, Yale University, New Haven, CT 06520, USA. 4Department of Chemistry, Yale University, P.O. Box 208107, New Haven, CT 06520-8107, USA. 5Departamento de Ciencias Integradas y Centro de Estudios Avanzados en Física, Matemáticas y Computación, Universidad de Huelva, Huelva 21071, Spain. 6Instituto Carlos I de Física Teórica y Computacional, Universidad de Granada, Fuentenueva s/n, 18071 Granada, Spain. ✉email: lea.santos@uconn.edu ARTICLE OPEN Spectral kissing and its dynamical consequences in the squeeze-driven Kerr oscillator Jorge Chávez-Carlos 1, Talía L. M. Lezama 2, Rodrigo G. Cortiñas 3, Jayameenakshi Venkatraman 3, Michel H. Devoret 3, Victor S. Batista 4, Francisco Pérez-Bernal 5,6 and Lea F. Santos 1✉ Jorge Chávez-Carlos 1, Talía L. M. Lezama 2, Rodrigo G. Cortiñas 3, Jayameenakshi Venkatraman 3, Michel H. Devoret 3, Victor S. Batista 4, Francisco Pérez-Bernal 5,6 and Lea F. Santos 1✉ Transmon qubits are the predominant element in circuit-based quantum information processing, such as existing quantum computers, due to their controllability and ease of engineering implementation. But more than qubits, transmons are multilevel nonlinear oscillators that can be used to investigate fundamental physics questions. Here, they are explored as simulators of excited state quantum phase transitions (ESQPTs), which are generalizations of quantum phase transitions to excited states. We show that the spectral kissing (coalescence of pairs of energy levels) experimentally observed in the effective Hamiltonian of a driven SNAIL- transmon is an ESQPT precursor. We explore the dynamical consequences of the ESQPT, which include the exponential growth of out-of-time-ordered correlators, followed by periodic revivals, and the slow evolution of the survival probability due to localization. These signatures of ESQPT are within reach for current superconducting circuits platforms and are of interest to experiments with cold atoms and ion traps. npj Quantum Information (2023) 9:76 ; https://doi.org/10.1038/s41534-023-00745-1 www.nature.com/npjqi Published in partnership with The University of New South Wales INTRODUCTION Solid lines are for the even parity sector and dashed lines for odd parity. The bright orange line in (b) marks the energy of the ESQPT, as given in Eq. (3). c–e Normalized density of states and f–h participation ratio for the eigenstates in the Fock basis for the values of ξ indicated in (c–e); even parity sector. Numerical (shade) and analytical (solid line) data are shown in (c–e). The vertical dashed line in (c–h) is the ESQPT energy from Eq. (3). i–l Husimi functions for different eigenstates and ξ = 180. parameter increases, the coalescence of a pair of adjacent eigenvalues, each level belonging to a different parity sector, happens at a higher energy. This spectral kissing becomes better visible in Fig. 1b, where larger values of ξ are used. For a given value of the control parameter, the spectral kissing happens at the critical energy of the ESQPT, E0 ESQPT, which is marked with a solid line in Fig. 1b and is obtained analytically [see Eq. (3) below]. justiﬁcation for these apparently opposite behaviors lies in the classical limit of the system. At the origin of the phase space, (q = 0, p = 0), there is a stationary but unstable point that is associated with the ESQPT. At this point, the evolution is dominated by the squeezing part of the Hamiltonian. The experimental capability of reconstruction of the full phase- space distribution59 motivates our analysis of the dynamics in phase space, which reveals features that were missed by previous works on ESQPTs and that are of interest to studies of nonequilibrium quantum dynamics. Depending on the initial state, the exponentially fast spread in phase space can be followed by the onset of complicated interference patterns or yet by periodic revivals that persist for long times. Our analysis also elucidates why states with exactly the same energy may exhibit different dynamics. y y In addition to the exponential approach of the energies in each pair, the eigenvalues cluster at E0 ESQPT (Supplementary Note 2). This produces the peak of the DOS displayed for different values of the control parameter in Fig. 1c–e. The peak diverges for ξ →∞, which is a main signature of the ESQPT17. INTRODUCTION ESQPT is a generalization of this phenomenon to excited states15–18, which can take place independently of the presence of QPTs19,20 and can be triggered by anharmonicities21–23. In an ESQPT, the separation of the states in two phases24 occurs at a point that depends on both the value of the energy and of the control parameter. There is a vast literature on the subject, which is reviewed in ref. 18. ESQPTs are associated with enhanced decoherence25,26, localized eigenstates27–29, very slow27–29 or accelerated30–32 quantum quench dynamics, speciﬁc dynamical As we explain here, the exponential approach of pairs of adjacent levels (spectral kissing) recently observed in the spectrum of the superconducting Kerr resonator as a function of the amplitude of a squeezing drive59, and previously discussed in10, marks the presence of an ESQPT. The dynamical counterpart of this transition presents a seeming paradoxical behavior, which can, in principle, be observed in a system such as the one in ref. 59. For Glauber coherent states close to the ESQPT, the initial decay of the survival probability (overlap of the initial and the evolved state) is slower than for coherent states away from the ESQPT, while the ﬁdelity out-of-time-ordered correlator (FOTOC) grows exponentially fast for the ﬁrst and slower for the latter. The Published in partnership with The University of New South Wales J. Chávez-Carlos et al. 2 a b c d e f g h i j k l Fig. 1 Spectral kissing and localization. a Energy levels as a function of the control parameter reproducing the experimental data59 with K/(2π) = 0.32 MHz and b E0=ð_KÞ for larger values of ξ. Solid lines are for the even parity sector and dashed lines for odd parity. The bright orange line in (b) marks the energy of the ESQPT, as given in Eq. (3). c–e Normalized density of states and f–h participation ratio for the eigenstates in the Fock basis for the values of ξ indicated in (c–e); even parity sector. Numerical (shade) and analytical (solid line) data are shown in (c–e). The vertical dashed line in (c–h) is the ESQPT energy from Eq. (3). i–l Husimi functions for different eigenstates and ξ = 180. a b j h g Fig. 1 Spectral kissing and localization. a Energy levels as a function of the control parameter reproducing the experimental data59 with K/(2π) = 0.32 MHz and b E0=ð_KÞ for larger values of ξ. INTRODUCTION The presence of the ESQPT gets reﬂected in the structure of the eigenstates, ψ j i ¼ P nCn n j i, written in the Fock basis, ^ay^a n j i ¼ n n j i. The eigenstates at the vicinity of the ESQPT are highly localized in the Fock state 0j i27–29. This can be quantiﬁed with the participation ratio, PR ¼ 1= PN 1 n¼0 jCnj4, where N is the size of the truncated Hilbert space. PR is large for an extended state and small for a localized state. In Fig. 1f–h, we show the participation ratio as a function of E0. An abrupt dip in the value of PR happens for E0 E0 ESQPT and the analysis of the components of the eigenstate at this energy conﬁrms its localization at 0j i. Equivalently to PR, the plot of the occupation number hψj^ay^ajψi as a function of energy exhibits a dip at E0 E0 ESQPT (Supplementary Note 2). npj Quantum Information (2023) 76 Published in partnership with The University of New South Wales Quantum system The system that we investigate was implemented in a super- conducting circuit59 based on driven SNAIL60 transmons. The static effective Hamiltonian of this system is given by (Supple- mentary Note 1) ^Hqu _ K ¼ ^nð^n 1Þ ξ ^ay2 þ ^a2 ; (1) (1) ESQPT The localization at the ESQPT critical point is also detected with the Husimi function61 obtained by writing the eigenstates in the basis of Glauber coherent states [see Eq. (13)]. The Husimi function gives the distribution of the quantum state in the phase space of canonical variables (q, p). As seen in Fig. 1k, the eigenstate closest to the ESQPT energy is highly concentrated in the origin of the phase space. This contrasts with the eigenstates below the ESQPT [Fig. 1i, j], which present two separated ellipses, and the eigenstates above it [Fig. 1l]. The localization in the phase space mirrors the localization where ^n ¼ ^ay^a, K is the Kerr nonlinearity, ξ = ϵ2/K is the control parameter, and ϵ2 is the squeezing amplitude. The system conserves parity, ½^Hqu; ð1Þ^ay^a ¼ 0. ^ q We study the spectrum of ^Hqu as a function of the control parameter ξ in Fig. 1a–e. The plots display the excitation energies, E0 ¼ ðE E0Þ, where E are the eigenvalues of ^Hqu and E0 its ground state energy. The numerical data in Fig. 1a reproduce the experimental data in Fig. 3A of ref. 59. One sees that as the control J. Chávez-Carlos et al. h F k b i i h h i h ( 0 0) I h i i h ξ 0 Th h SNAPSHOTS OF THE HUSIMI FUNCTIONS a b c d e Fig. 2 Phase space and quantum dynamics. a Energy curves in the phase space obtained with Eq. (2). The hyperbolic point is denoted as O, the center points are represented with blue diamonds, and the solid line intersecting at O is the separatrix. Points O, A–E mark the centers of the initial coherent states chosen for the quantum dynamics. b Evolution of the FOTOC, c Husimi entropy, and d survival probability as a function of time. The exponential [linear] curve with rate [slope] given by the Lyapunov exponent in Eq. (4) are indicated in (b)[c]. Quantum system e Snapshots of the Husimi functions; each row refers to one of the six initial coherent states investigated, and each column to a different time, as indicated. 3 a a b c d b a d SNAPSHOTS OF THE HUSIMI FUNCTIONS e SNAPSHOTS OF THE HUSIMI FUNCTIONS e SNAPSHOTS OF THE HUSIMI FUNCTIONS e SNAPSHOTS OF THE HUSIMI FUNCTIONS e HUSIMI FUNCTIONS Fig. 2 Phase space and quantum dynamics. a Energy curves in the phase space obtained with Eq. (2). The hyperbolic point is denoted as O, the center points are represented with blue diamonds, and the solid line intersecting at O is the separatrix. Points O, A–E mark the centers of the initial coherent states chosen for the quantum dynamics. b Evolution of the FOTOC, c Husimi entropy, and d survival probability as a function of time. The exponential [linear] curve with rate [slope] given by the Lyapunov exponent in Eq. (4) are indicated in (b)[c]. e Snapshots of the Husimi functions; each row refers to one of the six initial coherent states investigated, and each column to a different time, as indicated. in the Fock basis, since the coherent state with (q = 0, p = 0) coincides with the Fock state 0j i. It presents three stationary points when ξ > 0. They are the two center points fq; pg ¼ f ± ﬃﬃﬃﬃﬃ 2ξ p ; 0g with the minimal energy of the system Emin ¼ Hclðq; pÞ ¼ Kξ2, and the hyperbolic point {q, p} = {0, 0} with energy Ehyp ¼ 0. In the plot of the energy contours in Fig. 2a, the hyperbolic point is indicated as O, the red line that intersects at this point is the separatrix, and the two blue diamonds are the center points. npj Quantum Information (2023) 76 Published in partnership with The University of New South Wales Quantum dynamics: localization in Fig. 2c, where M2(t) is the integral of the square of the Husimi function (Supplementary Note 5.1). Both quantities, Fotoc(t) and SH2(t), measure how an evolving state spreads in the phase space. Snapshots of the evolution of the Husimi functions for ΨA;B;Cð0Þ (left) and for ΨO;D;Eð0Þ (right) are presented in Fig. 2e (more snapshots are in Supplementary Note 5.1 and videos are available in69). The results are as follows. in Fig. 2c, where M2(t) is the integral of the square of the Husimi function (Supplementary Note 5.1). Both quantities, Fotoc(t) and SH2(t), measure how an evolving state spreads in the phase space. Snapshots of the evolution of the Husimi functions for ΨA;B;Cð0Þ (left) and for ΨO;D;Eð0Þ (right) are presented in Fig. 2e (more snapshots are in Supplementary Note 5.1 and videos are available in69). The results are as follows. While the fastest and longest scrambling happens for the initial coherent state ΨOð0Þ j i, this state also presents the slowest decay of the survival probability, SpðtÞ ¼ hΨð0ÞjΨðtÞi j j2: (7) (7) SpðtÞ ¼ hΨð0ÞjΨðtÞi j j2: The survival probability for all other initial coherent states, with energy above or below the ESQPT, decays faster than SðOÞ p ðtÞ, as seen in Fig. 2d. (O): After the parabolic increase in t, that happens for very short times Kt < Kτ ¼ ð ﬃﬃﬃ 8 p ξÞ 1 (Supplementary Note 6), FðOÞ otocðtÞ [SðOÞ H2 ðtÞ] for the initial coherent state at the hyperbolic point, ΨOð0Þ j i, grows exponentially [linearly] fast with a rate proportional to the classical Lyapunov exponent given in Eq. (4), that is, FðOÞ otocðtÞ / e2λt [SðOÞ H2 ðtÞ / λt]. The snapshot of the Husimi function for a time as small as Kt = 0.013 indicates that ΨOðtÞ j i is already very spread out in phase space, covering an area larger than that for the other ﬁve states, even those with larger energies. Indeed, around Kt = 0.013, FðOÞ otocðtÞ [SðOÞ H2 ðtÞ] reaches the highest value among the states considered, as seen in Fig. 2b[c]. The maximum value happens at the Ehrenfest time, T lnðξÞ=λ (Supplementary Note 7). Classical limit The Hamiltonian of the Kerr oscillator in Eq. (1) develops two wells when ξ > 0. The depth of the wells and their energy levels grow as ξ increases, bringing the system closer to the classical limit. Experimentally, the value of ξ can be increased by reducing the impedance of the circuit, increasing the microwave power of the squeezing drive, or approaching the Kerr-free point (Supplemen- tary Note 1). The properties of the quantum system ﬁnd a parallel in the classical limit. The energy difference Ehyp Emin marks the separatrix in Fig. 2a and determines the energy of the ESQPT, (3) E0 ESQPT Kξ2; (3) E0 ESQPT Kξ2; The grounds for the onset of the ESQPT are found in the classical limit. The classical Hamiltonian is derived in Methods and is given by which is indicated with a bright orange line in Fig. 1b. The equality in Eq. (3) holds in the classical limit. Below this energy, the pairs of stable periodic orbits with equal energy are analogous to the degenerate states of the quantum system, and above that line the degeneracy is lost. The stationary point at the origin of the phase Hcl K ¼ 1 4 ðq2 þ p2Þ 2 ξðq2 p2Þ: (2) (2) Published in partnership with The University of New South Wales Published in partnership with The University of New South Wales J. Chávez-Carlos et al. 4 the initial distribution (see the Husimi function at Kt = 0.14). In the absence of dissipation, this yo-yo process of spreading and contraction persists for a long time (Supplementary Note 5.2). This behavior is the quantum counterpart of the classical dynamics at the vicinity of the hyperbolic (saddle) point O, which is both a repellor and an attractor (Supplementary Note 4), resulting in trajectories that move both towards and away from O. We also note that despite reaching the highest value at t T , the inﬁnite- time average of FðOÞ otocðtÞ is actually smaller than the saturation value for FðD;EÞ otoc ðtÞ (Supplementary Note 7). This result shows that the degree of spreading quantiﬁed by OTOCs depends not only on the initial state and system, but also on the timescale. space, (q, p) = (0, 0), justiﬁes the localization at the Fock state 0j i of the eigenstate with energy at the ESQPT. λ ¼ 2Kξ: The system described by Eq. (2) is regular, so the Lyapunov exponent for any initial condition is zero, except for the unstable point O32,64,65. y (A): The initial coherent state ΨAð0Þ j i is very close to a center point, so the evolution is very slow, FðAÞ otocðtÞ [SðAÞ H2 ðtÞ] never reaches large value, and the Husimi function remains close to the point A. (B) and (C): State ΨBð0Þ j i [ ΨCð0Þ j i] is slightly below [above] the ESQPT. Instead of the conﬁnement around the center point imposed to the classical orbit B, quantum effects allow ΨBðtÞ j i to escape and evolve similarly to ΨCðtÞ j i. The spread of the Husimi distributions for both states is comparable, reaching regions of the phase space with + q and −q (see snapshots in Fig. 2e and in Supplementary Note 5.2). In addition, since B and C are in the vicinity of the unstable point O, quantum ﬂuctuations trigger the exponential [linear] growth of FðB;CÞ otoc ðtÞ [SðB;CÞ H2 ðtÞ] observed in Fig. 2b, c. This behavior is at odds with the classical limit, where the positive Lyapunov exponent emerges only at the hyperbolic point and not close to it. As ξ increases and one approaches the classical limit, the duration of the exponential behavior for FðB;CÞ otoc ðtÞ decreases. (6) (6) SH2ðtÞ ¼ ln M2ðtÞ; Published in partnership with The University of New South Wales npj Quantum Information (2023) 76 Quantum dynamics: instability The instability associated with the hyperbolic point is manifested in the quantum domain with the exponential growth of out-of- time-ordered correlators (OTOCs)32,64–66. These quantities, deﬁned y yp p in the quantum domain with the exponential growth of out-of- time-ordered correlators (OTOCs)32,64–66. These quantities, deﬁned as Otoc ¼ h½ ^WðtÞ; ^Vð0Þ 2i, measure the spread (scrambling) of quantum information by assessing how the operators ^W and ^V fail to commute due to the evolution of ^W67. A particular example of OTOCs is the FOTOC, which corresponds to having the operator ^V ¼ Ψð0Þ j i Ψð0Þ h j, for the initial state Ψð0Þ j i, and ^W ¼ eiδϕ^G, where δϕ is a small perturbation and ^G is a Hermitian operator. In the perturbative limit, δϕ ≪0, the FOTOC is the variance σ2 GðtÞ ¼ h^G 2ðtÞi h^GðtÞi 268. as Otoc ¼ h½ ^WðtÞ; ^Vð0Þ 2i, measure the spread (scrambling) of quantum information by assessing how the operators ^W and ^V fail to commute due to the evolution of ^W67. A particular example of OTOCs is the FOTOC, which corresponds to having the operator as Otoc ¼ h½ ^WðtÞ; ^Vð0Þ 2i, measure the spread (scrambling) of quantum information by assessing how the operators ^W and ^V fail to commute due to the evolution of ^W67. A particular example of OTOCs is the FOTOC, which corresponds to having the operator ^V ¼ Ψð0Þ j i Ψð0Þ h j, for the initial state Ψð0Þ j i, and ^W ¼ eiδϕ^G, where δϕ is a small perturbation and ^G is a Hermitian operator. In the perturbative limit, δϕ ≪0, the FOTOC is the variance quantum information by assessing how the operators ^W and ^V fail to commute due to the evolution of ^W67. A particular example of OTOCs is the FOTOC, which corresponds to having the operator ^V ¼ Ψð0Þ j i Ψð0Þ h j, for the initial state Ψð0Þ j i, and ^W ¼ eiδϕ^G, where δϕ is a small perturbation and ^G is a Hermitian operator. In the perturbative limit, δϕ ≪0, the FOTOC is the variance σ2 GðtÞ ¼ h^G 2ðtÞi h^GðtÞi 268. ^V ¼ Ψð0Þ j i Ψð0Þ h j, for the initial state Ψð0Þ j i, and ^W ¼ eiδϕ^G, where δϕ is a small perturbation and ^G is a Hermitian operator. Classical limit g gy The existence of a non-degenerate hyperbolic point implies the logarithmic discontinuity of the level density, as shown in refs. 18,62, and explains the peak at E0 ESQPT in Fig. 1c–e. Using the smooth component of the Gutzwiller trace formula63, we obtain a semiclassical approximation for the DOS (Supplementary Note 3). This curve outlines the numerical data in Fig. 1c–e. g Another consequence of the hyperbolic point is the onset of a positive Lyapunov exponent (Supplementary Note 4), (4) λ ¼ 2Kξ: Quantum dynamics: instability In the perturbative limit, δϕ ≪0, the FOTOC is the variance σ2 GðtÞ ¼ h^G 2ðtÞi h^GðtÞi 268. otoc ð Þ (D) and (E): States ΨDð0Þ j i and ΨEð0Þ j i have the same high energy, but evolve differently. In terms of scrambling, ΨEð0Þ j i combines the best of both worlds, because in addition to high energy, which leads to the largest saturation value for FðD;EÞ otoc ðtÞ (Supplementary Note 7), it partially overlaps with the separatrix (see the snapshot of the Husimi function at t = 0 in Fig. 2e), so FðEÞ otocðtÞ [SðEÞ H2ðtÞ] in Fig. 2b, c presents an exponential [linear] growth analogous to that seen for ΨB;Cð0Þ , which is absent for ΨDð0Þ j i. The spread of the Husimi distribution for ΨEð0Þ j i happens simultaneously inside and outside the separatrix (Supplementary Note 5.2), leading to complicated quantum interference effects, as those observed in the snapshot of the Husimi function at Kt = 0.14. G We analyze the evolution of the FOTOC given by the variance of p and q, FotocðtÞ ¼ σ2 pðtÞ þ σ2 qðtÞ; (5) FotocðtÞ ¼ σ2 pðtÞ þ σ2 qðtÞ; (5) because the initial coherent states that we consider spread in both canonical coordinates32. These states are centered at the points O, A–E, marked in Fig. 2a, and are denoted as Ψjð0Þ with j = O, A, …, E. State ΨAð0Þ j i has the lowest energy, followed by ΨBð0Þ j i (negative energy close to zero), ΨOð0Þ j i (zero energy), and ΨCð0Þ j i (positive energy close to zero). States ΨDð0Þ j i and ΨEð0Þ j i have equal and high positive energy (see Methods). q g p gy We compare the growth of Fotoc(t) in Fig. 2b with the Husimi entropy, SH2ðtÞ ¼ ln M2ðtÞ; (6) Published in partnership with The University of New South Wales METHODS (12) In the Supplementary Note 1, we describe how the original driven Hamiltonian leads to the static effective Hamiltonian, where ^a α j i ¼ α α j i, N is the truncation of the Hilbert space, where ^a α j i ¼ α α j i, N is the truncation of the Hilbert space, ^Hqu _ ¼ K^ay2^a2 þ ϵ2ð^ay2 þ ^a2Þ; (8) (8) α ¼ ﬃﬃﬃ 1 2 r ðq þ ipÞ and how the parameters can be experimentally controlled. In the main text, we changed the sign of the Hamiltonian in Eq. (1) for convenience, so that we could say that E0 in E0 ¼ E E0 is the ground state energy of ^Hqu, instead of its highest energy. Regardless of the sign convention, dissipation will bring the experimental system to the attractors (stable nodes) in the bottom of the wells, which deﬁne unambiguously the ground state of the system. and Neff = 1, the Husimi function is given by Qψðq; pÞ ¼ 1 2π X N n¼0 Cneðq2þp2Þ 4 ðq ipÞn ﬃﬃﬃﬃﬃﬃﬃﬃﬃ 2nn! p 2 : (13) (13) DISCUSSION leads to the classical Hamiltonian (with ħ = 1), leads to the classical Hamiltonian (with ħ = 1), Hcl ¼ Kcl 4 ðq2 þ p2Þ 2 þ Kclξclðq2 p2Þ; (11) (11) where K ¼ Kcl=N2 eff and ξ ¼ ξclNeff: K ¼ Kcl=N2 eff and ξ ¼ ξclNeff: In the main text, we ﬁxed In the main text, we ﬁxed Neff ¼ 1; We conclude with a brief discussion about the static effective Hamiltonian, ^Hqu, investigated here and used to describe the driven SNAIL transmon in ref. 59. As the drive amplitude and nonlinearities of the experimental system increase, ^Hqu ceases to be valid, the ESQPT melts away, and chaos eventually sets in. The emergence of chaos, which could be captured experimentally and may affect the development of quantum devices, cannot be described by any static effective Hamiltonian9,14,59 obtained for systems with only one degree of freedom. The analysis of chaos, which will be the subject of our forthcoming papers, has to rely entirely on the original time-dependent Hamiltonian. and used large values of ξ. and used large values of ξ. and used large values of ξ. The experimental system admits an approximate classical description if it is initialized in a coherent state and for as long as the Hamiltonian phase space surface produces only a linear force (a quadratic Hamiltonian) over the spread of the evolving state. Quantum dynamics: localization The apparent paradox of the fast spread of ΨOðtÞ j i, as measured by FðOÞ otocðtÞ and SðOÞ H2 ðtÞ, and the slow decay of SðOÞ p ðtÞ is naturally resolved in view of the classical limit and from the analysis of the Husimi functions. The instability associated with the hyperbolic point O is the source of the exponentially fast spread of the variance of the phase-space distribution, but O is also a stationary point (the gradient of the Hamiltonian at this point is zero), so ΨOð0Þ j i is strongly localized in the eigenstate at the ESQPT [see Fig. 1k]. In other words, the width of the energy distribution for ΨOð0Þ j i, given by ﬃﬃﬃ 2 p Kξ, is the smallest one among the six states (Supplementary Note 6). Close to the origin of the phase space, the evolution is dominated by the squeezing, ^Hqu ϵ2ð^q2 ^p2Þ. This leads to the rapid stretching of ΨOðtÞ j i, while part of the ð Þ The fast scrambling of quantum information for ΨOðtÞ j i, which happens for τ < t < T , is later followed by partial reconstructions of J. Chávez-Carlos et al. 5 become very deep and the number of levels inside the wells become macroscopic, so ^Hqu exhibits properties comparable to the classical Hamiltonian. However, to derive the classical Hamiltonian for any depth of the wells, that is, to approach a continuous spectrum for a ﬁxed and not necessarily large value of the control parameter, we introduce the parameter Neff, whose reciprocal is related with the size of the zero point ﬂuctuations. We write population remains for some time in the vicinity of the origin. These two aspects of the dynamics become evident in the snapshot of the Husimi function for ΨOðtÞ j i at Kt = 0.0075. The small green ellipse in those panels indicates the size of the initial coherent state. One sees that the Husimi distribution for ΨOðtÞ j i at Kt = 0.0075 is stretched out, but part of it remains inside the green ellipse. Husimi function For an eigenstate written in the basis of the Glauber coherent states, α j i ¼ e1 2jαj2 X N n¼0 αn ﬃﬃﬃﬃ n! p n j i; (12) Published in partnership with The University of New South Wales DISCUSSION ^a ¼ ﬃﬃﬃﬃﬃﬃﬃﬃ Neff 2 r ^q þ i^p ð Þ; (9) ^a ¼ ﬃﬃﬃﬃﬃﬃﬃﬃ Neff 2 r ^q þ i^p ð Þ; (9) This work bridges communities working on superconducting circuits, ESQPTs, and nonequilibrium quantum dynamics. The squeeze-driven Kerr oscillator is an addition to the list of nuclear, molecular, and condensed matter systems that exhibit ESQPTs. Its advantage is to be experimentally realizable in an available superconducting circuit platform, where both frequency and time domain measurements can be done simultaneously, the control parameter can be tuned to approach the classical limit, arbitrary initial states can be prepared, and the dynamics can be studied in phase space. We expect superconducting circuits to become versatile quantum simulators for ESQPTs and related phenomena, such as isomerization, where the separation between neighboring energy levels decreases close to the isomerization barrier height70,71. and ½^q; ^p ¼ i Neff ; so the classical limit can be reached by taking Neff →∞, since ^q ! q and ^p ! p. This way, the quantum Hamiltonian, so the classical limit can be reached by taking Neff →∞, since ^q ! q and ^p ! p. This way, the quantum Hamiltonian, Hqu _ ¼ KN2 eff 4 ^q i^p ð Þ2 ^q þ i^p ð Þ2 þ ξ KNeff 2 ½ ^q i^p ð Þ2 þ ^q þ i^p ð Þ2; (10) (10) The dynamical consequences of ESQPTs that we presented should also appeal to experimental platforms, where long-range couplings can be tuned to approach models with collective interactions, such as those with cold atoms72 and trapped ions73. Of interest to those experiments is the demonstration of the exponential growth of OTOCs, which we showed to emerge for different initial states placed close to the separatrix that marks the ESQPT. Other highlights include the later revivals of a coherent state initially centered at the phase-space origin, the combined effects of fast scrambling and subsequent interferences for a high- energy state close to the separatrix, and the different dynamics for states with the same energy but initially located in different regions of the phase space. npj Quantum Information (2023) 76 Classical limit The six initial coherent states that we consider are obtained by using in Eq. (12) the values of p and q speciﬁed below. These are the points marked in Fig. 2a. Their classical energies E are given For large values of the control parameter, ξ = ϵ2/K ≫1, the double wells created by the quantum Hamiltonian in Eq. (8) J. Chávez-Carlos et al. 6 for ξcl = 180. Point O : q ¼ 0; p ¼ 0; E=Kcl ¼ 0: Point A : q ¼ 16:9143; p ¼ 0; E=Kcl ¼ 3:1034 ´ 104: Point B : q ¼ 1:2533; p ¼ 0; E=Kcl ¼ 0:0282 ´ 104: Point C : q ¼ 1:2506; p ¼ 0; E=Kcl ¼ 0:0282 ´ 104: Point D : q ¼ 0; p ¼ 8:4443; E=Kcl ¼ 1:4106 ´ 104: Point E : q ¼ 28:1302; p ¼ 0; E=Kcl ¼ 1:4106 ´ 104: (14 6 16. Cejnar, P., Heinze, S. & Macek, M. Coulomb analogy for non-hermitian degen- eracies near quantum phase transitions. Phys. Rev. Lett. 99, 100601 (2007). 17. Caprio, M., Cejnar, P. & Iachello, F. Excited state quantum phase transitions in many-body systems. Ann. Phys. 323, 1106 (2008). 18. Cejnar, P., Stránský, P., Macek, M. & Kloc, M. Excited-state quantum phase tran- sitions. J. Phys. A 54, 133001 (2021). 19. Stránský, P., Cejnar, P. & Filip, R. Stabilization of product states and excited-state quantum phase transitions in a coupled qubit-ﬁeld system. Phys. Rev. A 104, 053722 (2021). 20. Corps, A. L. & Relaño, A. 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https://openalex.org/W4313448844	https://zenodo.org/records/7502055/files/7.%20EVOLUCI%C3%93N%20DE.pdf, https://zenodo.org/record/7392254/files/8-Evoluci%C3%B3n%20de%20las%20entradas%20de%20inversi%C3%B3n.pdf	es		EVOLUCIÓN DE LAS ENTRADAS DE INVERSIÓN EXTRANJERA DIRECTA (IED) Y SU PATRON DE DISTRIBUCIÓN TERRITORIAL EN EL ESTADO DE VERACRUZ, 2000-2021	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	7,034	Economía EVOLUCIÓN DE LAS ENTRADAS DE INVERSIÓN EXTRANJERA DIRECTA (IED) Y SU PATRON DE DISTRIBUCIÓN TERRITORIAL EN EL ESTADO DE VERACRUZ, 2000-2021 THE INFLOWS OF FOREIGN DIRECT INVESTMENT (FDI) AND IT’S PATTERN OF TERRITORIAL DISTRIBUTION IN THE STATE OF VERACRUZ, 2000-2021 Ángel Toledo Tolentino* SUMARIO: Introducción, 1. Enfoques teóricos sobre la inversión extranjera directa (IED), La nueva geografía económica (NGE), El modelo ecléctico, Determinantes que favorecen la entrada de IED, 2. Evolución de la inversión extranjera directa (IED) a nivel nacional, 3.La inversión extranjera directa en el estado de Veracruz, 4. Distribución territorial de la inversión extranjera directa en Veracruz, Conclusión, Referencias ABSTRACT RESUMEN Este trabajo analiza la evolución de las This paper analyzes the evolution of Foreign entradas de la inversión extranjera directa Direct Investment (FDI) inflows and their (IED) y su patrón de distribución territorial territorial distribution pattern in the state of en el estado de Veracruz, del año 2000 al Veracruz, from 2000 to 2021. A quantitative 2021. Se utilizó un enfoque cuantitativo, approach was used, based on data from the sustentado en datos del gobierno mexicano Mexican Government of Foreign Direct de inversión extranjera directa (IED), con un Investment. (FDI), with a longitudinal alcance de corte longitudinal y de fuentes scope and documentary sources, which documentales, que sirvieron para realizar served to carry out a descriptive statistical un análisis estadístico de tipo descriptivo. analysis. Three groups of municipalities Se identificaron tres grupos de municipios with presence of FDI were identified: the con presencia de IED: el primero son first, are the central municipalities of the los municipios centrales de las zonas metropolitan areas of the state and in those metropolitanas del estado y en aquellos con with maritime vocation, with significant vocación marítima, exhibiendo economías presence of agglomeration economies; *Doctor en Estudios del Desarrollo (UAZ), maestro en Estudios Urbanos (El Colegio de México), licenciado en Economía (UCC). Docente de la Universidad de Xalapa, México. https://orcid.org/0000-0002-5693-6719; correo electrónico: atoledot@gmail. com. Universita Ciencia año 10, número 29 sep-dic 2022 87 RECIBIDO: 22 de agosto, 2022 ACEPTADO: 27 de septiembre, 2022 de aglomeración; el segundo, municipios the second, peripheral municipalities periféricos de las zonas metropolitanas, of the metropolitan areas, but have an pero tienen una importante relación con important relationship with the central los municipios centrales y un tercero, municipalities and a third, municipalities municipios que presentan importantes that have important natural resources. recursos naturales. PALABRAS CLAVE: inversión extranjera KEYWORDS: foreign direct investment, directa, ubicación geográfica, Veracruz, geographic location, Veracruz, agglomeration economías de aglomeración economies DOI: 10.5281/ZENODO.7392254 Universita Ciencia año 10, número 29 sep-dic 2022 88 INTRODUCCIÓN Para lo anterior, el artículo se estructura de la siguiente forma: primero, con base Este artículo pretende responder en la nueva geografía económica (NGE) a la siguiente pregunta ¿Cómo han y en el modelo OLI se establecen los evolucionado las entradas de inversión elementos teóricos del estudio; segundo, extranjera directa (IED) en el estado se analiza el comportamiento de la IED de Veracruz y el patrón de distribución a nivel internacional y nacional; tercero, territorial en el período comprendido se estudia la evolución del indicador en de 2000 a 2021? Para tal fin, se utiliza la el período de análisis, sus tendencias y información proveniente de la Dirección sectores económicos involucrados en el General de Inversiones Extranjeras (DGIE) estado de Veracruz; la cuarta, se identifica y del Registro Nacional de Inversiones a los municipios que son receptores de Extranjeras (RNIE), pertenecientes a la IED en el estado, los sectores económicos Secretaría de Economía (SE) de México. El beneficiados con su arribo y se delinean trabajo sigue un enfoque cuantitativo, de algunos elementos que facilitan su arribo; corte longitudinal, descriptivo y con fuentes por último, se establecen las conclusiones documentales, con los cuales se elaboraron y recomendaciones del trabajo. gráficos y variaciones porcentuales que sirvieron para analizar la información y dar 1. ENFOQUES TEÓRICOS SOBRE LA una respuesta a la pregunta planteada. INVERSIÓN EXTRANJERA DIRECTA (IED) Se tiene como objetivo ubicar cuáles son los municipios que son receptores de esa Se considera a la inversión extranjera inversión, en qué sectores de sus economías directa (IED) como “Una operación que se localizan y explorar qué elementos involucra una relación de largo plazo en la determinan su arribo; bajo el entendido que cual una persona física o jurídica residente la IED la realizan empresas multinacionales de una economía (inversor directo) tiene (EMNS) con la finalidad de lograr ganancias el objetivo de obtener una participación y que aprovechan condiciones económicas, duradera en una empresa o entidad político-institucionales y la utilización de residente de otra economía” (García y recursos naturales y geográficos para López, 2020, p. 6). Por tanto, la IED se asentarse en el territorio. El trabajar a entiende como una cantidad de dinero que nivel municipal es un aspecto novedoso de se invierte por una persona o empresa en esta investigación, ya que por lo regular la un negocio en el extranjero con el fin de información a este nivel de desagregación obtener beneficios económicos de largo no es tan completa. En consecuencia, los plazo, la empresa inversora puede o no resultados son un insumo para futuras tener el control total de la compañía local. investigaciones y para los tomadores de Dentro de la IED se pueden encontrar dos decisiones que se encuentran vinculados formas de cómo se realiza la inversión: la con la atracción de la inversión extranjera. primera, denominada de inicio greenfield investment, que consiste en tener una Universita Ciencia año 10, número 29 sep-dic 2022 89 operación nueva en el extranjero; la La NGE pretende explicar los procesos segunda, por fusiones y adquisiciones, la de concentración (fuerzas centrípetas) cual se materializa al comprar acciones o y dispersión (fuerzas centrífugas) que se fusionarse con una empresa local que ya producen en una región. En consecuencia, la NGE genera modelos matemáticos registra operaciones (Hill, 2011). que hacen posible entender la dinámica La IED la llevan a cabo empresas de una región en el contexto de toda la multinacionales (EMN): “Las cuales poseen economía. Bajo esta perspectiva, a una una casa matriz (headquarter) en su país escala más específica busca explicar porque de origen y filiales localizadas en distintos las compañías tienden a concentrarse países extranjeros” (García y López, en lugares donde existan más empresas 2020). La EMN lleva a otros países partes previamente localizadas, para lo cual toma de sus operaciones (producción, diseño, dos tipos de externalidades como lo son las mercadotecnia y comercialización de sus economías de aglomeración (Cuadradoproductos o servicios), donde la matriz Roura, 2014). juega un papel trascendente en la toma de Este enfoque es consciente, en línea decisiones de las filiales. con la causación acumulativa, que las Una vez definido el concepto de IED desigualdades regionales prevalecerán y sus elementos conceptuales más como resultado de las economías de relevantes, es necesario abordar los aglomeración, las cuales resultan de principales argumentos teóricos, para tal ganancias en la productividad por fin se considera el tema de la localización ubicarse en centros muy poblados o mediante la nueva geografía económica con muchas actividades productivas, (NGE) y las motivaciones que tiene una que suelen producirse en las regiones EMN para invertir en el exterior desde el más desarrolladas, en perjuicio de las punto de vista estratégico con el modelo menos favorecidas, por lo tanto no hay convergencia regional (Moncayo Jiménez, OLI o paradigma ecléctico. 2002; Polése, 1998). La nueva geografía económica (NGE) La nueva geografía económica (NGE) De acuerdo con Bracamonte Jaraba et al retomó aspectos teóricos postulados (2018), las economías de aglomeración con anterioridad: el lugar central y la se descomponen en tres vías: economías organización jerárquica de las actividades internas a la empresa, relacionados con en los centros urbanos (Losh y Christaller), el hecho de concentrar la producción en las economías de aglomeración (Marshall), una sola unidad económica; economías de de las ciencias regionales (Isard), y la localización, que se relacionan con ubicar causación circular acumulativa de (Myrdal la producción en industrias particulares y; economías de urbanización, que responden y Kaldor) (Moncayo Jiménez, 2002). al tamaño o diversificación de funciones de la ciudad. Estas últimas son relevantes Universita Ciencia año 10, número 29 sep-dic 2022 90 porque son ganancias que logran las Multinacionales (EMNS). Para Dunning, empresas al ubicarse en una ciudad y por las multinacionales tienen ventajas de el uso que hacen de los servicios públicos propiedad que buscan explotarlas en e infraestructura existentes, mano de terceros países mediante las siguientes obra calificada y diversidad de clientes y opciones: exportar bienes y servicios al proveedores a su disposición (Polése, 1998). extranjero, conceder licencias e internalizar Las economías aglomeración aumentan dichas ventajas mediante la construcción o cuando una empresa disfruta de adquisición de instalaciones en otros países, rendimientos crecientes de escala (a es decir, IED (Padilla-Pérez & Gomesmedida que va aumentando la producción Nogueira, 2015). los costos medios totales van disminuyendo hasta alcanzar un mínimo, si una empresa Invertir en un país extranjero involucra se encuentra en esta etapa se considera que riesgos y desembolsos importantes de tiene economías de escala) en un centro o recursos, por lo tanto, es mejor producir los región determinados, lo cual puede deberse bienes en casa y exportarlo al país destino; o a distintas causas: la disponibilidad de bien, conceder licencias a empresas locales recursos naturales o de localización (que no para que fabriquen y vendan los productos están dispersos); la posición monopolística de la multinacional en otro país a cambio o cuasi monopolística de una o varias de regalías, en este sentido, la empresa empresas (condiciones de competencia local asume los riesgos. No obstante, el imperfecta); una decisión política tomada exportar desde casa presenta obstáculos en el pasado; y otras posibles razones que como las barreras comerciales y los costos puedan plantearse al respecto (Cuadrado- de transporte; mientras que la emisión Roura, 2014). Lo anterior es relevante, ya de licencias enfrenta el peligro que la que la IED se ve favorecida en territorios empresa local asimile la transferencia de donde se producen estas economías de conocimientos y se vuelva un competidor local de importancia para la empresa aglomeración. (Hill, 2011). En consecuencia, dado que tanto la exportación y las licencias tienen El modelo ecléctico Se toma como base los aportes teóricos de problemas, Dunning manifiesta que una John H. Dunning, que desarrolló un modelo empresa local invierta en otro país (IED), para estudiar qué motiva a una empresa a sobre esta base él desarrolló el modelo OLI. invertir en otro país, los determinantes y las estrategias, a esto se le conoce Con base en lo anterior, la ventaja de como “paradigma ecléctico” o modelo propiedad (O) afirma que para conquistar Ownership-Location-Internalisation (OLI, los mercados extranjeros una empresa por sus siglas en inglés); este modelo toma multinacional requiere obtener ventajas elementos de la teoría de la organización competitivas derivadas de los siguientes industrial, de la localización de actividades elementos intangibles (tecnología, marcas, y de la internalización con el fin de poder patentes, mercadotecnia, de administración comprender el actuar de la Empresas y diferenciación de productos) y tangibles Universita Ciencia año 10, número 29 sep-dic 2022 91 (mano de obra, capital y recursos naturales) se enfocan en conseguir capacidades (Padilla-Pérez y Gomes-Nogueira, 2015; tecnológicas, de conocimiento del García y López, 2020). mercado y de gestión administrativa para mantenerse competitivas. En la práctica, Respecto a la ventaja de ubicación (L), se encuentran combinaciones entre los es necesario que el país extranjero diferentes tipos aquí mencionados (Padillacuente con elementos que impulsen la Pérez y Gomes-Nogueira, 2015; García y atracción de EMNS como el tamaño del López, 2020). mercado, recursos naturales y los costos de transporte, además de un entramado Determinantes que favorecen la entrada jurídico e institucional que dé certeza a de IED las nuevas inversiones y se traduzca en No existe un solo factor que determine la beneficios económicos. Por último, las IED, por el contrario, hay muchos elementos ventajas de internalización (I) explican que inciden que una multinacional porque las multinacionales buscarían decida invertir en un país, como son los realizar inversiones en el extranjero (IED) siguientes: a) condiciones económicas o internalizar sus operaciones. Si se tienen generales: el tamaño y potencial del mayores beneficios con IED que al exportar mercado interno, disponibilidad de los al exterior o al otorgar licencias, aumentan factores de la producción (recursos las posibilidades que una empresa prefiera naturales, capital y mano de obra), producir sus bienes o servicios en otro país estabilidad macroeconómica, economías (Padilla-Pérez y Gomes-Nogueira, 2015; de aglomeración, infraestructura de Hill, 2011). transporte y comunicaciones, capacidades tecnológicas y de innovación locales; Dunning identificó cuatro formas por las b) condiciones político-institucionales: cuales las multinacionales realizan IED: claridad en las reglas políticas y jurídicas buscadoras de recursos, de mercados, para los inversionistas, normas regulatorias, de eficiencia y de activos estratégicos. impuestos, liberalización comercial y Las EMNS buscadoras de recursos, acuerdos regionales con terceros países; c) invierten con el fin de explotar recursos instrumentos para incidir en las decisiones naturales (minerales, energéticos, de inversión: cercanía geográfica, idioma productos agrícolas) que se encuentran similar, aspectos culturales y de horarios en un determinado país. Las buscadoras (García & López, 2020). de mercados realizan las inversiones con el objetivo de producir y vender bienes En síntesis, el utilizar los aportes de la NGE y servicios en el mercado local y en los y del modelo OLI brinda la base teórica países vecinos. En cambio, las buscadoras para comprender porque las empresas de eficiencia buscan reducir sus costos deciden primero en qué país van a invertir y de producción a fin de lograr economías después en que parte del territorio lo harán, de escala y de alcance. Por último, las por tanto, estos enfoques teóricos con buscadoras de activos estratégicos elementos complementarios para nuestro trabajo. Universita Ciencia año 10, número 29 sep-dic 2022 92 2. EVOLUCIÓN DE LA INVERSIÓN que es Estados Unidos de América (EUA), EXTRANJERA DIRECTA (IED) A NIVEL mano de obra calificada, un mercado NACIONAL interno creciente y por las ventajas que ofrece el T-MEC para las mercancías El proceso de globalización económica que que se producen en suelo mexicano, es se presenta desde la segunda mitad del decir, existen determinantes que atraen siglo pasado depende de la liberalización la inversión como lo manifiestan García de las economías nacionales en favor del y López (2020): condiciones económicas libre mercado, el desarrollo e innovación generales, instrumentos para decidir en de nuevas tecnologías y, en particular, la las decisiones de inversión y condiciones desregulación en favor de la atracción político-institucionales. Sin embargo, de IED. En este sentido, nuestro país no en este último aspecto, el Gobierno de estuvo ajeno a esta situación, este proceso México canceló la construcción del Nuevo de liberalización inició con la entrada Aeropuerto Internacional de la Ciudad de de nuestro país al Acuerdo General de México (NAICM) en 2018 y ha modificado Aranceles y Comercio (GATT) en 1986 y aspectos legales en el sector energético, que tuvo su manifestación en materia de elementos que han creado incertidumbre atracción de IED con los cambios legales entre los inversionistas extranjeros, lo que en la década de los noventa (Lascurain ha inhibido la llegada de mayores flujos de Fernández, 2018). dinero. Dado que la atracción de IED es un En la Gráfica 1 se aprecia una tendencia elemento distintivo de la globalización decreciente en las entradas de IED al país, económica y, en particular, de la apertura pues pasó de los 35 mil 250 mdd en el año de las economías nacionales, en esta 2000 a 21 mil 456 mdd en 2021, con una sección se muestra cómo se comportó TCMA de -2.3%. Si se suma la IED recibida la IED en nuestro país desde principios anualmente se logra una cifra acumulada de del presente siglo. Para esta sección se 706 mil 705 mdd, donde el año 2001 fue el tomaron datos de la Dirección General de mejor con 54 mil 765 mdd seguido del 2013 Inversión Extranjera (DGIE), perteneciente con 48 mil 255 mdd; mientras que el peor a la Secretaría de Economía, del año 2000 fue 2020 con 20 mil 395 mdd resultado de al 2021 en términos de millones dólares la pandemia del Covid-19. americanos (mdd) a precios constantes de 2013. Nuestro país en 2021 se ubicó, según datos de la Conferencia de las Naciones Unidas para el Comercio y el Desarrollo (UNCTAD), en el lugar 11 a nivel mundial como receptor de IED, consecuencia de ser vecino del mayor mercado del mundo Universita Ciencia año 10, número 29 sep-dic 2022 93 Gráfica 1. Entradas de Inversión Extranjera Directa (IED) hacia México en millones de dólares, 20002021 (dólares constantes 2013) Fuente: Elaboración propia con base en datos de la Dirección General de Inversión Extranjera (DGIE, 2022a) México registra la IED en tres categorías de acuerdo con su financiamiento: nuevas inversiones (empresas nuevas), reinversión de utilidades y cuentas entre compañías (transferencias de la matriz a la filial y viceversa). En el período de 2000 a 2021, 331 mil 034 mdd (46.5%) fueron nuevas inversiones, 211 mil 812 mdd (30.0%) de reinversión de utilidades y 163 mil 858 mdd (23.2%) provinieron de cuentas entre compañías. Universita Ciencia año 10, número 29 Después de analizar los datos se ubicaron dos períodos de análisis. El primero, que va del año 2000 al 2013, donde el 52.6% de la IED provino de nuevas inversiones, 24.3% reinversión de utilidades y 23.1% de cuentas entre compañías. El segundo, que fue de 2014 a 2021, en él la reinversión de utilidades alcanzó el 43.2%, seguido del 33.4% de nuevas inversiones y 23.4% de cuentas entre compañías. Esta recomposición en la estructura de los flujos de inversión hacia el país fue producto sep-dic 2022 94 de una disminución en el crecimiento de 31 mil 274 mdd a 7 mil 913 mdd, explica económico, incertidumbre por eventos la contracción de la IED en los últimos políticos y las tendencias a la baja de los años (ver Gráfica 2). La baja en la IED de la flujos de inversión a nivel internacional, industria manufacturera se produjo por sin dejar de lado los efectos de la pandemia la disminución en los subsectores de las (Salles Sainz Grant Thornton, 2021). industrias: de las bebidas y del tabaco, la industria química y de la fabricación de La DGIE utiliza la clasificación de las equipo de transporte. actividades de acuerdo con el Sistema de Clasificación Industrial de América del La DGIE registró en el período de estudio Norte, versión 2013 (SCIAN 2013)1 para a 51 países con inversiones en el territorio contabilizar la IED por sector de actividad. nacional, no obstante, tres de ellos El 70% de la IED total en el lapso de análisis representan casi el 70% del total. EUA llegó a los siguientes sectores: la industria encabezó la lista (48.9%), le siguió España manufacturera (46.5%); la industria de (12.9%) y Canadá (6.7%). En el caso del los servicios financieros y de seguros primer y tercer lugar, un factor clave que lo (16.9%) y el comercio (7.4%), el resto de las explica es que ambos son parte del T-MEC actividades sumó un 29.2%. junto con México y su localización. En el caso de España, el tamaño del mercado, el T-MEC, La IED en la industria manufacturera es la ubicación geográfica, beneficios fiscales y relevante para el país, pues fue la que más los acuerdos comerciales con varios países dinero recibió, en consecuencia, la fuerte son los elementos centrales que atraen a las caída que se dio después de 2013 2, al pasar EMS españoles al país (México: primero la 1 Los sectores son los siguientes: 11 inversión, 2019). Agricultura, cría y explotación de animales, aprovechamiento forestal, pesca y caza; 21 Minería; 22 Generación, transmisión y distribución de energía eléctrica, suministro de agua y de gas por ductos al consumidor final; 23 Construcción; 3133 Industrias manufactureras; 43 y 46 Comercio; 48 y 49 Transportes, correos y almacenamiento; 51 Información en medios masivos; 52 Servicios financieros y de seguros; 53 Servicios inmobiliarios y de alquiler de bienes muebles e intangibles; 54 Servicios profesionales, científicos y técnicos; 56 Servicios de apoyo a los negocios y manejo de residuos y desechos, y servicios de remediación; 61 Servicios educativos; 62 Servicios de salud y de asistencia social; 71 Servicios de esparcimiento culturales y deportivos, y otros servicios recreativos; 72 Servicios de alojamiento temporal y de preparación de alimentos y bebidas; 81 Otros servicios excepto actividades gubernamentales. de la empresa belga AB InBev, por un monto de 2 El fuerte incremento de la IED en 2013 13 mil 249 millones de dólares («Venta de Modelo resultó de la compra de grupo Modelo por parte impulsa 147% la IED», 2013). Universita Ciencia año 10, número 29 sep-dic 2022 95 Gráfica 2. Entradas de Inversión Extranjera Directa (IED) hacia México en millones de dólares por sector de actividad económica, 2000-2021 (dólares constantes 2013) Fuente: Elaboración propia con base en datos de la Dirección General de Inversión Extranjera [DGIE] (2022a) 1. LA INVERSIÓN EXTRANJERA la Ciudad de México (22.1%), en segundo DIRECTA EN EL ESTADO DE lugar se ubicó a Nuevo León (9.5%), el VERACRUZ Estado de México en tercer lugar (9.1%), en cuarto lugar Chihuahua (5.7%) y Jalisco Gran parte de la IED que llegó a México (5.7%). En esta línea, cabe resaltar que el en estas dos décadas se concentró en los estado de Veracruz de Ignacio de la Llave estados del centro y norte del país, como (estado de Veracruz en adelante) se colocó respuesta principal a la cercanía de Estados en el lugar 11 entre los 32 estados del país, Unidos y las ventajas por el T-MEC, lo que con un 2.9%, el mejor lugar para cualquier coadyuva a las desigualdades regionales, estado ubicado en el sur del país. como lo explica la NGE. Lo anterior, queda de manifiesto si uno analiza el comportamiento La Gráfica 3 presenta la forma en que se de la inversión extranjera por estado, pues la comportó el flujo de IED en el estado de IED se concentró en los siguientes lugares: Veracruz de 2000 a 2021. La IED acumulada Universita Ciencia año 10, número 29 sep-dic 2022 96 en el período alcanzó los 20 mil 811 mdd. La nacional e internacional, en particular en inversión pasó de 602 mdd en 2000 a 635 la última fase, puesto que a partir de 2013 mdd en 2021, lo que significó una TCMA a 2021 se presenta una importante caída del 0.3%. No obstante, se pueden apreciar de la inversión, que se profundizó por los tres fases marcadas en la evolución de la efectos del Covid-19. inversión: la primera, del año 2000 al 2006 con una TCMA de -8.9%; la segunda, que En la Gráfica 4 siguiente se aprecia cuánto comprende de 2006 a 2013 con una TCMA pesó la IED en relación con el Producto de 16.0%; y la tercera, de 2013 a 2021, Interno Bruto Estatal (PIBE) entre 2003 a con un ritmo de crecimiento del -11.6%; 2018. La IED para la economía veracruzana en dos de las tres fases se presentaron representó 1.9% promedio, mientras que tendencias decrecientes marcadas. El para el país fue de 2.7%, es decir, para el año 2013 registró la mayor entrada de caso veracruzano la inversión extranjera inversión del período con mil 703 mdd, en representó mucho menos que para el país en cambio, el mínimo de inversión se dio en su conjunto. Aunque, al observar el gráfico 2006 con 345 mdd. La tendencia estatal este valor ha venido en ascenso desde el año tiene parecido con el comportamiento 2010, donde logró su valor más alto con un 3.0% en 2015. Gráfica 3. Entradas de Inversión Extranjera Directa (IED) hacia el estado de Veracruz en millones de dólares, 2000-2021 (dólares constantes 2013) Fuente: Elaboración propia con base en datos de la Dirección General de Inversión Extranjera (DGIE, 2022 b) Universita Ciencia año 10, número 29 sep-dic 2022 97 De la IED total que presentó el estado, el financiamiento de las casas matrices 47.6% se registró como nuevas inversiones, ubicadas en el extranjero. un 31.5% como reinversión en utilidades y un 21% en cuentas entre compañías. Al Al igual que a nivel país, para Veracruz el igual que a nivel país se exhibieron dos fases sector que más IED recibió en el periodo en la composición de la IED: la primera del fue el relacionado con el relacionado con año 2000 a 2010, con la mayor parte de la industria manufacturera (44.5%), seguido la IED en el rubro de nuevas inversiones del sector de servicios financieros y de (61.7%); mientras que la segunda, 2010 a seguros (14.4%), en tercer lugar el comercio 2021, el panorama fue menos claro, pues (8.8%) y del sector de la minería (7.1%). la reinversión de utilidades concentró el Estos sectores recibieron en el período el 38.2%, seguida de nuevas inversiones con 75% de la IED total. La inversión que llegó al el 35.8% y el 26.0% como cuentas entre sector de las manufacturas explica en buena compañía. El comportamiento tuvo una parte la evolución de la IED estatal, su fuerte tendencia similar al que se presentó en el impulso en buena parte de la década pasada país, donde se da una caída en la atracción repercutió en los niveles altos de inversión, de nuevos capitales que se compensó así también su severa caída después de 2017 con las empresas instaladas en el estado marcó la tendencia a la baja del indicador en reinvirtiendo utilidades y recibiendo los últimos años; sin embargo, el desplome Gráfica 4. Inversión Extranjera Directa (IED) como porcentaje del Producto Interno Bruto (PIB) anual del estado de Veracruz, 2003-2020 Nota: Para los años 2019 y 2020 las cifras son preliminares. Fuente: Elaboración propia con base en datos de la Dirección General de Inversión Extranjera (DGIE, 2022 b) y del Instituto Nacional de Estadística y Geografía (INEGI, 2022) Universita Ciencia año 10, número 29 sep-dic 2022 98 se vio amortiguado por el crecimiento del con fuerza a partir de la década anterior y sector minero. Dentro de las manufacturas, se concentró en la industria petroquímica. se destacaron las ramas de la industria de Respecto al número de empresas que la bebida y de la fabricación de productos manifestaron tener flujos de IED hacia el químicos como receptoras de capitales estado de Veracruz por lo menos una vez al año. El primero, que va de 2000 a 20014, se internacionales. Gráfica 5. Entrada de Inversión Extranjera Directa (IED) en el estado de Veracruz por sector de actividad económica en millones de dólares, 2000-2021 (dólares constantes 2013) Fuente: Elaboración propia con base en datos de la Dirección General de Inversión Extranjera (DGIE, 2022 b) Son tres países los que proveyeron el 69% de la IED de 2000 a 2021: Estados Unidos de América (EUA) con el 39.2%, España (16.7%) y Brasil (13%). Tanto EUA y España reflejaron la misma importancia que a nivel nacional, la inversión de Brasil se presentó Universita Ciencia año 10, número 29 pasó de 350 empresas a 230, una caída de 120 empresas. El segundo, de 2015 a 2021, va de 214 a 192 compañías, aunque aquí empujado por los efectos de la pandemia Covid-19. No obstante, en los dos períodos se aprecia una marcada disminución, síntoma de que las empresas extranjeras sep-dic 2022 99 están menos interesadas en invertir en el de las empresas registradas. Le siguieron estado, situación similar a lo que pasa en el los municipios de Coatzacoalcos, Xalapa, país en su conjunto. Poza Rica, Orizaba y Tuxpan. El resto de los municipios que se presentan en el 1. DISTRIBUCIÓN TERRITORIAL DE LA gráfico son municipios cercanos a ciudades INVERSIÓN EXTRANJERA DIRECTA relevantes o con características muy EN VERACRUZ particulares. Esta sección busca establecer en que municipios3 se asentó la IED en el estado de 2000 a 2021. El estado de Veracruz se integra por 212 municipios, pero solo en 48 (22.6%) de ellos se registró la presencia de 421 EMNS. Los municipios que encabezan la lista (Veracruz, Boca del Río, Coatzacoalcos, Xalapa, Poza Rica, Córdoba y Orizaba) son los municipios centrales de las zonas metropolitanas (ZMS) que reciben el mismo nombre, lugares que ofertan bienes y servicios que no se producen en La Gráfica 6 muestra el 87% que de las los municipios vecinos, con infraestructura empresas multinacionales que invirtieron de comunicación y servicios públicos en los municipios veracruzanos, la mayor importantes, además de ser puntos con parte se ubicaron en los municipios que una mayor especialización y diversificación engloban las ciudades más importantes económica, lo que les concede la presencia del estado. Se destaca la marcada significativa de economías de aglomeración concentración de empresas multinacionales y, sobre todo, de urbanización. Lo anterior en los municipios contiguos de Veracruz y se vuelve clave para la atracción de EMNS, Boca del Río, ya que juntos sumaron 238 en particular en la manufactura, el comercio empresas que equivalen al 56.5% del total y los servicios. 3 En este trabajo se considera a las empresas multinacionales que invierten en Veracruz como: las empresas mexicanas con inversión extranjera en su capital social, donde se considera su domicilio fiscal, por lo cual solo se mencionan solo una ocasión, de acuerdo con el Registro Nacional de Inversión Extranjera (RNIE). Además de las empresas extranjeras que reportaron actividades en los municipios de Veracruz, se tomaron datos de los informes de Gobierno de Veracruz desde el año 2000 a 2016, los años posteriores no se encontró información alguna, así también, información de las empresas listadas en el Panorama minero del estado de Veracruz 2020 (SGM, 2020). Para ser compatibles los datos del Gobierno de Veracruz y del Panorama Minero solo se toma el primer año que la empresa realizó una inversión en un determinado municipio. Universita Ciencia año 10, número 29 Veracruz alberga el principal puerto del país en el Golfo de México4 con comunicación con EUA y Europa y con carreteras y vías férreas en el interior del país, lo que le da una ventaja competitiva frente al resto de los municipios, por tanto, explica la llegada del mayor número de empresas, además de contar con un aeropuerto y el parque industrial Bruno Pagliai, que ha beneficiado el arribo de empresas de tipo industrial y de servicios vinculados a ellas. Boca del Río, es un municipio concentrado en sectores económicos relacionados con el comercio y los servicios, elementos que son complementarios a las actividades del sep-dic 2022 100 Gráfica 6. Empresas multinacionales que invierten a nivel municipal en el estado de Veracruz, 2000-2021 (porcentaje) Fuente: Elaboración propia con base en datos del Registro Nacional de Inversiones Extranjeras (RNIE, 2022), el Servicio Geológico Mexicano (SGM, 2020) e Informes de Gobierno del estado de Veracruz de Ignacio de la Llave de 2000 a 2016. municipio de Veracruz; así también funge Coatzacoalcos y Tuxpan, además de como un importante centro turístico. Veracruz, son puertos importantes en Veracruz atrajo IED en las manufacturas, el el Golfo de México, ofrecen ventajas de comercio y los servicios; mientras que Boca ubicación. Coatzacoalcos se especializa en del Río lo hizo en el comercio y los servicios. el sector de las manufacturas, el comercio y los servicios, se destaca la actividad petrolera que se desarrolla desde décadas 4 Por su ubicación, tiene una gran vinculación con 5 el centro del país, infraestructura y tecnología. Es atrás, en concreto la industria química un puerto de altura que es la mejor opción logística (Barcelata-Chávez, 2012a; Programa de las para la importación y exportación de bienes hacia Estados Unidos, Canadá, Europa y Sudamérica. Tiene relación con 150 puertos extranjeros mediante 27 navieras y 54 rutas marítimas. Moviliza el 70% de las importaciones y el 84% de las exportaciones hacia Estados Unidos. Se ubica como líder nacional en la maniobra de autos y de graneles agrícolas, y como uno de los puertos punteros en el movimiento de contenedores en el país (La situación actual, 2019, pp. 20-23) Universita Ciencia año 10, número 29 El principal productor de petroquímicos básicos localizado en Coatzacoalcos es la empresa Braskem Idesa, que emplea a más de 100 trabajadores y que inició operaciones en 2017 con una producción de más de 923 550 toneladas de polietileno, de las cuales el 56 % se destinaron al mercado nacional y el resto para mercados extranjeros (ONU-Habitat, 2021, p. 356). sep-dic 2022 101 Naciones Unidas para los Asentamientos le concede un carácter particular; mientras Humanos [ONU-Habitat], 2021). Ofrece un que Orizaba y Córdoba son municipios con servicio de ferrobuque, ocupa la segunda importantes vías de comunicación hacia posición en el manejo de productos el centro del país; los tres municipios se petroquímicos y el tercer lugar en granel encuentran especializados en sectores agrícola («La situación actual. Perspectiva relacionados con el comercio y lo servicios portuaria de México a nivel internacional», (Vela Martínez y Barcelata Chávez, 2014). 2019). Como resultado de lo anterior, atrajo Poza Rica muestra una concentración de a multinacionales en las manufacturas, los actividades en el comercio y los servicios, no obstante, destaca su especialización en la servicios y la construcción. minería: procesamiento de gas, exploración Tuxpan destaca por su vocación marítima, se y extracción de petróleo (Barcelata-Chávez, especializa en las actividades del comercio, 2012 b). En el lapso del estudio, Córdoba los servicios y registra la presencia de y Orizaba atrajeron multinacionales en empresas pesqueras y de la construcción. el comercio y las manufacturas; a su vez, Es un centro nodal para el abastecimiento Xalapa y Poza Rica revieron empresas de bienes y servicios para los habitantes relacionadas en el comercio y los servicios. de los municipios vecinos (Visión global Una característica importante de los para la acción local [Glocal], 2022). Es un siguientes municipios es que pertenecen puerto multipropósito que se enfoca en a importantes ZMS del estado, por el manejo de combustibles petrolíferos, lo cual están vinculados económica y manejo a granel de productos agrícolas territorialmente con los municipios y minerales, además de carga general; centrales que dan nombre a la zona así también, brinda apoyo logístico a la metropolitana (ZM): Manlio Fabio industria petrolera costa afuera. Es el Altamirano con la ZM de Veracruz, puerto más cercano al centro del país («La Amatlán de los Reyes con la ZM de situación actual. Perspectiva portuaria Córdoba, Ixtaczoquitlán con la ZM de de México a nivel internacional», 2019). Orizaba y Tihuatlán con la ZM de Poza Gracias a estas características, Tuxpan Rica. Caso particular es Pánuco, municipio recibió capital extranjero en los sectores que pertenece a la ZM de Tampico, zona de la construcción, las manufacturas y el que comparten los estados de Veracruz y Tamaulipas. transporte. Córdoba, Orizaba, Poza Rica y Xalapa son los Dentro del resto de municipios se municipios centrales de las ZMS del mismo destacan los de Actopan, Las Minas, Alto nombre, por lo tanto brindan actividades Lucero y Tatatila que se encuentran con especializadas y de mayor complejidad a los una importante presencia de mineras municipios cercanos. El municipio de Xalapa canadienses 6 que pretenden explotar es la capital del estado, lugar de los poderes legales de la entidad y de importantes 6 Source Exploraction Corp, proyecto La Miqueta instituciones de educación superior, lo que en Las Minas; Mexican Gold Corp, proyecto Las Minas en el municipio del mismo nombre; Universita Ciencia año 10, número 29 sep-dic 2022 102 recursos del subsuelo como oro, cobre, y de cuentas entre compañías; el sector plata y hierro. En el lapso de estudio se económico más relevante es la inversión registraron siete empresas, aunque todas que se hizo en la industria manufacturera en fase de exploración (SGM, 2020). Aquí pero que en los últimos años ha tenido una la IED se localizó como consecuencia de importante contracción que explica la caída los recursos naturales que se encuentran del indicador en el período de análisis. en un lugar específico, lo que ofrece a las empresas ventajas de ubicación y atrae a De forma importante, el trabajo mostró empresas multinacionales buscadoras de la manera en cómo las empresas recursos. multinacionales se ubicaron en el territorio veracruzano a escala municipal, lo que CONCLUSIÓN evidenció un proceso de concentración en ciertos lugares del espacio. Los municipios México se ha convertido en un destino que mayor IED recibieron fueron aquéllos importante para la IED global por su que fungen como los nodos centrales cercanía con los Estados Unidos, al ofrecer de las zonas metropolitanas del estado mano de obra calificada, facilidades en (Veracruz, Boca del Río, Coatzacoalcos, materia comercial con el T-MEC (antes Xalapa, Poza Rica, Córdoba, Orizaba) TLCAN) y el potencial del mercado y Tuxpan, puerto clave para exportar mexicano en términos de ingreso y a EUA y Europa. Estos territorios se población. No obstante, como pasó en especializan en actividades relacionadas el escenario mundial, los últimos años con la manufactura, el comercio y los ofrecen una tendencia decreciente de los servicios, por lo tanto, las multinacionales flujos de entrada al país, resultado de un invirtieron en estos rubros. Esto obedece escenario complejo internacional y de las a que en estos lugares se encuentran políticas gubernamentales seguidas por economías de aglomeración y, en particular, esta administración, en particular en el de urbanización que facilitan su arribo y sector energético. continuidad en el tiempo. Especial atención se muestra en los municipios de Veracruz, Por su parte Veracruz, manifestó un Coatzacoalcos y Tuxpan, con fuerte crecimiento importante en los influjos de presencia de actividades de transporte IED hasta 2013, a partir de ese momento la marítimo, los cuales ofertan ventajas de tendencia es a la baja como en el escenario ubicación para las empresas buscadoras nacional y con un cambio en su estructura: de mercados. Los municipios de Córdoba con una mayor presencia de inversión en y Orizaba atrajeron multinacionales en el su modalidad de reinversión de utilidades comercio y las manufacturas; mientras que Xalapa y Poza Rica registraron empresas en el comercio y los servicios. Chesapeake Gold Corp, proyecto Tatatila en Tatatila; Goldgruop Mining Corp, proyecto de Caballo Blanco en Alto Lucero; Azucarminerals, proyecto El Cobre en Actopan (Servicio Geológico Mexicano [SGM], 2020, p.19). Universita Ciencia año 10, número 29 Por otra parte, se encontró un segundo grupo de municipios receptores de IED sep-dic 2022 103 que son municipios periféricos de las REFERENCIAS ZMS, pero que guardan estrecha relación con los municipios centrales: Manlio Barcelata Chávez, H. (2012a). Fabio Altamirano, Amatlán de los Reyes, Coatzacoalcos. Economía local Ixtaczoquitlán Tihuatlán y Pánuco. Por y problemática social: Vol. IV. último, un tercer grupo son los municipios Universidad de Málaga. https:// relacionados con el uso de los recursos www.uv.mx/ofd/files/2014/05/ naturales, en específico, los municipios COATZACOALCOSEconomiayprobl ematicasocial.pdf que revieron IED en el sector minero: Actopan, Las Minas, Alto Lucero y Tatatila, con ventajas de ubicación por empresas Barcelata Chávez, H. (2012b). Poza Rica. buscadoras de recursos. Economía local y problemática social: Vol. III. Universidad de Dado los resultados obtenidos, para los Málaga. https://uv.mx/personal/ futuros estudios será conveniente analizar hbarcelata/files/2014/05/ sus implicaciones a nivel municipal y en PozaRicaEconomialocalyproblemati casocial.pdf particular de zona metropolitana, por especialización económica, los factores que favorecen el arribo de las EMNS, las Bracamonte Jaraba, M. A., Hernández relaciones con el comercio internacional González, D. F., & Astudillo Miller, del estado, además de ampliar las fuentes M. X. (2018). Economías de de información sobre la IED que llega nivel aglomeración en servicios hoteleros: municipal. los casos de Acapulco, Guerrero y Boca del Río Veracruz en México. Interconectando Saberes, 5, 95115. http://dx.doi.org/10.25009/ is.v0i5.2563 Cuadrado-Roura, J. R. (2014). ¿Es tan “nueva” la “Nueva Geografía Económica”? Sus aportaciones, sus límites y su relación con las políticas. EURE, 40(120), 5-28. http://dx.doi.org/10.4067/S025071612014000200001 García, P. M., & López, A. (2020). La inversión extranjera directa: Definiciones, determinantes, impactos y políticas públicas (IDB-TN-1995; p. 44). Banco Interamericano de Desarrollo Universita Ciencia año 10, número 29 sep-dic 2022 104 [BID]. https://publications.iadb.org/ publications/spanish/document/ La-inversion-extranjera-directaDefiniciones-determinantesimpactos-y-politicas-publicas.pdf Hill, C. W. L. (2011). Negocios internacionales. Competencia en el mercado global (8a ed). McGraw Hill/Interamericana Editores, S.A. de C.V. h t t p s : // w w w. i c ex . e s / i c ex /e s / Navegacion-zona-contacto/ revista-el-exportador/mundo/ REP2019819184.html Moncayo Jiménez, E. (2002). Nuevos enfoques teóricos, evolución de las políticas regionales e impacto territorial de la globalización (No 27; Gestión pública, pp. 1-78). Instituto Latinoamericano y del Caribe de Planificación Económica y Social - ILPES, CEPAL. https:// repositorio.cepal.org/bitstream/ handle/11362/7277/S0212982_ es.pdf?sequence=1&isAllowed=y Instituto Nacional de Estadística y Geografía (INEGI). (2022). PIB de las actividades económicas por entidad federativa [Registros de bases de datos]. Producto Interno Bruto por Entidad Federativa. Padilla-Pérez, R., & Gomes-Nogueira, Año base 2013. https://www.inegi. C. (2015). Determinantes de la org.mx/app/tabulados/default. salida de IED y efectos en el país aspx?pr=17vr=6&in=2&tp=20&wr=1 emisor. Evidencia de América Latina (N.o 166; Estudios y Lascurain Fernández, M. (2018). Direct perspectivas, p. 75). Comisión Foreign Investment in The State Económica para América Latina y of Veracruz, Mexico: Analysis el Caribe [CEPAL]. https://www. and Perspective. Dimensión cepal.org/es/publicaciones/39224Empresarial, 16(2), 177-190. https:// determinantes-la-salida-ieddoi.org/10.15665/dem.v16i2.1381 efectos-pais-emisor-evidenciaamerica-latina La situación actual. Perspectiva portuaria de México a nivel internacional. Polése, M. (1998). Economía regional y (2019). Prospectus. Tendencias y urbana: Introducción a la relación escenarios para la educación superior, entre territorio y desarrollo. Libro 2, 15-32. https://www.uv.mx/ Universitario Regional. secretaria-desarrolloinstitucional/ files/2019/07/PROSPECTIVA- Programa de las Naciones Unidas para VOL2.pdf los Asentamientos Humanos (ONUHabitat). (2021). Atlas prospectivo México: Primero la inversión. (2019). territorial-industrial para la El Exportador. Revista para atracción de inversiones. Sectores la internacionalización, 14. con proyección en México (p. 525). Universita Ciencia año 10, número 29 sep-dic 2022 105 Registro Nacional de Inversiones Extranjeras (RNIE). (2022). Listado del registro de Sociedades mexicanas con inversión extranjera en su capital social [Registro de bases de datos]. Inversión Extranjera Directa. https://www.gob.mx/se/accionesy-programas/competitividad-ynormatividad-inversion-extranjeradirecta?state=published mx/wp-content/uploads/2022/ transparencia/ley_875/Art16/F_II/ I n c _ a _ P L A N _ M U N I C I PA L _ D E _ DESARROLLO_TUXPAN_PUERTO_ DE_LA_ESPERANZA_2022_2025. pdf Salles, Sainz Grant Thornton. (2021). Boletín de economía (N.o 3). Salles Sainz – Grant Thornton S.C. https://www.grantthornton.mx/ globalassets/1.-member-firms/ mexico/pdf/boletin-de-economiamarzo2021.pdf Servicio Geológico Mexicano (SGM). (2020). Panorama minero del estado de Veracruz (Panorama minero de los estados, p. 42). Subsecretaría de minería. http://www.sgm.gob.mx/ pdfs/VERACRUZ.pdf Vela Martínez, R., & Barcelata Chávez, H. (2014). Zonas metropolitanas del estado de Veracruz: OrizabaCórdoba-Xalapa. Universidad Veracruzana. Venta de Modelo impulsa 147% la IED. (2013). Expansión. https://expansion. mx/economia/2013/08/21/ventade-modelo-impulsa-ied-a-mexico Visión global para la acción local (Glocal). (2022). Plan municipal de desarrollo de Tuxpan 20222025. https://tuxpanveracruz.gob. Universita Ciencia año 10, número 29 sep-dic 2022 106
https://openalex.org/W3098986756	https://www.researchsquare.com/article/rs-50747/v1.pdf?c=1597738407000	English	null	Present and future suitability of the Lake Tana Biosphere Reserve in Ethiopia for the Nile monitor (Varanus niloticus) using the MaxEnt model	Environmental systems research	2,020	cc-by	7,333	Present and Future Suitability of the Lake Tana Biosphere Reserve (LTBR) in Ethiopia for the Nile monitor (Varanus niloticus) using the MaxEnt Mode Dessalegn Ejigu (  dessalegn_ejigu@yahoo.com ) Bahir Dar University https://orcid.org/0000-0002-5672-4484 Conclusions The potential distribution map for V. niloticus can help in planning land use management around its existing habitat range, discover new populations or set priorities to restore its natural habitat for more effective conservation. Extensive reductions in the amount of suitable areas under future climate scenarios suggest that the species may become threatened in future if effective conservation measures are not implemented. Results on average 2750 individuals were recorded within the LTBR. Mean annual temperature, precipitation and temperature were the most important predictors that limit the potential distribution of V. niloticus. Most of its suitable habitats were mainly predicted in the northern and southern parts of the Lake. The ecological niche model produced an average AUC of 0.85. Notable records of the species were found in the vicinity of the lake and wetlands nearby. Future projection of potential suitable areas revealed that the currently available suitable area will decline in both 2050 and 2070 under both RCP 6.5 and RCP 8.5, of which the decline in suitable area under the business as usual scenario is the greatest. DOI: https://doi.org/10.21203/rs.3.rs-50747/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 at Environmental Systems Research on November 9th, 2020. See the published version at https://doi.org/10.1186/s40068-020-00197-y. Page 1/26 Page 1/26 Page 1/26 Background The Nile monitor (Varanus niloticus) is a reptile native to Sub-Saharan Africa along the Nile River. The species inhabits a wide variety of habitats including woodlands, grasslands, mangroves, and swamps. Although the practice is not common in the Lake Tana Biosphere Reserve (LTBR) , the species is being hunted in Sahelian Africa for its leather, food, pet trade and fat-content. Consequently, the species is listed under the Convention on International Trade in Endangered Species. Methods Data collection was based on original onsite GIS aided presence recording. Each record of the species was first vetted for data quality. A multicollinearity analysis was conducted before fitting the MaxEnt model to the 19 bioclimatic variables. Since it provides good coverage for Africa, the Hadley Global Environment Model 2-Atmosphere Ocean (HadGEM2-AO) model was used for extracting future climate scenarios. The jackknife test was selected to measure the contribution of each environmental variable to the MaxEnt model for the species. Area under the curve of the receiver operating characteristic was used to evaluate the performance of MaxEnt model. Background The Nile monitor (Varanus niloticus: Linnaeus 1758) is a reptile native to Sub-Saharan Africa along the Nile River. Its range encompasses most of sub-Saharan Africa extending northward along the Nile River all the way to Egypt occuring along the periphery of deserts and from grasslands to rainforests in the Page 2/26 vicinity of rivers, swamps, ponds, lakes, and seashores (Enge et al. 2004). However, currently the species occurs in North America as a result of the pet trade, and if introduced, it will likely spread into many regions in the Americas, the Caribbean, Madagascar, Southeast Asia, and Australia (Bevan 2016). In its introduced range in Florida, the most suitable habitats include mangrove swamps, edges of freshwater and saltwater marshes, river banks, canals, and lakes (Enge et al. 2004). It has the largest geographic distribution capable of reaching high population density of up to 50/km2. The ecology of V. niloticus is highly related with the ecology of Crocodylus niloticus. However, it seems very unlikely that a true interspecific competition could occur between the two species as they remarkably differ in size and the area where they forage. Crocodiles entirely forage in aquatic habitats whereas Varanids prefer to both terrestrial and aquatic habitats with permanent water bodies to forage and open rooftops and streets to bask (Dowell et al. 2015). Besides, Varanids inhabit a wide variety of habitats including woodland, dry savanna, scrub, evergreen thickets, swamps, mangroves, marshes, creeks, rivers, lakes and in disturbed areas near canals (Luiselli et al. 1999). The Nile monitor has the potential to disperse into ecologically sensitive areas where it could threaten different wildlife species (Enge et al. 2004). Nile monitor can swim, climb, run, and dig, feed on a wide array of aquatic, terrestrial and arboreal prey, and it is also known to hunt cooperatively (Campbell 2005). Stomach content analyses of the species revealed that its diet is extremely broad including many taxa of invertebrates and vertebrates (Bevan 2016; Campbell 2005). Nile monitor mainly feeds on arthropods, the eggs of birds, alligators, crocodiles, and turtles and could impact many threatened and endangered species, including, owls and sea turtles. Juveniles may be insectivorous (Bennett 2002; Bevan 2016; Enge et al. 2004). The lack of fat accumulation in these animals suggests they do not undergo extended fasting periods (Bennett 2002). Background Morphologically, Nile monitor is gray-brown or dark olive with darker reticulation on its dorsal side and with six to nine bands of yellow-golden ocelli in adults, while juveniles are with black and yellow patterns. The tongue is blue (Bennett 1998), and it has large, strong claws. The neck is longer than the narrow- snouted head, and it has a laterally compressed tail. It can grow up to 2.4 m with a body mass of up to 7 kg (Campbell 2005; Enge et al. 2004). Though Nile monitor is poikilothermic, it can tolerate the ecological niche thermal range of beyond expected limits by developing adaptation of living in underground burrows (Bennett 2002; Campbell 2005). The Nile monitor reaches sexual maturity at one to two years of age and about 50% of mature females reproduce each year (Ahmed et al. 2018; Enge et al. 2004). In Africa, female Nile monitors oviposit in burrows or active termite mounds from August to January (Bennett 2002; Enge et al. 2004). Females oviposit 50 to 60 eggs, and eggs apparently take six to ten months to hatch in the wild (Ahmed et al. 2018; Campbell 2005; Ciliberti et al. 2012; Enge et al. 2004). On average females reach sexual maturity at two years of age. Page 3/26 Page 3/26 Page 3/26 Although the practice is not common in the Lake Tana Biosphere Reserve (LTBR), the species is being hunted in Sahelian Africa for its leather, food, pet trade and for some medical treatments (Ahmed et al. 2018). Consequently, the species is listed under the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES, Appendix II) (Dowell et al. 2015). This study, therefore, has tried to estimate population size, identify potential hotspots, and model the spatiotemporal distribution of Nile monitor (Varanus niloticus) within the LTBR, Ethiopia. Description of the study area The Lake Tana Biosphere Reserve (LTBR) is located in the Amhara National Regional State approximately 565 km northwest of Addis Ababa. Lake Tana surface area is 3156 km² which is stretching approximately 84 km north-south and 66 km east-west. The Lake is located within the watershed, which consists of 137 Administrative Kebeles, 10 Districts, and four Administration Zones (Fig. 1). It is located at 11°25'07” N − 12°29'18”N latitude, and 36°54'01”E − 37°47’20”E longitude with altitude ranging from 1788 to 3712 m a.s.l (Friedrich zur Heide 2012). The total surface area of the reserve is 695,885 ha of which the core area comprises 22,841 ha (terrestrial: 7,699 ha). Its buffer area is 187,567 ha (terrestrial: 30,969 ha), and the transition zone is 485,477 ha (terrestrial: 354,297 ha). Lake Tana has been recognized as UNESCO Biosphere Reserve since 2014. Lake Tana, the source of the Blue Nile River, is a shallow lake with an average depth of 9 m and its maximum depth is 14 m with a decreasing trend due to siltation and lowering water level. The lake is surrounded by lagoons and wetlands and has more than 40 tributaries of which Gilgel Abay, Ribb, Gumara and Megech Rivers accounting for 93% of the total inflow. The climate around the lake is warm with a mean temperature of 21.7 °C. Rainfall is strongly seasonal with a dry season between November and May and a pronounced rainy season between July and September. The mean annual rainfall ranges from 800 to 2000 mm. Lake Tana is the largest national freshwater body, accounting for 50% of the total inland water of the country. The Biosphere Reserve is an important fish resource and is home to different species of fish of which 70% are endemic. The barbus species of Lake Tana constitute the only remaining intact species of large cyprinid fish in the world. A large number of wetlands are located all around Lake Tana, some of them being the largest and ecologically most important units in Ethiopia and in the Horn of Africa. These wetlands, dominated by papyrus and typha stands are breeding, nesting and feeding grounds for various bird populations, and provide a source of animal feed, domestic water supply, building material, fuel and food for local communities. Lake Tana is the largest national freshwater body, accounting for 50% o country. Description of the study area The Biosphere Reserve is an important fish resource and is hom which 70% are endemic. The barbus species of Lake Tana constitute the large cyprinid fish in the world. A large number of wetlands are located a them being the largest and ecologically most important units in Ethiopia wetlands, dominated by papyrus and typha stands are breeding, nesting bird populations, and provide a source of animal feed, domestic water s food for local communities. The LTBR consists of four terrestrial and three freshwater ‘key biodiversity areas’. The area is internationally renowned as Important Bird Area and the highest abundances of wetland birds qualify areas around the lake as Ramsar site. Many Palaearctic migrant water birds depend on the lake as Page 4/26 Page 4/26 Page 4/26 feeding and resting grounds, including the common crane (Grus grus), Northern shoveller (Anas acuta), Black-tailed Godwit (Limosa limosa), and ruff (Philomachus pugnax) (Tassie and Bekele 2008). Few patches of original forest vegetation and mountain ecosystem remain in the biosphere reserve that has high plant endemism of global importance. Indigenous trees found in the biosphere reserve include but not limited to Albizia gummifera, Millettia ferryginea, and Cordia africana. The region is a gene centre for indigenous agricultural crops such as Guizotia abyssinica, and Eragrostis tef. Wild coffee (Coffee arabica) occurs naturally in the area, especially in the Zegie Peninsula. Four major wetland ecosystem types such as riverine freshwater wetlands, lacustrine freshwater wetlands, palustrine freshwater wetlands, and agricultural flooded freshwater wetlands have been identified. The LTBR comprises Lake Tana, which is the main source of the Blue Nile that provides important ecosystem services to the region. The LTBR is a hotspot of biodiversity, and it is part of the two biodiversity hotspots i.e., Eastern Afromontane and Horn of Africa biodiversity hotspots. It is internationally known as an Important Bird Area (Aynalem and Bekele 2008; Tassie and Bekele 2008), and is of global importance for genetic diversity. The area is characterized by an enormous heterogeneity of land uses and natural ecosystems. The reserve is known for mammal species including Hippopotamus (Hippopotamus amphibious), black and white colobus monkeys (Colobus guereza), aardvark (Orycteropus afer), crested porcupine (Hystrix cristata), Grimm’s duiker (Sylvicapra grimmia), leopard (Panthera pardus), honey badger (Mellivora capensis), African civet cat (Civettictus civetta), Bailey´s shrew (Crocidura baileyi), foxes, highland hyenas, rabbits and other rodents. Description of the study area Besides Nile monitor, other reptiles recorded within the biosphere reserve are crocodile (Crocodyla niloticus), and python (Python sebae). Data Collection The survey areas for spotting Nile monitor were selected based on the nature of potential habitats of the species. For example, habitats such as swamp forests, river banks, dry land forests in the vicinity of water bodies, shrub lands, farmlands, cultivations, and urban and suburb areas were considered during the survey. In the reserve, the Lake itself and associated wetlands and rivers that drain to the lake ( Fig. 2) and the lake shore areas suspected for the presence of V. niloticus were surveyed for the presence data. The study was carried out for four consecutive months, July to October 2018. Although the survey was conducted in the aforementioned habitats, focal group discussants and key informants from different woredas and kebeles within the survey area were also consulted prior to the commencement of the actual filed data collection. The study had invested all possible effort to address all known and suspected potential habitats of the species within the biosphere reserve. Distribution data Page 5/26 Distribution data collection was mainly based on original on site GIS aided locality recording for ground truth. The ground truth includes the aforementioned habitats. However, prior to GIS recording, the general whereabouts of the species was obtained by consulting key informants and focal group discussants from different woredas and kebeles within the study area. In addition to field observation, the locality records were collected from literature sources and correspondents in the field. Each record of the species was first vetted for data quality. Records lacking high range of uncertainty or insufficient information to consider credible were eliminated. In such cases, anecdotal observations were excluded and we focused on credible and confirmed observations for the analysis to prevent distortion in statistical analysis or over-fitting of models. A total of 307 presence records for V. niloticus in LTBR were used in subsequent analyses. Although the focus area of our survey was on the entire lake and its lush shore, every potential habitat of the species got due emphasis during the survey. Field trips carried out on either sunny or rainy days. The field observation was conducted from 08:00am to 6:00 pm. Materials and equipments used during this study include binoculars; digital photographing camera; GPS; data sheets; notebooks; Push-wheel switch counter; motor boats and papyrus boats. Random routes were followed to observe animals throughout each habitat type (Luiselli et al. 1999). The distribution map is mainly plotted on the basis of the field observation data (Fig. 3). Environmental data layers Bioclimatic variables are derived from the monthly temperature and rainfall values in order to generate more biologically meaningful variables. These are often used in species distribution modeling and related ecological modeling studies. Ninteen bioclimatic variables used in this study were downloaded from WorldClim database (www.worldclim.org) (Hijmans et al. 2005). To account for the effect of anthropogenic activities on the distribution of the target species (Pulliam 2000; Soberon 2007), the study also included variables that are associated with human influences on ecosystems or landscapes. In this regard we incorporated human population density figures from World Pop database (http://www.afripop.org/) and land use classes for Ethiopia which were downloaded from http://due.esrin.esa.int/globcover/. Before fitting the MaxEnt model to the variables, a multicollinearity analysis was performed using ENM tools 1.44, to ensure that correlated variables were removed in the same model. For pairs of variables that had a high correlation (≥ 0.75) (Stiels et al. 2015) only one of the variables was retained (Guisan and Thuiller 2005) based on its biological significance to the species. After correlation analysis only 6 variables were retained namely; Annual Mean Temperature (Bio1), Temperature Seasonality (Bio4), annual precipitation (Bio12), precipitation seasonality (Bio15), Human population density and land cover. When modelling species distribution, it is important to ensure that the environmental variables have the same spatial extent and resolution. However, the downloaded environmental layers had different resolutions; consequently, this mismatch in resolution was corrected, using GIS such that all Page 6/26 Page 6/26 environmental variables had a resolution of 1 km2. Processing of all the environmental layers was done using ArcGIS 10.5.1. Climate scenarios To extract future climate scenarios the Hadley Global Environment Model 2-Atmosphere Ocean (HadGEM2-AO) from Worldclim database was used (Collins 2008). This climate projection model was chosen for this study because it provides good coverage for Africa (Davis et al. 2012; Jaramillo et al. 2011). From this model four climate scenarios were extracted – 2050 RCP6.0, 2050 RCP8.5, 2070 RCP6.0 and 2070 RCP8.5. The climate scenarios for 2050 represent averages for 2041–2060 while the scenarios for 2070 represent averages for 2061–2080 (IPCC2013). The RCPs used in this study signify two possible greenhouse emission scenarios ranging from moderate (RCP 6.0) to high (RCP 8.5); corresponding to increases in global radiative values in the year 2100 relative to preindustrial values (6.0 and 8.5 w/m2, respectively) (Wei et al. 2017). This study assumed that human population density and land cover will remain constant in the future as such they were not projected to 2050 and 2070, however all the other variables were projected to 2050 and 2070. The assumption made for population density and land cover classes has limitations as it is expected that human population density and land cover classes will change in future. To extract future climate scenarios the Hadley Global Environment Model 2-Atmosphere Ocean (HadGEM2-AO) from Worldclim database was used (Collins 2008). This climate projection model was chosen for this study because it provides good coverage for Africa (Davis et al. 2012; Jaramillo et al. 2011). From this model four climate scenarios were extracted – 2050 RCP6.0, 2050 RCP8.5, 2070 RCP6.0 and 2070 RCP8.5. The climate scenarios for 2050 represent averages for 2041–2060 while the scenarios for 2070 represent averages for 2061–2080 (IPCC2013). The RCPs used in this study signify two possible greenhouse emission scenarios ranging from moderate (RCP 6.0) to high (RCP 8.5); Modelling the distribution of Nile monitor u Applications of SDMs include predicting impacts of climate change and habitat loss, identification of corridors and reserve areas for conservation, and predicting the spread of invasive species (Elith et al. 2011). To date, no studies have applied SDMs to study the distribution of V. niloticus in the LTBR. Predictions of potential current and future distribution of V. niloticus were made using MaxEnt version 3.3.3; a software based on maximum entropy method (Phillips et al. 2006). MaxEnt was chosen for this study because it has proven to perform better among species distribution modeling algorithms using presence only datasets (Elith et al. 2006; Wei et al. 2017). MaxEnt models for all the species were calibrated with similar settings. We used a regularization value of 1 which has been shown to perform well across a variety of organisms and regions (Phillips and Dudik 2008). The jackknife test was selected to measure the contribution of each environmental variable to the MaxEnt model for the species. The default convergence threshold value was adopted, while the maximum number of iterations was set to 5000 and maximum number of background points was set to 10000. We selected sub-sample run type and we performed 10 replicate runs and averaged the results. During the runs, 75% of the species occurrence records were used for training the model and the remaining 25% for validation. Population size estimate of Nile monitor Field survey and interview with the local communities about the population size estimate of Nile monitor within the Hotspots of the LTBR showed that the northern part of Lake Tana harbours more Nile monitors, while relatively the least number of individuals were recoreded in the southern part of the Lake. The average population was estimated at 2750 individuals (Fig. 4). Boolean maps for suitable and unsuitable areas for the species Page 7/26 Page 7/26 Classification of suitable and unsuitable areas for the species was done by converting the probability distribution values which range from 0 to 1 (Pearson et al. 2007). A 10th percentile training presence logistic threshold value obtained from MaxEnt model results was used to classify suitable and unsuitabl areas for the species (Hao et al. 2012; Liu et al. 2005). Pixels with values above the 10th percentile training presence logistic threshold were classified as suitable areas while pixels with values below the threshold value were classified as unsuitable areas (Hao et al. 2012). All the suitability maps were produced and calculation of the amount of suitable areas under current and future climate scenarios were done using ArcGIS 10.5.1. MaxEnt model performance evaluation Area under the curve (AUC) of the receiver operating characteristic (ROC) was used to evaluate the performance of MaxEnt model. High AUC values imply that the model performance is good; in general AUC values within the range 0.5–0.7 signify poor model performance, while values ranging between 0.7 and 0.9 indicate good performance, and values greater than 0.9 indicate excellent performance (Wei et al. 2017). Distribution of Nile monitor Most suitable habitat for V. niloticus was mainly predicted in the northern and southern parts of the Lake and the eastern and western parts of the Lake were also predicted to be suitable for the species (Fig. 5). Besides, fragmented distributions are predicted following the presence of tributaries of the Lake Tana. The Maxent model’s internal jackknife test of variable importance showed that mean annual temperature (bio1), Precipitation seasonality (coefficient of variation) (bio15), and temperature seasonality (standard deviation) (bio4) were the three most important predictors that limit the potential distribution of V. niloticus (Table 1). These variables presented the highest gain that is contained most information compared to other variables (Fig. 6). Page 8/26 Table 1 Mean AUC value and percent contribution of the most important variables influencing the distribution of the species Species Mean AUC Variable Contribution to MaxEnt model (%) Remark Varanus niloticus 0.85 Annual Mean Temperature 45.9 Precipitation Seasonality 28.4 Temperature Seasonality 15.8 Population density 6.3 Land use 3.4 Annual precipitation 0.1 Table 1 The ecological niche model constructed from this species occurrence records (n = 307) produced an average test AUC of 0.85 (Table 1). Projecting the species onto the study area showed that the vast majority of the currently suitable areas is predicted to be highly unsuitable. Aggregated observation records illustrate water body centered occurrence of V. niloticus in the study area. Notable records of the species were found in the vicinity of the lake and wetlands nearby. Likewise, the northern and eastern parts of the Lake are observed to host large number of individuals of the species. There were also clustered sightings closer to ponds. Model performance and variable importance for the specie’s distribution The prediction accuracy of the MaxEnt models for the species was good, as the mean AUC values is greater than 0.8. The highest AUC value observed here indicates that the model performed well in predicting potential suitable habitats for the species. Jackknife analysis of the environmental predictor variables suggests Annual Mean Temperature to be the greatest predictor of V. niloticus presence. Precipitation seasonality, averaged across replicate runs, is responsible for a 28.4% contribution to the output of the model, identifying it as the most significant predictor. Temperature seasonality was calculated to contribute 15.8% to the model, followed in relative importance by the population desnsity 6.3%, land use 3.4%, and Annual Precipitation 0.1%. The last three variables were found to be of less important in the predictions of the Maxent model. The sensitivity of the species to the variables that greatly influenced the distribution varied greatly. For example, the probability of occurrence for the species increased with increase in annual mean temperature from 12 to 21 °C, with an optimum annual temperature of 21 °C that favored the species to Page 9/26 occur in the study area, while the probability of occurrence rapidly declined as the temperature goes above 21 °C (Fig. 7). Precipitation seasonality, the Coefficient of Variation, is the standard deviation of the monthly precipitation estimates expressed as a percentage of the mean of those estimates (i.e. the annual mean). The larger the standard deviation, the greater the variability. It is the tendency of a place to have more precipitation in certain months or seasons than in others. Large values show high seasonality and small values low. For this species, precipitation seasonality was found to be the second important factor to determine its distribution. This index provides percentage of precipitation variability where larger values indicate greater variability of precipitation. In other words, while increase in precipitation seasonality up to 118% led to an increase in the probability of occurrence for the species, but seasonality increase above 130% led to a decrease in the probability of occurrence (Fig. 8). This result shows the presence of high precipitation variability in the study area where precipitation amounts that favored the occurrence of the species ranged from 118–130%. Likewise, precipitation seasonality was found to be the third important factor to determine V. niloticus distribution. Model performance and variable importance for the specie’s distribution If there is a high variation then the seasonality is high, but if low, then there is less extreme difference through the year, resulting in no pronounced season, but rather an even and mild climate range. So it is not indicating when the season is. This index provides percentage of temperature variability where larger values indicate greater variability of temperature. While increase in temperature seasonality up to 11% led to an increase in the probability of occurrence for the species, a seasonality increase above 14% led to a decrease in the probability of occurrence. This result shows the presence of high temperature variability in the study area where temperature amounts that favored the occurrence of the species ranged from 11–14% (Fig. 9). Since intensity of land-use change in Ethiopia is expected to increase in the future, the assumption of constant land-use classes over time gives conservative estimates of changes in the species distributions. Nevertheless, the species favored more land use category of water bodies coded as 210 followed by rain fed, post flooding, irrigated or aquatic croplands coded as 14 and mosaic vegetation coded as 30 (Fig. 10). This finding is counter intuitive because it is commonly expected that change in land use type should lead to a change in the probability of occurrence of the species. Current and future distribution of V. niloticus MaxEnt model predictions for the current distribution of suitable areas for the species indicated that the species had a wide range of suitable areas across the study area. Despite the species having a wide range of current suitable areas, future projections revealed that the amount of suitable areas will reduce greatly. Generally, the more extreme global climate change scenario (RCP 8.5) predicted a larger loss of suitable area than the other (RCP 6.0). The area predicted to be suitable for V. niloticus as a whole became more restricted under future climate projections, with the more extreme scenarios (RCP 8.5) showing extremely smaller predicted ranges (Fig. 11). Largely, our findings revealed that areas very close to water bodies such as the Lake and rivers were predicted to be suitable for the species or the species is Page 10/26 Page 10/26 least affected by range losses as a result of climate change, thus it can be said that the ranges for the species are shifting towards areas where water is available. For all RCP scenarios, areas of probable presence suggest reduction of suitable areas. The findings are not surprising as it was observed that the species was very sensitive to increases in temperature, and since the species might use water bodies to regulate the body temperature such findings are logical. Nile monitors are usually found near water, including rivers and lakes. They are both terrestrial and aquatic which allows them for great adaptability to different environments (Szczepaniuk, 2011). Generally, the range loss for the species is huge under future climate change scenarios; it ranged from 54.93 to 98.70% (Table 2). The highest percentage of range loss was observed in 2070 under the business as usual climate change scenario (2070RCP8.5), while the least range loss was observed in 2050RCP6.0. These results indicate that potentially suitable areas for this species are declining as time goes by in years and climate changes under scenarios used. Table 2 Current and projected suitable area left and percent loss of the specie’s suitable area under different climate scenarios Current and future climate scenarios Species Area category Current 2050 RCP 6.0 2050 RCP 8.5 2070 RCP 6.0 2070 RCP 8.5 V. niloticus Suitable area(Km2) 3011 1357 474 277 38 Range loss (%) 0 54.93 84.26 90.80 98.70 Discussion The distribution of records suggests water bodies are corridors for V. niloticus. Indeed, the riverine areas are home to a notable proportion of records. GPS coordinates place an additional majority of records in wetlands that physically flank habitats of the riverine areas and the Lake. V. niloticus prefers to live near permanent bodies of water in its native range (Pianka et al. 2004) confirming that the species’ home range contains at least a permanent source of water. It inhabits mainly vegetated spots and gallery forests in the vicinities of rivers and water bodies (Luiselli et al. 1999). As has been noted during our observations, rivers serve the species as convenient means of retreat, feeding and breeding ground. Furthermore, river banks and sidewalks lining rivers offer attractive basking sites. MaxEnt predictions for the current distribution of V. niloticus revealed that overall this species occurs in the immediate vicinity of water bodies. Thus, suggesting that the MaxEnt models correctly predicted the current distribution of the species as the species tends to choose aquatic habitats compared with Page 11/26 Page 11/26 Page 11/26 terrestrial (Noah 2017). Predictably, the entire lush shore of the Lake Tana is identified by Maxent as the area with the highest probability of V. niloticus presence in the study area. More interesting is the identification of rivers that flow to the lake as sites of highly probable presence. Credible sighting of V. niloticus on areas outside of rivers have been rare, which might further confirm the preference of the species to aquatic habitats. However, since the species is not entirely aquatic, it is possible that the lack of records from the terrestrial areas is the result of survey efforts that mainly focused on the shore of the lake and rivers that drain to the Lake. The mapping of potential distribution of species offers a number of advantages for conservation (Elith et al. 2011). The present study showed that SDMs can be used to predict current and potential species distribution in current and future climatic condition in an area of high biodiversity using species occurrence and environmental variables. The AUC value was above 0.80 for the species Maxent models provided new information about the future distribution of the species with a fairly good accuracy.. Future projections of the distribution of the species portrayed huge losses in the amount of suitable areas for the species. Discussion The species had a maximum range loss of greater than 50% in 2050 and greater than 90% in 2070 under the two scenarios used (Table 2). Since the species was less sensitive to population density, land use change and annual precipitation than temperatures, it can be supposed that the declines in suitable areas are due to increases in annual mean temperatures and seasonality under future climate scenarios (Parmesan 2006). Nevertheless, the potential effects of these factors cannot be ignored as population density leads to loss of habitats or change in land use and rainfall affects the availability of water resources and consequently food productivity for this species. Average temperatures in Ethiopia for 2070 under RCP 8.5 will increase by 4.10C ( Tassie 2016), as such huge declines in the amount of suitable areas for the species as observed in this study can be expected. Increased temperatures have the potential of not only affecting energy expenditure but also egg production (Pendlebury et al. 2004), consequently threatening the survival of a given species. Therefore, our findings suggest that even though this species is presently under least concern category (DeLisle 1996) it may become threatened or endangered soon, due to habitat loss which will result from increases in temperatures. Further, the results showed that human population density, land use and annual precipitation played no important role in predicting the distribution of the species. The lack of response to the human population density might be attributed to the ability of the species to adapt areas where human population density is high. For example, the species is observed to feed on cow milk. Such feeding behavior implies that the presence of humans provides vast opportunities to this species for finding food hence occurrence near human settlement is expected. Monitor Lizards are well known to people inhabiting small villages and towns (Angelici and Luiselli 1999). Moreover, our observation and interviews with the local community had confirmed that the species is known for sucking cow milk directly from cow’s nipple in rural villages where cattle are being reared. Additionally, the species has been reported to regularly visit towns and villages in association with churches, large buildings, roads and bridges, for instance the species is Page 12/26 Page 12/26 enormous at the junction where Nile River leaves Lake Tana in the southern part of the Lake. Discussion These findings suggest that the species have adapted to human or anthropogenic landscapes either through increased encroachment of their habitats by humans or through the species frequent visits to human habitats in search for food. Therefore, it is not surprising that some of the variables selected to predict the spatiotemporal distribution of the species are not invaluable for predicating the potential suitable areas of the species. Availability of data and materials The authors declare that data is available on the hands of the corresponding author, and can be avilable on request for the corresponding author. Consent for Publication Not applicable Ethics approval Not applicable Conclusion In this study we modeled the current and potential future distribution of V.niloticus under RCP 6.0 and RCP 8.5 scenarios in 2050 and 2070. The findings of this study indicate that the most important factors that determine the distributions of these species are annual mean temperature, temperature and precipitation seasonality. Future projection of potential suitable areas revealed that the currently available suitable geographic area will decline in both 2050 and 2070 under both climate scenarios of RCP 6.5 and RCP 8.5, of which the decline in suitable area under the business as usual scenario is the greatest. The potential habitat distribution map for V. niloticus can help in planning land use management around its existing populations, discover new populations, identify top-priority survey sites, or set priorities to restore its natural habitat for more effective conservation. Extensive reductions in the amount of suitable areas under future climate scenarios suggest that the species may become threatened or endangered in future if effective and sustainable conservation measures are not implemented. Although the general characteristics of the distribution of V. niloticus is addressed quite satisfactorily from the present study, more detailed surveys throughout the study area are necessary to reliably depict the distribution of the species. Authors’ contributions Ejigu D and Tassie N conceptualized this study, collected, analyzed, interpreted the data and wrote, read and approved the final manuscript. Competing interests The Authors declare that there is no competing interest in publishing this manuscript. Page 13/26 Dessalegn Ejigu Bahir Dar University, Department of Biology, P. O. Box 79, Bahir Dar, Ethiopia Nega Tassie Nega Tassie Aknowledgenments We would like to thank community elders, Citizen Scientists, key informants, focus group discussion participants and local administrators of the study area for their kindness and cooperation during the field work. The Authors would also like to thank Nature and Biodiversity Conservation Union (NABU) for financially suporting the study. Finally we are also thankful to the WorldClim data center for offering free acces to the environmental data. Affilations Bahir Dar University, Department of Biology, P. O. Box 79, Bahir Dar, Ethiopia Bahir Dar University, Department of Biology, P. O. Box 79, Bahir Dar, Ethiopia Dessalegn Ejigu Bahir Dar University, Department of Biology, P. O. Box 79, Bahir Dar, Ethiopia Nega Tassie References Ahmed YA., Mohammed A Elsaysed M (2018) Histological insight into the hepatic tissue of the Nile monitor (Varanus niloticus). J Exp Appl Anim Sci (3): 240-250 Angelici FM Luiselli L(1999) Aspects of the ecology of varanus nilot/cus (reptilia, varanidae) in southeastern nigeria, and their contribution to the knowledge of the evolutionary history of v. Niloticus species complex’. 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Ecol Lett, 3: 349–361. https://doi.org/10.1007/bf00379666 Pulliam RH (2000) On the relationship between niche and distribution. Ecol Lett, 3: 349–361. https://doi.org/10.1007/bf00379666 Page 16/26 Soberón J (2007) Grinnellian and Eltonian niches and geographic distributions of species. Ecol Lett10(12): 1115–1123. https://doi.org/10.1111/j.1461-0248.2007.01107.x Stiels D Gaißer B Schidelko K Engler JO Rödder D (2015) Niche shift in four non-native estrildid finches and implications for species distribution models. Ibis 157(1), 75–90. https://doi.org/10.1111/ibi.12194. Szczepaniuk K 2011 "Varanus niloticus" (On-line), Animal Diversity Web. Accessed April 14, 2019 at https://animaldiversity.org/accounts/Varanus_niloticus/ Tassie N (2016) Climate change and Bird distribution with a focus on Ethiopia: Implications for conservation. Dissertation, National University of Singapore. Tassie N Bekele A (2008) Diversity and Habitat association of birds of Dembia plain wetlands. Lake Tana, Ethiopia. SINET:Ethiop.J.Sci, 31(1): 1–10. Wei J Zhang H Zhao W Zhao Q (2017) Niche shifts and the potential distribution of Phenacoccus solenopsis (Hemiptera: Pseudococcidae) under climate change. PLoS One, 12(7), 1–17. https://doi.org/10.1371/journal.pone.018091 Figures Page 17/26 Figure 1 Lake Tana Biosphere Reserve Lake Tana Biosphere Reserve Page 18/26 Page 18/26 Figure 2 Tributaries of Lake Tana. Page 19/26 Page 19/26 Figure 3 Sightings of the Nile monitor (n = 307) in the study area. Sightings of the Nile monitor (n = 307) in the study area. Page 20/26 Figure 4 Population size estimate of Nile monitor within the LTBR Population size estimate of Nile monitor within the LTBR Page 21/26 Page 21/26 Page 22/26 Figure 5 Distribution map of the Nile monitor within the LTBR Figure 5 Distribution map of the Nile monitor within the LTBR Page 22/26 Figure 6 Jackknife test of variable importance in modeling distribution of Nile monitor Figure 7 The dependence of predicted suitability of the species on annual mean temperature Figure 6 Jackknife test of variable importance in modeling distribution of Nile monitor Jackknife test of variable importance in modeling distribution of Nile monitor Figure 7 The dependence of predicted suitability of the species on annual mean temperature The dependence of predicted suitability of the species on annual mean temperature The dependence of predicted suitability of the species on annual mean temperature The dependence of predicted suitability of the species on annual mean temperature Page 23/26 Page 23/26 Figure 8 The dependence of predicted suitability of the species on precipitation seasonality Figure 9 Figure 8 The dependence of predicted suitability of the species on precipitation seasonality Figure 8 Figure 8 Figure 8 The dependence of predicted suitability of the species on precipitation seasonality The dependence of predicted suitability of the species on precipitation seasonality Page 24/26 p p y p p p y Figure 9 Figure 9 Page 24/26 Page 24/26 The dependence of predicted suitability of the species on temperature seasonality Figure 10 The dependence of predicted suitability of the species on land use types of the study area. The dependence of predicted suitability of the species on temperature seasonality Figure 10 The dependence of predicted suitability of the species on land use types of the study area. The dependence of predicted suitability of the species on land use types of the study area. Page 25/26 Page 25/26 Figure 11 Current and potential suitable areas of Nile monitor under different clim Figure 11 Current and potential suitable areas of Nile monitor under different climate scenarios. Current and potential suitable areas of Nile monitor under different climate scenarios. Current and potential suitable areas of Nile monitor under different climate scenarios. Page 26/26
W4237817011.txt	http://benthamopen.com/contents/pdf/CCGTM/CCGTM-1-1.pdf	null		EDITORIAL	Current chemical genomics	2,008	cc-by	546	Editorial Current Chemical Genomics, 2008, Volume 1 1 EDITORIAL We are pleased to launch this inaugural issue of Current Chemical Genomics. As an open access online journal, all the papers from this journal can be read free of charge by our readers without any restrictions to access of the full content. This peerreviewed journal publishes research letters/articles, reviews, and technology notes on all aspects of the research and development focusing on integrative approaches at the interface of chemistry and genomics. The journal reports the newly identified small molecule probes and their applications in chemical genomic research. It also reports on the methods and technologies used to identify and optimize chemical probes that interact with proteins of unknown function and with proteins of cellular signaling pathways. Coverage also includes reports on the new chemo- informatics methods for chemical genomics. We hope that these approaches will facilitate the identification of new drug targets and the development of novel therapeutic strategies leading to a new generation of medicines. In the last decade, a number of academic screening centers have been established aimed in the discovery of small molecule probes for academic research. The small molecule probes can serve as an alternative tool to unlock the functions of unknown genes and play an important role in genome research. In 2005, the National Institutes of Health (NIH) of the United States launched a probe screening and development initiative named the Molecular Libraries Screening Center Network (MLSCN). These NIH-funded centers carry out high-throughput screenings to identify compounds active in target- and phenotype-based assays; optimize the identified hits into chemical probes, and deposit screening data to a freely accessible public database, PubChem. NIH also established an Imaging Probe Development Center (IPDC) to discover and produce new imaging probes as well as to improve probe detection sensitivity for biomedical researches and clinical applications. Therefore, through all of these efforts, chemical genomics research has moved to a new level. More probes will become available and easier to access for all researchers in the near future. We will publish the selected probes identified from these centers and others including both small molecule and imaging probes. Open access to all scientific literature is an irresistible trend for the future of scientific publishing. A comparison between the open access and non-open access research papers revealed that the open access publications are more quickly recognized and cited than these non-open ones (Eysenbach G, Plos Biol. 2006,4(5): e157; MacCallum and Parthasarathy, Plos Biol. 2006,4(5): e176). Free online access of scientific articles enables researchers to quickly and effectively search and obtain scientific information and thus efficiently facilitate their research work. To meet the challenge of the electronic era, Bentham Scientific Publishers has decided to launch a series of open access journals including Current Chemical Genomics. With this open access format, we believe that Current Chemical Genomics will become an important resource for the information of new probes, methods and technologies on chemical genomics research as well as serve as an alternative journal for quickly publishing research related to chemical genomics. We welcome and encourage our readers to submit suggestions and feedbacks to support us on fulfilling the missions of this new journal. Wei Zheng National Institutes of Health National Human Genome Research Institute NIH Chemical Genomics Center Tel: 301-217-5720 E-mail: wzheng@mail.nih.gov
https://openalex.org/W4313327783	https://www.mdpi.com/2072-6694/15/1/186/pdf?version=1672227885	English	null	Clinical Significance of Glycolytic Metabolic Activity in Hepatocellular Carcinoma	Cancers	2,022	cc-by	11,140	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/). cancers cancers cancers Article Joann Jung 1,†, Sowon Park 1,†, Yeonwoo Jang 1, Sung-Hwan Lee 2,3, Yun Seong Jeong 1, Sun Young Yim 3,4,* and Ju-Seog Lee 1,* , , 2 Division of Hepatobiliary and Pancreatic Surgery, Department of Surgery, Yonsei University College of Medicine, Yonsei 03722, Republic of Korea 3 Division of Hepatobiliary and Pancreas, Department of Surgery, CHA Bundang Medical Center, CHA University, Seongnam 46371, Republic of Korea 4 Department of Internal Medicine, Korea University College of Medicine, Seoul 02841, Republic of Korea * Correspondence: eug203@korea.ac.kr (S.Y.Y.); jlee@mdanderson.org (J.-S.L.) † These authors contributed equally to this work. Simple Summary: Hepatocellular carcinoma (HCC) is among the most common cancers and causes about 830,000 deaths annually in the world. Metabolic reprogramming is a critical hallmark of HCC, enabling HCC cells to adapt to the high energy demands necessary for fast growth. However, the clinical relevance of metabolic alteration in HCC has not been systematically assessed. By performing cross-species comparison of genomic data from mouse and human tissues, we identiﬁed three distinct metabolic subtypes of HCC and uncovered clinical and molecular characteristics associated with three subtypes. Importantly, we showed that the high metabolic subtype is less susceptible to immunotherapy and uncovered a potential mechanism associated with resistance to immunotherapy. Abstract: High metabolic activity is a hallmark of cancers, including hepatocellular carcinoma (HCC). However, the molecular features of HCC with high metabolic activity contributing to clinical outcomes and the therapeutic implications of these characteristics are poorly understood. We aimed to deﬁne the features of HCC with high metabolic activity and uncover its association with response to current therapies. By integrating gene expression data from mouse liver tissues and tumor tissues from HCC patients (n = 1038), we uncovered three metabolically distinct HCC subtypes that differ in clinical outcomes and underlying molecular biology. The high metabolic subtype is characterized by poor survival, the strongest stem cell signature, high genomic instability, activation of EPCAM and SALL4, and low potential for beneﬁtting from immunotherapy. Interestingly, immune cell analysis showed that regulatory T cells (Tregs) are highly enriched in high metabolic HCC tumors, suggesting that high metabolic activity of cancer cells may trigger activation or inﬁltration of Tregs, leading to cancer cells’ evasion of anti-cancer immune cells. In summary, we identiﬁed clinically and metabolically distinct subtypes of HCC, potential biomarkers associated with these subtypes, and a potential mechanism of metabolism-mediated immune evasion by HCC cells. Citation: Jung, J.; Park, S.; Jang, Y.; Lee, S.-H.; Jeong, Y.S.; Yim, S.Y.; Lee, J.-S. Clinical Signiﬁcance of Glycolytic Metabolic Activity in Hepatocellular Carcinoma. Cancers 2023, 15, 186. https://doi.org/ 10.3390/cancers15010186 Citation: Jung, J.; Park, S.; Jang, Y.; Lee, S.-H.; Jeong, Y.S.; Yim, S.Y.; Lee, J.-S. Clinical Signiﬁcance of Glycolytic Metabolic Activity in Hepatocellular Carcinoma. Cancers 2023, 15, 186. https://doi.org/ 10.3390/cancers15010186 Academic Editor: Filippo Oliveri Received: 31 October 2022 Revised: 17 December 2022 Accepted: 23 December 2022 Published: 28 December 2022 Received: 31 October 2022 Revised: 17 December 2022 Accepted: 23 December 2022 Published: 28 December 2022 Keywords: liver cancer; hepatocellular carcinoma; cancer metabolism; glycolysis; transcriptome; survival; stem cells; immunotherapy; Tregs 1. Introduction Hepatocellular carcinoma (HCC) is among the most common cancers worldwide and causes about 830,000 deaths annually [1]. The incidence of HCC in the United States has increased over the past 25 years, to an estimated 41,260 new cases in 2022 [2]. Less than one-third of HCC patients are eligible for potentially curative treatments [3–7]; the vast majority of HCC patients present with advanced disease not amenable to curative https://www.mdpi.com/journal/cancers Cancers 2023, 15, 186. https://doi.org/10.3390/cancers15010186 Cancers 2023, 15, 186 2 of 14 treatments. Current standard ﬁrst-line treatments for advanced HCC include targeted therapy with kinase inhibitors such as sorafenib and lenvatinib, which have antiangiogenic and antiproliferative effects, and immunotherapy with atezolizumab combined with be- vacizumab [8–11]. However, kinase inhibitors appear to prolong HCC patients’ survival by only a few months, and immunotherapy only beneﬁts patients who have HCC with viral etiologies [12]. Thus, there is a clear need to enhance our insight into the molecular development of HCC, which could lead to the discovery of new targeted therapies for HCC and/or effective strategies to extend the survival of HCC patients. Metabolic reprogramming is a critical hallmark of cancer [13,14], that enables cancer cells to adapt to the high energy demands necessary for fast growth. Indeed, many cancer cells acquire deregulated high metabolic activity that enables them to prosper even in a resource-limited microenvironment [15]. The best example is the surge in consuming glucose through anaerobic glycolysis, even in the presence of oxygen [16]. Considering that the liver is the primary site of metabolism in the body, it is not surprising to see highly dysregulated metabolism in HCC cells compared to that of normal hepatocytes [17,18]. However, the clinical relevance of metabolic alteration, particularly in glycolytic pathway, in HCC tumors has not been systematically assessed and clearly demonstrated. In a previous study [19], we showed that genomic signatures from mouse models are similar to those from human tumors and developed the approach known as “comparative systems genomics” that performs cross-species comparison of genomic data from mouse and human tissues to classify patients according to deﬁned conditions from preclinical mouse models. In the current study, we adopted this method to uncover the clinical signiﬁcance of metabolic alteration in HCC. 2.2. Gene Expression and Clinical Data from Human HCC Gene expression data and clinical data were described in earlier studies [21–26]. Brieﬂy, gene expression data from the Fudan cohort were obtained from the GEO database (acces- sion number GSE14520) [21]. Gene expression data from the Korean cohort were generated using the Illumina microarray platform human-6 v2 and v4 (accession numbers GSE16757, GSE43619) [22,23]. Gene expression data from the Modena cohort were obtained from GEO databases (accession number GSE54236) [24]. Gene expression data from Zhongshan hospital cohort were obtained from National Omics Data Encyclopedia (NODE, accession number OEP000321) [25]. We also included gene expression data from The Cancer Genome Atlas (TCGA) HCC project in this analysis [26]. Table S1 shows the summary of data sets in all ﬁve cohorts. All patients had undergone surgical resection as their primary treatment for HCC. 2.1. Gene Expression Proﬁle Data from Mouse Liver Tissues Gene expression proﬁle data from mouse liver tissues were generated as described previously [20]. Eight week-old C57B/l6 male mice were fed a regular diet (ad libitum) with or without 20% glucose or fructose for 24 h in drinking tap water (n = 6 per group and 18 in total). Mice were euthanized for collection of RNA at 14 h after the start of light period in the animal housing unit. Total RNA was extracted from liver tissues of mice and used to generate gene expression data via the Agilent microarray platform (SurePrint G3 Mouse GE v2 8x60K Microarray). Data are available in the National Center for Biotechnology Information’s Gene Expression Omnibus (GEO) database (GSE92502). 2. Materials and Methods 2.1. Gene Expression Proﬁle Data from Mouse Liver Tissues 2.4. Data Analysis Collected gen 2.4. Data Analysis Collected gene expression data were transformed and normalized as described pre- viously [20]. BRB-ArrayTools ,v4.6, a freeware program from the National Cancer Insti- tute (https://brb.nci.nih.gov/BRB-ArrayTools/ accessed on 11 June 2022), was used for an- alyzing the data and building a predictive model [27]. Cluster (v 3.0) and TreeView (v 1.6) were used to generate a heatmap of gene expression data [28]. R language (http://www.r- project.org, v 4.1.1 accessed on 15 September 2021) was used for statistical analysis. So- matic copy number alterations in TCGA data were determined by profiling HCC on Affy- metrix SNP 6.0 arrays and analysis by GISTIC 2.0 [29]. B f li d h i d t f f i i Collected gene expression data were transformed and normalized as described pre- viously [20]. BRB-ArrayTools, v4.6, a freeware program from the National Cancer In- stitute (https://brb.nci.nih.gov/BRB-ArrayTools/ accessed on 11 June 2022), was used for analyzing the data and building a predictive model [27]. Cluster (v 3.0) and Tree- View (v 1.6) were used to generate a heatmap of gene expression data [28]. R language (http://www.r-project.org, v 4.1.1 accessed on 15 September 2021) was used for statistical analysis. Somatic copy number alterations in TCGA data were determined by proﬁling HCC on Affymetrix SNP 6.0 arrays and analysis by GISTIC 2.0 [29]. Before pooling mouse and human gene expression data for performing cross-species analysis, expression data of orthologous genes in both data sets were independently con- verted to z-scores (z = (x − mean)/standard deviation) [19]. A Bayesian compound covari- ate prediction (BCCP) algorithm was used to estimate the probability that a particular human HCC sample would have a given gene expression pattern from mouse tissue [19,30]. Gene expression data from mouse tissue (training sets) were combined to form a predictor according to a BCCP model. The robustness of the predictor was estimated by a misclassification rate determined using leave-one-out cross-validation during training. Sensitivity and specificity of predicting sugar-fed liver tissue in the mouse training set were 1.0 and 1.0, respectively. The BCCP model estimated the probability that an y y y y Before pooling mouse and human gene expression data for performing cross-species analysis, expression data of orthologous genes in both data sets were independently con- verted to z-scores (z = (x −mean)/standard deviation) [19]. 2.3. Identiﬁcation of Hepatic Glycolytic Gene Expression Signature from Mouse Liver To identify genes reﬂecting high glycolytic activity in mouse liver tissue, we ﬁrst selected genes whose expression is signiﬁcantly induced by fructose-feeding or glucose- feeding in mouse livers, yielding 1960 genes for fructose-speciﬁc induction and 2022 genes for glucose-speciﬁc induction. By comparing the two gene lists, 416 overlapping genes were identiﬁed as glycolytic genes, and their expression patterns were considered to be Cancers 2023, 15, 186 3 of 14 lucose- 2 genes the hepatic glycolytic gene expression signature (Figure 1). Later, identiﬁed gene sets were subjected to ingenuity pathway analysis (September release 2022), which revealed a myriad of affected signaling pathways and functional categories. were identified as glycolytic genes, and their expression patterns were considered to be the hepatic glycolytic gene expression signature (Figure 1). Later, identified gene sets were subjected to ingenuity pathway analysis (September release 2022), which revealed a myr- iad of affected signaling pathways and functional categories. the hepatic glycolytic gene expression signature (Figure 1). Later, identiﬁed gene sets were subjected to ingenuity pathway analysis (September release 2022), which revealed a myriad of affected signaling pathways and functional categories. were identified as glycolytic genes, and their expression patterns were considered to be the hepatic glycolytic gene expression signature (Figure 1). Later, identified gene sets were subjected to ingenuity pathway analysis (September release 2022), which revealed a myr- iad of affected signaling pathways and functional categories. Figure 1. Hepatic glycolytic gene expression signature from mouse liver. (A) Venn diagram of genes selected by a two-sample t test. The red circle (gene list X) represents genes differentially expressed between liver tissues from mice fed with tap water and those fed with fructose water. The blue circle (gene list Y) represents genes differentially expressed between liver tissues of mice fed with tap water and those fed with glucose water. We applied a cut-off p-value of less than 0.01 to retain genes whose expression is significantly different between the two groups of tissues. (B) Expression pat- terns of selected genes in the Venn diagram. Gene expression data from livers of mice fed with fructose, glucose or control tap water were selected from 416 overlapping genes. Figure 1. Hepatic glycolytic gene expression signature from mouse liver. (A) Venn diagram of genes selected by a two-sample t test. The red circle (gene list X) represents genes differentially expressed between liver tissues from mice fed with tap water and those fed with fructose water. 2.3. Identiﬁcation of Hepatic Glycolytic Gene Expression Signature from Mouse Liver The blue circle (gene list Y) represents genes differentially expressed between liver tissues of mice fed with tap water and those fed with glucose water. We applied a cut-off p-value of less than 0.01 to retain genes whose expression is signiﬁcantly different between the two groups of tissues. (B) Expression patterns of selected genes in the Venn diagram. Gene expression data from livers of mice fed with fructose, glucose or control tap water were selected from 416 overlapping genes. Figure 1. Hepatic glycolytic gene expression signature from mouse liver. (A) Venn diagram of genes selected by a two-sample t test. The red circle (gene list X) represents genes differentially expressed between liver tissues from mice fed with tap water and those fed with fructose water. The blue circle (gene list Y) represents genes differentially expressed between liver tissues of mice fed with tap water and those fed with glucose water. We applied a cut-off p-value of less than 0.01 to retain genes whose expression is significantly different between the two groups of tissues. (B) Expression pat- terns of selected genes in the Venn diagram. Gene expression data from livers of mice fed with fructose, glucose or control tap water were selected from 416 overlapping genes. Figure 1. Hepatic glycolytic gene expression signature from mouse liver. (A) Venn diagram of genes selected by a two-sample t test. The red circle (gene list X) represents genes differentially expressed between liver tissues from mice fed with tap water and those fed with fructose water. The blue circle (gene list Y) represents genes differentially expressed between liver tissues of mice fed with tap water and those fed with glucose water. We applied a cut-off p-value of less than 0.01 to retain genes whose expression is signiﬁcantly different between the two groups of tissues. (B) Expression patterns of selected genes in the Venn diagram. Gene expression data from livers of mice fed with fructose, glucose or control tap water were selected from 416 overlapping genes. 3.1. Gene Expression Signature Reﬂecting Glycolytic Activity from Mouse Liver Tissue We examined the glycolytic activity of HCC tumors by using a comparative cross- species genomic approach that integrates genomic data from the well-deﬁned experimental conditions of animal models into those from human HCC. To do this, genes whose expres- sion is signiﬁcantly correlated with glycolytic activity in mouse liver were identiﬁed by applying a Student’s t-test to gene expression proﬁle data from liver tissues of mice fed with fructose or glucose versus control tap water. Overlapping expression of 416 genes was identiﬁed as a hepatic glycolytic signature (p < 0.01, Figure 1, and Table S2). As expected, the upregulated genes included metabolic genes such as Psat1, Fut1, Gpi1, Rpia, Acaca, and Pklr, suggesting that the signature well reﬂect high metabolism in the liver. Hereafter, we refer to the deﬁned signature as the glycolysis metabolic (GM) signature. To further reveal the underlying biological activity of the GM signature in the liver, we next performed gene network analysis of the GM signature by applying Ingenuity Pathway Analysis. Not surprisingly, it revealed the glycolysis pathway as one of the most activated pathways in the GM signature (Table S3). Other activated pathways included the mTOR pathway, reﬂecting high energy consumption, and the cell growth pathway, suggesting that highly glycolytic activity leads to high cellular energy production and cell growth. Interestingly, the ferroptosis signaling pathway was also activated by high glycolysis, suggesting that high metabolic activity may increase oxidative stress, which is the foundation of ferroptotic cell death [35,36]. In agreement with this, the NRF2-mediated oxidative stress response pathway was also activated by high glycolysis. 2.4. Data Analysis Collected gen 2.4. Data Analysis A Bayesian compound co- variate prediction (BCCP) algorithm was used to estimate the probability that a particular human HCC sample would have a given gene expression pattern from mouse tissue [19,30]. Gene expression data from mouse tissue (training sets) were combined to form a predictor according to a BCCP model. The robustness of the predictor was estimated by a misclassiﬁ- cation rate determined using leave-one-out cross-validation during training. Sensitivity and speciﬁcity of predicting sugar-fed liver tissue in the mouse training set were 1.0 and 1.0, respectively. The BCCP model estimated the probability that an individual human HCC sample would have high or low glycolytic activity and trichotomized tumors according to Bayesian probability (cutoff of 0.8 and 0.2). Cancers 2023, 15, 186 4 of 14 To generate the hepatic stem cell (HSC) probability of HCC tumors, we applied a previously established HSC signature to gene expression data from HCC tumors as described previously [31,32]. 2.5. Gene Expression Data from HCC PDX Models HCC PDX tumors were established by Crown Bioscience as described earlier [33,34]. mRNA expression data from PDX tumors were generated by Illumina HiSeq2500 platform. For bioinformatics analysis of transcriptome sequencing data, RNAseq raw data were ﬁrst cleaned up by removing contamination mouse mRNA reads that preferentially mapped to mouse genome (UCSC MM10). Clean reads were mapped to reference genes (ENSEMBL GRCh37.66) by Bowtie (v 1.2.3), and gene expression was calculated by MMSEQ (v 1.0.10). The hepatic glycolytic gene expression signature was applied to gene expression data from PDX model to stratify them to 3 subtypes. 3. Results 3.1. Gene Expression Signature Reﬂecting Glycolytic Activity from Mouse Liver Tissue 3.2. Association of Hepatic Metabolic Activity with Prognosis of Patients with HCC Having generated a gene expression signature reﬂecting high metabolic activity in liver, we next tested the clinical relevance of hepatic glycolytic activity in primary HCC tumors from patients by extracting the expression of GM signature genes from patients’ tumors and comparing them with the GM signature from mouse liver tissues. To validate clinical association of GM signature in HCC, we built a stratifying prediction model with the mouse GM signature and directly applied it to the genomic data from HCC tumors. Expression data from the mouse GM signature (training set) were used to generate a BCCP that estimated the probability of high metabolic activity in each HCC tumor (Figure 2A). Patients in the Fudan HCC cohort (n = 242) were trichotomized according to Bayesian probability (<0.2, 0.2 to 0.8, >0.8), which classiﬁed 69, 108, and 65 patients into high, middle, and low metabolic activity subgroups, respectively (Figure 2B). Kaplan–Meier plots for overall survival (OS) of patients in the Fudan cohort showed signiﬁcant differences in OS after treatment (p = 1.6 × 10−6 by log-rank test) among the three subgroups (Figure 2C), strongly indicating that high glycolytic activity in HCC signiﬁcantly contributes to patients’ clinical outcome after treatment. 5 of 14 g n- Cancers 2023, 15, 186 rank test g g g y y y p Figure 2. Clinical association of metabolic activity in hepatocellular carcinoma (HCC). (A) Schematic diagram of the prediction model. (B) Heatmap of glycolysis metabolic (GM) gene expression signa- ture in patients from the Fudan cohort. (C) Kaplan–Meier plots of overall survival (OS) of HCC patients in the Fudan cohort stratified by GM subtype. TCGA, The Cancer Genome Atlas. Figure 2. Clinical association of metabolic activity in hepatocellular carcinoma (HCC). (A) Schemat diagram of the prediction model. (B) Heatmap of glycolysis metabolic (GM) gene expression signatur in patients from the Fudan cohort. (C) Kaplan–Meier plots of overall survival (OS) of HCC patien in the Fudan cohort stratiﬁed by GM subtype. TCGA, The Cancer Genome Atlas. Figure 2. Clinical association of metabolic activity in hepatocellular carcinoma (HCC). (A) Schematic diagram of the prediction model. (B) Heatmap of glycolysis metabolic (GM) gene expression signa- ture in patients from the Fudan cohort. (C) Kaplan–Meier plots of overall survival (OS) of HCC Figure 2. Clinical association of metabolic activity in hepatocellular carcinoma (HCC). (A) Schema diagram of the prediction model. 3.2. Association of Hepatic Metabolic Activity with Prognosis of Patients with HCC (B) Heatmap of glycolysis metabolic (GM) gene expression signatu in patients from the Fudan cohort. (C) Kaplan–Meier plots of overall survival (OS) of HCC patien sociation of metabolic activity in hepatocellular carcinoma (HCC). (A) Schematic iction model. (B) Heatmap of glycolysis metabolic (GM) gene expression signa- m the Fudan cohort. (C) Kaplan–Meier plots of overall survival (OS) of HCC n cohort stratified by GM subtype. TCGA, The Cancer Genome Atlas. Figure 2. Clinical association of metabolic activity in hepatocellular carcinoma (HCC). (A) Schematic diagram of the prediction model. (B) Heatmap of glycolysis metabolic (GM) gene expression signature in patients from the Fudan cohort. (C) Kaplan–Meier plots of overall survival (OS) of HCC patients in the Fudan cohort stratiﬁed by GM subtype. TCGA, The Cancer Genome Atlas. We next examined the correlation of glycolytic activity with patients’ prognosis in four additional HCC cohorts (ZhongShan cohort, n = 159; TCGA cohort, n = 371; Korean cohort, n = 188; and Modena cohort, n = 78, Figure 2A and Table S1). When the BCCP used in the Fudan cohort was applied to the four additional cohorts, Kaplan–Meier plots of all cohorts showed signiﬁcant differences in OS among the three GM subtypes (p = 1.0 × 10−5 for ZhongShan, p = 0.005 for TCGA, p = 0.02 for Korean, and p = 0.002 for Modena by log-rank test, Figure 3). Together, the results from all ﬁve cohorts (n = 1038) clearly demonstrated a strong association between the high glycolytic activity and poor OS rates in HCC. Cancers 2023, 15, 186 6 of 14 Figure 3. Validation of clinical association of low, middle, and high glycolysis metabolic subtypes in hepatocellular carcinoma. Kaplan–Meier plots of overall survival (OS) in patients in the valida tion cohorts. Zhongshan cohort (n = 159), Korea cohort (n = 188), The Cancer Genome Atlas (TCGA cohort (n = 371), Modena cohort (n = 78), and pool of five cohorts (n = 1038). 3 3 Prognostic Significance of GM Subtypes Figure 3. Validation of clinical association of low, middle, and high glycolysis metabolic subtypes in hepatocellular carcinoma. Kaplan–Meier plots of overall survival (OS) in patients in the validation cohorts. Zhongshan cohort (n = 159), Korea cohort (n = 188), The Cancer Genome Atlas (TCGA) cohort (n = 371), Modena cohort (n = 78), and pool of ﬁve cohorts (n = 1038). 3.3. 3.2. Association of Hepatic Metabolic Activity with Prognosis of Patients with HCC Prognostic Signiﬁcance of GM Subtypes To quantify the prognostic weight of glycolytic activity in combination with other of clinical association of low, middle, and high glycolysis metabolic subtypes cinoma. Kaplan–Meier plots of overall survival (OS) in patients in the valida- han cohort (n = 159), Korea cohort (n = 188), The Cancer Genome Atlas (TCGA) dena cohort (n = 78), and pool of five cohorts (n = 1038). Figure 3. Validation of clinical association of low, middle, and high glycolysis metabolic subtypes in hepatocellular carcinoma. Kaplan–Meier plots of overall survival (OS) in patients in the validation cohorts. Zhongshan cohort (n = 159), Korea cohort (n = 188), The Cancer Genome Atlas (TCGA) cohort (n = 371), Modena cohort (n = 78), and pool of ﬁve cohorts (n = 1038). of clinical association of low, middle, and high glycolysis metabolic subtypes cinoma. Kaplan–Meier plots of overall survival (OS) in patients in the valida- han cohort (n = 159), Korea cohort (n = 188), The Cancer Genome Atlas (TCGA) dena cohort (n = 78), and pool of five cohorts (n = 1038). Figure 3. Validation of clinical association of low, middle, and high glycolysis metabolic subtypes in hepatocellular carcinoma. Kaplan–Meier plots of overall survival (OS) in patients in the validation cohorts. Zhongshan cohort (n = 159), Korea cohort (n = 188), The Cancer Genome Atlas (TCGA) cohort (n = 371), Modena cohort (n = 78), and pool of ﬁve cohorts (n = 1038). 3.4. Mutations and Genomic Alterations Associated with GM Subtypes We next assessed the association of genomic characteristics with GM subtypes in the TCGA cohort, for which genomic data were available. to gain additional insight into each subtype’s biology. We found no differences in mutation burden among the three GM subtypes (Figure 4A). However, alterations of genomic copy number differed signiﬁcantly among the subtypes, with the high GM subtype having the most (Figure 4B). We next sought to identify somatic mutations signiﬁcantly associated with the subtypes (Figure S2). TP53 mutations were associated with the high and middle GM subtypes (Figure 4C). FAM47A mutations were associated with the high GM subtype, and CTNNB1 (encoding β-catenin) mutations were associated with the low GM subtype. ALB (coding albumin) mutations were signiﬁcantly less frequent in the high GM subtype, suggesting a potential connection of loss of albumin activity in regulation of the glycolytic pathway. ( ) p 3.3. Prognostic Signiﬁcance of GM Subtypes nificance of GM Subtypes he prognostic weight of glycolytic activity in combination with other tures, we performed univariate Cox proportional analyses with clinical Zhongshan cohort, which had the most complete set of clinical data. In size and Barcelona Clinics Liver Cancer (BCLC) stage, which are well- ssociated with OS, the GM signature was a statistically significant pre- To quantify the prognostic weight of glycolytic activity in combination with other critical clinical features, we performed univariate Cox proportional analyses with clinical features from the Zhongshan cohort, which had the most complete set of clinical data. In addition to tumor size and Barcelona Clinics Liver Cancer (BCLC) stage, which are well-known variables associated with OS, the GM signature was a statistically signiﬁcant predictor of OS (Table 1). In multivariate analysis with analyzed variables together, the high GM subtype was independent prognostic predictor for OS as evidenced by high hazard ratio of 2.97 (95% conﬁdence interval, 1.72−5.12 and p = 8.5 × 10−5). Cancers 2023, 15, 186 7 of 14 Table 1. Univariate and multivariate Cox regression analyses of overall survival in Zhongshan cohort. Table 1. Univariate and multivariate Cox regression analyses of overall survival in Zhongshan cohort. Characteristic Univariate Multivariate Hazard Ratio (95% CI) p Value Hazard Ratio (95% CI) p Value Patient sex (M or F) 0.75 (0.4–1.41) 0.381 Age (>60 years or not) 0.8 (0.44–1.45) 0.47 AFP (>300 ng/mL or not) 3.12 (1.83–5.34) <0.001 2.75 (1.53–4.91) <0.001 Cirrhosis (yes or no) 1.28 (0.69–2.35) 0.42 Tumor size (>6 cm or not) 3.53 (1.97–6.32) <0.001 5.26 (1.86–14.8) 0.001 BCLC stage (B/C/D or 0/A) 2.77 (1.51–5.09) 0.001 0.57 (0.23–1.4) 0.23 GM signature (high or mid/low) 2.97 (1.72–5.12) <0.001 1.84 (1.04–3.25) 0.033 CI, conﬁdence interval; AFP, alpha-fetoprotein; BCLC, Barcelona Clinic Liver Cancer; GM, glycolytic metabolism. We next estimated how GM subtypes are independent across the standard clinical stages. When the GM signature was applied to patients with BCLC stage A, which is con- sidered early stage HCC [37], patients with the high GM subtype had worse OS outcomes than patients with the middle and low GM subtypes (Figure S1). Taken together with Cox analysis, this observation suggests that GM signature retains its prognostic signiﬁcance even after the classic clinicopathological variables have been taken into account. 3.5. Potential Sensitivity to Immunotherapy among GM Subtypes The combination of bevacizumab, which targets VEGF, and atezolizumab, an immune checkpoint inhibitor that selectively targets PD-L1, has yielded encouraging results in HCC patients [11]. Therefore, we estimated each GM subtype’s potential response to immunotherapy using tumor immune dysfunction and exclusion (TIDE) scores, which reﬂect resistance to immune checkpoint inhibitors [38]. Interestingly, most tumors (86.8%) in the high GM subtype showed high TIDE scores (Figure 5A), suggesting that HCC patients with high metabolic activity would not have substantial beneﬁt from immunotherapy. Moreover, the GM probability was positively correlated with TIDE score (r = 0.3381, p = 1.98 × 10−11) (Figure 5B), further supporting the association of the high GM subtype with resistance to immunotherapy. To uncover the biology underlying the low response of high metabolic HCC tumors to immunotherapy, we explored the percentage of immune cells in tumors by analyzing their gene expression data using the previously established CIBERSORT algorithm [39] (Figure 5C). Interestingly, the fraction of immunosuppressive regulatory T cells (Tregs) was signiﬁcantly higher in the high GM subtype (Figure 5D), suggesting that high metabolic activity in the tumor microenvironment may trigger activation of Tregs, leading to the low response to immunotherapy in high GM HCC tumors. Furthermore, the fraction of naïve M0 macrophages was also higher in high GM HCC tumors (Figure 5E), suggesting that that absence of active anti-cancer macrophages may contribute to the poor response of high GM tumors to immunotherapy. In agreement with these observations, the estimated level of myeloid-derived suppressor cells from the TIDE analysis was signiﬁcantly higher in Cancers 2023, 15, 186 8 of 14 8 of 14 high GM subtype than in the other subtypes (Figure S3A). Similarly, expression of major inhibitors of immune checkpoints CTLA-4 and PD-1 were signiﬁcantly higher in the high GM subtype (Figure S3B), further supporting the notion that high metabolic activity may suppress immune activity. IEW 8 of 15 Figure 4. Genomic alterations in the glycolysis metabolic (GM) subtypes. (A) Bee swarm box plots for number of nonsynonymous mutations in GM subtypes (n = 367). No significant difference is observed among the GM subtypes. In the box plots, the boundary of the box indicates the 25th to 75th percentile, and the black line within the box marks the mean. Whiskers above and below the box indicate the 10th and 90th percentiles. Circles represent the number of mutations in each tumor. 3.5. Potential Sensitivity to Immunotherapy among GM Subtypes Mutation rates of each gene are presented as fraction within subtypes. Red, light blue, and dark blue represent the high, middle, and low GM subtypes, respectively. 3.5. Potential Sensitivity to Immunotherapy among GM Subtypes Figure 4. Genomic alterations in the glycolysis metabolic (GM) subtypes. (A) Bee swarm box plots for number of nonsynonymous mutations in GM subtypes (n = 367). No signiﬁcant difference is observed among the GM subtypes. In the box plots, the boundary of the box indicates the 25th to 75th percentile, and the black line within the box marks the mean. Whiskers above and below the box indicate the 10th and 90th percentiles. Circles represent the number of mutations in each tumor. (B) The fraction of the genome altered by copy number gain and loss was estimated by GISTIC2 analysis in each tumor (n = 367). The high GM subtype has signiﬁcantly higher alterations than the other two subtypes (all p < 0.05 by Student t test). (C) Somatic mutations associated with GM subtypes in TCGA cohort. Mutation rates of each gene are presented as fraction within subtypes. Red, light blue, and dark blue represent the high, middle, and low GM subtypes, respectively. Figure 4. Genomic alterations in the glycolysis metabolic (GM) subtypes. (A) Bee swarm box plots for number of nonsynonymous mutations in GM subtypes (n = 367). No significant difference is observed among the GM subtypes. In the box plots, the boundary of the box indicates the 25th to 75th percentile, and the black line within the box marks the mean. Whiskers above and below the box indicate the 10th and 90th percentiles. Circles represent the number of mutations in each tumor. (B) The fraction of the genome altered by copy number gain and loss was estimated by GISTIC2 analysis in each tumor (n = 367). The high GM subtype has significantly higher alterations than the other two subtypes (all p < 0.05 by Student t test). (C) Somatic mutations associated with GM sub- types in TCGA cohort. Mutation rates of each gene are presented as fraction within subtypes. Red, light blue, and dark blue represent the high, middle, and low GM subtypes, respectively. 3.5. Potential Sensitivity to Immunotherapy among GM Subtypes Figure 4. Genomic alterations in the glycolysis metabolic (GM) subtypes. (A) Bee swarm box plots for number of nonsynonymous mutations in GM subtypes (n = 367). 3.5. Potential Sensitivity to Immunotherapy among GM Subtypes (B) The fraction of the genome altered by copy number gain and loss was estimated by GISTIC2 analysis in each tumor (n = 367). The high GM subtype has significantly higher alterations than the other two subtypes (all p < 0.05 by Student t test). (C) Somatic mutations associated with GM sub- types in TCGA cohort. Mutation rates of each gene are presented as fraction within subtypes. Red, light blue, and dark blue represent the high, middle, and low GM subtypes, respectively. 3 5 P t ti l S iti it t I th GM S bt Figure 4. Genomic alterations in the glycolysis metabolic (GM) subtypes. (A) Bee swarm box plot for number of nonsynonymous mutations in GM subtypes (n = 367). No signiﬁcant difference i observed among the GM subtypes. In the box plots, the boundary of the box indicates the 25th t 75th percentile, and the black line within the box marks the mean. Whiskers above and below th box indicate the 10th and 90th percentiles. Circles represent the number of mutations in each tumo (B) The fraction of the genome altered by copy number gain and loss was estimated by GISTIC analysis in each tumor (n = 367). The high GM subtype has signiﬁcantly higher alterations than the other two subtypes (all p < 0.05 by Student t test). (C) Somatic mutations associated with GM subtypes in TCGA cohort. Mutation rates of each gene are presented as fraction within subtype Red, light blue, and dark blue represent the high, middle, and low GM subtypes, respectively. Figure 4. Genomic alterations in the glycolysis metabolic (GM) subtypes. (A) Bee swarm box plots for number of nonsynonymous mutations in GM subtypes (n = 367). No significant difference is observed among the GM subtypes. In the box plots, the boundary of the box indicates the 25th to 75th percentile, and the black line within the box marks the mean. Whiskers above and below the box indicate the 10th and 90th percentiles. Circles represent the number of mutations in each tumor. (B) The fraction of the genome altered by copy number gain and loss was estimated by GISTIC2 analysis in each tumor (n = 367). The high GM subtype has significantly higher alterations than the other two subtypes (all p < 0.05 by Student t test). (C) Somatic mutations associated with GM sub- types in TCGA cohort. 3.5. Potential Sensitivity to Immunotherapy among GM Subtypes No signiﬁcant difference is observed among the GM subtypes. In the box plots, the boundary of the box indicates the 25th to 75th percentile, and the black line within the box marks the mean. Whiskers above and below the box indicate the 10th and 90th percentiles. Circles represent the number of mutations in each tumor. (B) The fraction of the genome altered by copy number gain and loss was estimated by GISTIC2 analysis in each tumor (n = 367). The high GM subtype has signiﬁcantly higher alterations than the other two subtypes (all p < 0.05 by Student t test). (C) Somatic mutations associated with GM subtypes in TCGA cohort. Mutation rates of each gene are presented as fraction within subtypes. Red, light blue, and dark blue represent the high, middle, and low GM subtypes, respectively. 9 of 14 Cancers 2023, 15, 186 py p = 1.9 with resistance to immunotherapy. Figure 5. Immune characteristics of the glycolysis metabolic (GM) subtypes. (A) Waterfall plots for the response rates to immunotherapy predicted by the tumor immune dysfunction and exclusion (TIDE) algorithm in The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA-LIHC) co- hort (n = 371). Numbers below waterfall plots represent the fraction of responders in the patients with each GM subtype. (B) Scatter plot for the correlation between TIDE score and GM probability in the TCGA cohort (n = 371). Blue line indicates locally weighted scatterplot smoothing (lowess) regression. (C) The pattern of infiltrations of 22 immune subsets according to GM subtype from fetal liver signatures predicted by the CIBERSORT algorithm in the TCGA cohort. (D,E) Box and scatter plots of fraction of regulatory T cells (Tregs) (D) and M0 macrophages (E) in GM subtypes. Relative fraction of each immune-subset was normalized by mean and standard deviation across the sam- ples. In the scatter plots, blue line indicates locally weighted scatterplot smoothing (lowess) regres- sion. In the box plots, the boundary of the box indicates the 25th to 75th percentile, and the black line within the box marks the mean Whiskers above and below the box indicate the 10th and 90th Figure 5. Immune characteristics of the glycolysis metabolic (GM) subtypes. (A) Waterfall plots for the response rates to immunotherapy predicted by the tumor immune dysfunction and exclusion (TIDE) algorithm in The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA-LIHC) cohort (n = 371). 3.5. Potential Sensitivity to Immunotherapy among GM Subtypes Numbers below waterfall plots represent the fraction of responders in the patients with each GM subtype. (B) Scatter plot for the correlation between TIDE score and GM probability in the TCGA cohort (n = 371). Blue line indicates locally weighted scatterplot smoothing (lowess) regression (C) The pattern of inﬁltrations of 22 immune subsets according to GM subtype from fetal liver signatures predicted by the CIBERSORT algorithm in the TCGA cohort. (D,E) Box and scatter plots of fraction of regulatory T cells (Tregs) (D) and M0 macrophages (E) in GM subtypes. Relative fraction of each immune-subset was normalized by mean and standard deviation across the samples. In the scatter plots, blue line indicates locally weighted scatterplot smoothing (lowess) regression. In the box plots, the boundary of the box indicates the 25th to 75th percentile, and the black line within the box marks the mean Whiskers above and below the box indicate the 10th and 90th percentiles Each e characteristics of the glycolysis metabolic (GM) subtypes. (A) Waterfall plots for s to immunotherapy predicted by the tumor immune dysfunction and exclusion in The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA-LIHC) co- umbers below waterfall plots represent the fraction of responders in the patients btype. (B) Scatter plot for the correlation between TIDE score and GM probability ort (n = 371). Blue line indicates locally weighted scatterplot smoothing (lowess) e pattern of infiltrations of 22 immune subsets according to GM subtype from fetal redicted by the CIBERSORT algorithm in the TCGA cohort. (D,E) Box and scatter f regulatory T cells (Tregs) (D) and M0 macrophages (E) in GM subtypes. Relative mmune-subset was normalized by mean and standard deviation across the sam- r plots, blue line indicates locally weighted scatterplot smoothing (lowess) regres- lots, the boundary of the box indicates the 25th to 75th percentile, and the black x marks the mean. Whiskers above and below the box indicate the 10th and 90th Figure 5. Immune characteristics of the glycolysis metabolic (GM) subtypes. (A) Waterfall plots for the response rates to immunotherapy predicted by the tumor immune dysfunction and exclusion (TIDE) algorithm in The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA-LIHC) cohort (n = 371). Numbers below waterfall plots represent the fraction of responders in the patients with each GM subtype. (B) Scatter plot for the correlation between TIDE score and GM probability in the TCGA cohort (n = 371). 3.6. Stem Cell Characteristics in GM Subtypes 3.6. Stem Cell Characteristics in GM Subtypes We next sought to correlate the GM subtypes with stem cell characteristics by applying a previously established HSC signature to gene expression data from HCC tumors [31]. The high GM subtype showed signiﬁcantly higher HSC probability than the middle and low GM subtypes (p < 0.001 by t test, Figure 6A), suggesting that high metabolic activity in HCC might be driven by genetic or genomic switches activated in HSCs. Consis- tently, HSC probability showed signiﬁcant correlation with GM probability (r = 0.6269, p = 4.9 × 10−49, Figure 6B), further supporting a close relationship between high metabolic activity and HSC features in HCC. We next examined the expression of cancer stem cell markers. Not surprisingly, expression of many stem cell markers were signiﬁcantly higher in the GM high subtype than in the other GM subtypes (Figure 6C). In particular, expression of well-known hepatic stem markers such as AFP, KRT19, and EPCAM was signiﬁcantly higher in high GM subtypes (Figure 6D), and SALL4 was the most signiﬁcantly correlated transcription regulator with the high GM subtype. IEW 11 of Figure 6. Stem cell characteristics of glycolysis metabolic (GM) subtypes. (A) Hepatic stem cell pro ability of hepatocellular carcinoma tumors in GM subtypes in The Cancer Genome Atlas (TCG cohort. In the box plots, the boundary of the box indicates the 25th to 75th percentile, and the bla line within the box marks the mean. Whiskers above and below the box indicate the 10th and 90 percentiles. Each circle represents the fraction of the indicated immune cells in each tumor. Stude t test. (B) Scatter plot for the correlation between hepatic stem cell probability and GM probabil in the TCGA cohort (n = 371). (C) Heatmap for expression of major stem-cell markers according G subtype in the TCGA cohort. (D) Box plots of expression of stem cell markers according to G subtype in the TCGA cohort. * p < 0.001 by Student t test. 3 7 GM Subtypes in Preclinical Models Figure 6. Stem cell characteristics of glycolysis metabolic (GM) subtypes. (A) Hepatic stem cell probability of hepatocellular carcinoma tumors in GM subtypes in The Cancer Genome Atlas (TCGA) cohort. In the box plots, the boundary of the box indicates the 25th to 75th percentile, and the black line within the box marks the mean. 3.5. Potential Sensitivity to Immunotherapy among GM Subtypes Blue line indicates locally weighted scatterplot smoothing (lowess) regression. (C) The pattern of inﬁltrations of 22 immune subsets according to GM subtype from fetal liver signatures predicted by the CIBERSORT algorithm in the TCGA cohort. (D,E) Box and scatter plots of fraction of regulatory T cells (Tregs) (D) and M0 macrophages (E) in GM subtypes. Relative fraction of each immune-subset was normalized by mean and standard deviation across the samples. In the scatter plots, blue line indicates locally weighted scatterplot smoothing (lowess) regression. In the box plots, the boundary of the box indicates the 25th to 75th percentile, and the black line within the box marks the mean. Whiskers above and below the box indicate the 10th and 90th percentiles. Each circle represents the fraction of indicated immune cells in each tumor. * p < 0.001 by Student t test. Cancers 2023, 15, 186 10 of 14 10 of 14 4. Discussion In the current study, by integrating gene expression proﬁle data from human HCC tumors with those from mouse models, we identiﬁed three metabolically distinct HCC subtypes that are signiﬁcantly different in prognosis and potential response to standard treatment with immunotherapy. Analysis of genomic data from multiple sources uncovered connections between high metabolic activity and several pathways that might account for poor prognosis in HCC patients and identiﬁed potential prognostic markers. Our results may lead to new opportunities in advancing molecular classiﬁcation of HCC patients and providing potential treatment guidance. p g p g To develop the gene expression signature reﬂecting hepatic metabolic activity and prognosis, we used a supervised approach combined with validation in multiple cohorts of HCC patients. This approach yielded several lines of evidence that support signiﬁcant association of metabolic activity with prognosis in HCC. First, its strong association with prognosis was tested and validated in ﬁve independent HCC cohorts. Second, the GM signature could identify patients at high risk of shorter OS among those with early stage HCC (BCLC A stage). Last, in multivariable Cox regression analysis, the GM signature was one of the most signiﬁcant predictors of OS. g p In our study, HCC tumors with high metabolic activity were characterized by high genomic instability, as reﬂected in their numerous copy number alterations and high frequency of TP53 mutation. This is in agreement with previous reports showing poor prognostic features of HCC with TP53 mutations [40]. Interestingly, the TIDE score, which reﬂects potential response to immunotherapy, showed that the high GM subtype would be the least responsive to immunotherapy. The strong connection of high genome instability with poor response to immunotherapy in the high GM subtype is consistent with previ- ous studies showing that chromosome instability is signiﬁcantly associated with immune evasion and with poor response to immunotherapy [41,42]. The predicted poor response to immunotherapy of the high GM subtype is further supported by a high expression of immune checkpoint regulatory genes, such as those encoding CTLA-4 and PD-1, in that subtype. Our observation of poor response of high metabolic tumors to immunotherapy is further supported by clinical analysis of 18F-FDG PET/CT imaging data to assess the response of patients with metastatic melanoma to immunotherapy [43]. 3.7. GM Subtypes in Preclinical Models 3.7. GM Subtypes in Preclinical Models We next examined whether the GM subtypes’ metabolic characteristics are preserved in preclinical models of HCC. We applied the BCCP GM predictor to the genomic data from 77 HCC patient-derived xenograft (PDX) tumors. GM gene expression patterns were well conserved in these tumors (Figure S4A), indicating that metabolic characteristics are well preserved in PDX tumors. Established PDX models appeared to be stable, as illustrated by the fact that there was no signiﬁcant difference in number of passages in PDX models among subtypes (Figure S4B), suggesting that metabolic features in primary tumors do not fade out over passages. 3.6. Stem Cell Characteristics in GM Subtypes Whiskers above and below the box indicate the 10th and 90th percentiles. Each circle represents the fraction of the indicated immune cells in each tumor. Student t test. (B) Scatter plot for the correlation between hepatic stem cell probability and GM probability in the TCGA cohort (n = 371). (C) Heatmap for expression of major stem-cell markers according GM subtype in the TCGA cohort. (D) Box plots of expression of stem cell markers according to GM subtype in the TCGA cohort. * p < 0.001 by Student t test. Figure 6. Stem cell characteristics of glycolysis metabolic (GM) subtypes. (A) Hepatic stem cell pro ability of hepatocellular carcinoma tumors in GM subtypes in The Cancer Genome Atlas (TCG cohort. In the box plots, the boundary of the box indicates the 25th to 75th percentile, and the bla line within the box marks the mean. Whiskers above and below the box indicate the 10th and 90 percentiles. Each circle represents the fraction of the indicated immune cells in each tumor. Stude t test. (B) Scatter plot for the correlation between hepatic stem cell probability and GM probabili in the TCGA cohort (n = 371). (C) Heatmap for expression of major stem-cell markers according G subtype in the TCGA cohort. (D) Box plots of expression of stem cell markers according to G subtype in the TCGA cohort. * p < 0.001 by Student t test. Figure 6. Stem cell characteristics of glycolysis metabolic (GM) subtypes. (A) Hepatic stem cell probability of hepatocellular carcinoma tumors in GM subtypes in The Cancer Genome Atlas (TCGA) cohort. In the box plots, the boundary of the box indicates the 25th to 75th percentile, and the black line within the box marks the mean. Whiskers above and below the box indicate the 10th and 90th percentiles. Each circle represents the fraction of the indicated immune cells in each tumor. Student t test. (B) Scatter plot for the correlation between hepatic stem cell probability and GM probability in the TCGA cohort (n = 371). (C) Heatmap for expression of major stem-cell markers according GM subtype in the TCGA cohort. (D) Box plots of expression of stem cell markers according to GM subtype in the TCGA cohort. * p < 0.001 by Student t test. Cancers 2023, 15, 186 11 of 14 11 of 14 4. Discussion A meta-analysis of 24 published reports showed that tumors’ high metabolic activity, reﬂected in baseline metabolic tumor volume, maximum standardized uptake value, and total lesion glycolysis, was signiﬁcantly associated with poorer OS of patients after immunotherapy. More inter- estingly, we found that the estimated Treg cell fraction in the tumor microenvironment was highest in high GM subtype, indicating that poor response of high metabolic HCC tumors to immunotherapy might result from the activation of Tregs that suppress anti-cancer immune activity [44]. High glycolytic activity of HCC tumors eventually leads to accumulation of the glycolysis by-product lactic acid in the tumor microenvironment [45]. A recent study showed that Tregs can effectively use lactic acid as metabolic fuel for proliferation [46], suggesting that lactic acid might account for the aggregation of Tregs in HCC tumors’ high metabolic microenvironment. The high GM subtype is also characterized by strong stem cell features, as reﬂected in their high stemness scores and high expression of HSC markers such as AFP, KRT19, Cancers 2023, 15, 186 12 of 14 12 of 14 EPCAM, and SALL4. While the gene expression patterns of high GM HCC tumors were substantially similar to fetal HSCs, it is currently unknown whether this high similarity reﬂects the origin of cancer cells or the high fraction of cancer stem cells in the tumor mass. Invasion is a common event in poor-prognosis tumors with stem cell features [47]. Since zinc-ﬁnger transcription factor SALL4 is a stem cell factor triggering invasion and migration of cancer cells [48], it might contribute to a metastatic phenotype in high metabolic HCC tumors. Interestingly, recent studies showed that SALL4 is a neosubstrate of the molecular glue thalidomide and its derivatives that degrade its target proteins via the E3 ligase complex system [49,50], suggesting that thalidomide and its derivatives could be used for treatment of high metabolic HCC tumors in the future. g The current study was a genomic analysis with limited exploration of the biology of high metabolic activity in association with poor prognosis and poor response to im- munotherapy. However, the GM signature had a solid association with clinical outcome in HCC patients. For validation of high metabolic activity’s association with resistance to immunotherapy in patients with HCC, more in vitro and in vivo study will be necessary. Nevertheless, the newly identiﬁed oncogenic pathways associated with metabolic activity will offer opportunities to identify novel therapeutic targets for HCC. 4. Discussion Moreover, the GM signature is well conserved in PDX models, offering a tool for selecting the best preclinical models for future study. 5. Conclusions In summary, our ﬁnding suggested that high glycolytic activity in HCC is signiﬁcantly associated with poor survival of patients. In depth analysis of metabolism associated genomic traits further suggested that high glycolytic activity in HCC may trigger activation of cancer stem cells and evasion of cancer cells from immune surveillance. Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/cancers15010186/s1, Figure S1: Prognostic signiﬁcance of glycolysis metabolic (GM) subtype in early-stage hepatocellular carcinoma (HCC); Figure S2: Proﬁle of somatic mutations in glycolysis metabolic subtypes in The Cancer Genome Atlas (TCGA) cohort; Figure S3: Myeloid-derived suppressor cells (MDSCs) and expression of immune checkpoint genes in glycolysis metabolic (GM) subtypes; Figure S4: Glycolysis metabolic (GM) subtypes in preclinical models for hepatocellular carcinoma (HCC); Table S1: Summary of HCC gene expression data sets; Table S2: Genes in glycolysis metabolic signature; Table S3: Canonical pathways activated or deactivated in GM. Author Contributions: J.-S.L. and S.Y.Y. conceived and supervised the study. S.P., Y.J. and J.J. ana- lyzed the data and prepared the ﬁgures. S.-H.L. and Y.S.J. processed and maintained data for analysis. S.Y.Y. and J.-S.L. wrote the manuscript and prepared the ﬁgures. All authors have read and agreed to the published version of the manuscript. Funding: This work is supported by the NIH/NCI under award numbers R01CA237327, P50CA217674, and P30CA016672; the Duncan Family Institute for Cancer Prevention and Risk Assessment Seed Funding Research Program at MD Anderson Cancer Center (2016 cycle); Institutional bridge fund from MD Anderson Cancer Center (2022 cycle); an Institutional Research Grant from MD Anderson Cancer Center (2021 cycle) to J.-S.L.; and Korea University Hospital Research Grant to S.Y.Y. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: All data used in current study are publicly available as shown in Table S1. Acknowledgments: The authors also thank Bryan Tutt, Scientiﬁc Editor, of the Research Medical Library at MD Anderson for editing the manuscript. Acknowledgments: The authors also thank Bryan Tutt, Scientiﬁc Editor, of the Research Medical Library at MD Anderson for editing the manuscript. 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. 13 of 14 13 of 14 Cancers 2023, 15, 186 References 2022, 12, 31–46. [CrossRef] 15. Feng, J.; Li, J.; Wu, L.; Yu, Q.; Ji, J.; Wu, J.; Dai, W.; Guo, C. Emerging roles and the regulation of aerobic glycolysis in hepatocellular carcinoma. J. Exp. Clin. Cancer Res. 2020, 39, 126. [CrossRef] [PubMed] p 16. Vander Heiden, M.G.; Cantley, L.C.; Thompson, C.B. Understanding the Warburg effect: The metabolic requirements of cell proliferation. Science 2009, 324, 1029–1033. [CrossRef] p 17. 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https://openalex.org/W4307454484	https://journal.uny.ac.id/index.php/diksi/article/download/45663/17402	English	null	Physical representation of female character in children’s novels by children	Diksi	2,022	cc-by-sa	4,875	ABSTRACT Literary work is a form and result of creative works of art whose objects are humans and their lives use language as a medium. Especially children's literature by children, basically has its own advantages. The storyline is unique and interesting and builds the expression of the child's world. This study aims to describe the physical aspects of female characters in children's novels by children. This paper is a qualitative research with a descriptive approach. The approach used in this study is a psychological literacy approach. The technique of collecting research data was done by reading carefully accompanied by marking. The analytical technique used is a symbolic hermeneutic technique. Based on the results of the study, the findings of this study relate to the physical aspects of female characters in children's novels which include physical aspects in terms of gender, physical aspects in terms of age, physical aspects in terms of facial characteristics, the physical aspect in terms of the clothes used, and the physical aspect in terms of the state of the body (senses). Key words: physical, representation, children's novel, and children's work Article history Submitted: 5 December 2021 Accepted: 25 March 2022 Published: 30 March 2022 Citation (APA Style): Muhsyanur, M., Suharti, S., & Sudikan, S. Y. (2022). Physical representation of female character in children’s novels by children. Diksi, 30(1), 66-73. https://doi.org/10.21831/diksi.v30i1.45663 Citation (APA Style): DIKSI Vol. 30, No. 1, pp. 66-73 https://journal.uny.ac.id/index.php/diksi DOI: https://doi.org/10.21831/diksi.v30i1.45663 DIKSI Vol. 30, No. 1, pp. 66-73 https://journal.uny.ac.id/index.php/diksi DOI: https://doi.org/10.21831/diksi.v30i1.45663 Physical representation of female character in children’s novels by children Muhsyanur1, Sri Suharti2 & Setya Yuwana Sudikan3 1Institut Agama Islam (IAI) As’adiyah, Indonesia 2Universities Bina Sarana Informatika, Indonesia 3Universitas Negeri Surabaya, Indonesia Corresponding Author; Email: muhsyanur.academic@gmail.com INTRODUCTION Character is a person who is equipped with moral qualities and character which is what the dialogue says and what actions support (Widyahening & Wardhani, 2016) . This is in line with Abrams' opinion (Burhan, 2013) that story characters (characters) are people who are shown in a narrative work, or drama that is liked to have certain moral qualities and tendencies expressed in speech and carried out in the form of action. Purba (2010) states that the characterizations include matters relating to naming, characterization, physical condition of the character (psychological aspect), social condition of the character (sociological aspect), and character of the character. However, the thing that is the main supporter and cannot be separated from the character is something related to psychology. Aderia et al. (2013) and Ibrasma et al. 2013) explains that psychology is a science that investigates and studies behavior and various activities as a manifestation of mental life. This study focuses on the physical aspects of female characters in several children's novels by children. The physical aspect of the female character is the same as the female character's self- image. A woman's self-image is a picture of a woman's world that is typical of herself and all kinds of behavior (Fahs, 2014 & Muhsyanur, 2021). Sugihastuti (2019) and Rokhmansyah (2016) states that women's self-image is a woman's state and view that comes from within herself, which includes physical and psychological aspects. It is also said that these aspects are a unity that has a relationship with one another in the construction of a self-image. Women are physically an individual figure formed by the biological process of a baby girl, who in the course of her age reaches the adult stage (O’Neal et al., 2020) and (Waynforth, 2001). In the physical aspect, it is said that women experience different conditions from men. For example, pregnant, giving birth, and breastfeeding children. Lestari et al. (2021) suggests that physically women have a reproductive role which is then constructed as women's duties and responsibilities. Meanwhile, according to Irfarettha et al. (2013) the image of women seen from the physical aspect is a picture of women shown based on physical or outward characteristics which include age, gender, body condition (five senses), facial features, and clothing. INTRODUCTION The essence of literary works based on content is a representation of all the activities of people's lives. This confirms that the literary work itself was born and created by the community. One of the goals of literary works created is as a form of pouring out the creative process of society itself. Therefore, there are two things that bind the essence of literary works, namely literary works as creative and imaginative works. First, it is said to be a creative work because it requires creative ideas, ranging from topic topics, expressing ideas, to the use of language, such as choosing the right words. Of course it requires creative spaces as well. That is, the process of creating a literary work is not instantaneous. Furthermore, secondly, it is said to be an imaginative work because basically, in the process of creating a literary work, it is not only supported by creative spaces, but also requires maximum empowerment of reasoning. The reasoning in question is the power of a person to interpret and interpret something imaginatively. The two binding processes of the literary creation process mentioned above also become spirits that cannot be separated from a literary work. Based on general observations, literary works reveal more aspects of the author's life experience. Therefore, a novel based on content is a representation of the life of the community or the author himself which is appointed based on factual life, then fictionalized in the work. Thus the novel can be called the book of the journey of human life. Nevertheless, it is still presented using imaginative language as a characteristic that literary works are works of fiction. Therefore, a literary work is also a work of art which contains the creative process of the author. Djojosuroto, (2006) and (Muhsyanur Muhsyanur, 2019) says that the essence of literature/literary work is a form and result of creative art work whose objects are humans and their lives by using language as the medium. 66 DIKSI, Vol. 30, No. 1 DIKSI, Vol. 30, No. 1 The main element in literary works, especially novels, is the character. Characterization in a literary work is an amalgamation of characters and character traits in a story. According to Atmazaki (2005), character is an important component in a story. INTRODUCTION Juanda & Azis (2018) adds that the physical image of women in a literary work is marked by the similarity between the characteristics of women in real situations and the images created by the author in literary works. Based on some of these opinions, the physical aspect of women can be said to be something that is in women, both in terms of reproductive roles, body conditions (five senses), facial features, clothes used, age, and gender that are different from men. If it is associated with the physical image of women in literary works, then the physical aspects of women can be said to be all physical characteristics that have similarities with the characteristics of women in real situations. Therefore, this study focuses on examining the physical description of the female character in the children's literature. The physical depiction in question relates to internal and external. Internal matters, such as voice, tone, expression, and so on. While with regard to the external, among others, such as body style, expression of acting or hanging out, and so on. Basically, children's literary works have advantages compared to other literary works or in general. This is in line with the statement by Apriliya et al., (2020) that children's literature has extraordinary potential. Physical Aspect in terms of Gender The physical aspect in terms of gender is found in five children's novels by children. The author of the novel is a girl, so the depiction of the main character as a girl is described according to what is in her. The following is an excerpt from the data in the novel (1) “Halo teman-teman! Namaku Farah, terima kasih.” Farah adalah gadis cantik dengan balutan jilbab yang menutupi rambutnya (I/LF/16). “Hello guys! My name is Farah, thank you.” Farah is a beautiful girl with a veil covering her hair (I/LF/16). (2) Gadis berambut cokelat pirang itu mengarahkan pandangannya ke sumber suara. Dia melempar seulas senyum sekenanya pada sosok yang kini ada di hadapannya. Lalu kembali merebahkan tubuhnya di sofa dengan malas (II/TWBL/11). The blonde brown haired girl turned her gaze to the source of the sound. He flashed a sly smile at the figure who was now in front of him. Then he lay back down on the couch lazily (II/TWBL/11). (3) Lizabeth adalah temanku. Dia gadis cantik, baik, periang, ramah, dan sangat pintar. Sama sepertiku dia juga punya rambut yang halus dan enak disisir (III/LBC/9). Lizabeth is my friend. She is a beautiful girl, kind, cheerful, friendly, and very smart. Just like me he also has smooth and easy to comb hair (III/LBC/9). (4) Aku duduk di kelas V di SDI permata indah, sekolah khusus perempuan (IV/TPS/13). I am in fifth grade at SDI Permata Indah, an all-girls school (IV/TPS/13). (5) Aku termasuk gadis yang sangat suka menggunakan bandana rambut dengan bunga di tengahnya (VI/RC/38). I'm one of those girls who really like to wear a hair bandana with a flower in the middle (VI/RC/38). Based on the data (1), (2), (3), and (5) the main character is described as a girl. The author's depiction of the main character in the story gives the reader the view that the character is female. In contrast to data (4), the author directly illustrates that the character is female by the actions she takes when she enters a special school for girls. Data (1) describes the main character named Farah as a girl, this statement is evidenced by the description in the narrative by the author regarding Farah's physical condition when introducing herself in front of her friends. Results The physical aspect of women's self-image in children's novels by children, based on the results of the study it was found that the image of women depicted through the main character in terms of gender was found in five children's novels by children, the depiction of the physical aspect in terms of age was found in four novels, the physical aspect in terms of characteristics The face is found in three novels, in terms of clothing it is found in four novels, and the depiction of the physical aspect in terms of body condition is found in five children's novels by children. Following are the results of the analysis based on the data in the novel. METHOD This type of research is a qualitative research using a descriptive approach. The data and sources of research data are in the form of texts related to the physical aspects of female characters contained in several children's novels by children. The source of the data for this research comes from children's novels by children. The data collection technique in this study used a reading technique with a marking system. This is done to produce accuracy in data collection. The data that has been collected is then selected and processed. The processing is based on data that is more in line with the research focus by using a grouping system. The data analysis technique used is a symbolic hermeneutic technique. The symbolic hermeneutic technique in question is interpreting and interpreting the data presented (Harris, 1997). Interpretation is giving the impression of an object (Wilson, 2011 & Klingler, 2021). In relation to this research, it is interpreting the data to answer the research focus. 67 DIKSI, Vol. 30, No. 1 DIKSI, Vol. 30, No. 1 Physical Aspect in terms of Age The physical aspect in terms of age is found in four children's novels by children. Through the depiction of the main character, the child writer describes the female character at a young age or in childhood, namely 11-12 years. The age is in accordance with the conditions experienced by the author, as has been said that child writers tend to describe themselves in story characters. The following is an excerpt from the data in the novel. (1) Viola Agneta Salsabila adalah sahabatku. Anak berumur 12 tahun sama denganku, yang sama-sama gemar bermain musik menggunakan biola, viola, piano, dan beberapa alat tradisional Indonesia seperti angklung dan gamelan (I/LF/83). Viola Agneta Salsabila is my best friend. A 12 year old child like me, who both likes to play music using the violin, viola, piano, and some traditional Indonesian instruments such as angklung and gamelan (I/LF/83). (1) Viola Agneta Salsabila adalah sahabatku. Anak berumur 12 tahun sama denganku, yang sama-sama gemar bermain musik menggunakan biola, viola, piano, dan beberapa alat tradisional Indonesia seperti angklung dan gamelan (I/LF/83). Viola Agneta Salsabila is my best friend. A 12 year old child like me, who both likes to play music using the violin, viola, piano, and some traditional Indonesian instruments such as angklung and gamelan (I/LF/83). (2) Saat ini umurku 11 tahun (IV/TPS/14). I am currently 11 years old (IV/TPS/14). (3) Hai, kenalkan namaku Vexia Reziella dan biasa dipanggil Vexia. Sekarang usiaku dua belas tahun dan sudah duduk di kelas Tab.VII di Chrysan College, sekolah pali terkenal di Chrysan(V/MDV/11). Hi, my name is Vexia Reziella and usually called Vexia. I am now twelve years old and in the Tab.VII class at Chrysan College, the most famous school in Chrysan(V/MDV/11). (4) Sabrina adalah reporter cilik dari kelas lima (VI/RC/37). Sabrina is a little reporter from fifth grade (VI/RC/37). Data (1) illustrates that the age of the main character I or the main character is 12 years. Through the depiction of the age of the main character, it can be concluded that the main character is a young woman. Data (2) confirms that the main character is also 11 years old, that is, the age at which a person is still in the stage of child development (not yet an adult). The author gives a direct description of the main character's physical condition based on age. Physical Aspect in terms of Age Data (3) is not much different from the previous data, the main character in children's novels by children is described as a girl who is 12 years old and sitting in Tab class. VII or equivalent to grade VI SD if in Indonesia. Data (5) describes the main character as a young girl when the author illustrates her as a child who is still in school at the fifth grade of elementary school. In Indonesia, in general, children who are in fifth grade of elementary school are 11-12 years old. Based on these data, it can be concluded that in children's novels, the main character is also described as a child, through the depiction of the age of the main character, namely children aged 11-12 years. However, according to the science of child development, at the age of 11-12 years, children have started the process of finding their identity so that there will be many problems experienced by children. Physical Aspect in terms of Gender Data (2) describing the main character named Elsie as a girl is done by the author through the use of the word girl and narrative descriptions related to the main character's physical characteristics at the beginning of the story. Data (3) describes the main character named Mery as a girl, in addition to using the word girl, also through the character's actions when doing a monologue and describing her condition, starting with the description of her friend's condition. Data (4) describes the main character as a girl in the story, apart from the use of the word girl, it is also 68 DIKSI, Vol. 30, No. 1 DIKSI, Vol. 30, No. 1 DIKSI, Vol. 30, No. 1 seen when the author illustrates that the character goes to an all-girls school. Data (5) describes the main character as a girl through the use of the word girl and is also reinforced by the character's actions when describing the attributes she wears. seen when the author illustrates that the character goes to an all-girls school. Data (5) describes the main character as a girl through the use of the word girl and is also reinforced by the character's actions when describing the attributes she wears. Based on these data, it can be concluded that female writers tend to describe the same gender as themselves, namely the female gender. This is done to get a true depiction of girls. Physical Aspects in terms of Facial Characteristics Physical aspects in terms of facial features are found in three children's novels by children. The facial features depicted through the main character in the novel conclude that the character is a woman. Girls writers emphasize the depiction of female facial features in the story. The depiction of male facial features is almost never depicted in all children's novels by girls. This problem occurs 69 DIKSI, Vol. 30, No. 1 DIKSI, Vol. 30, No. 1 because female writers tend to position male characters as merely complementary characters in the story, not characters who have an important influence in the story. Here are the data in the novel. because female writers tend to position male characters as merely complementary characters in the story, not characters who have an important influence in the story. Here are the data in the novel. (1) Gadis dengan bola matanya yang cokelat dan bulu mata yang lentik itu tertawa kecil ketika mendengar ucapan Bunda (II/TWBL/13). The girl with brown eyes and thick eyelashes laughed lightly when she heard Mother's words (II/TWBL/13). (2) Lizabeth adalah temanku. Dia gadis cantik, baik, periang, ramah, dan sangat pintar. Sama sepertiku dia juga punya rambut yang halus dan enak disisir (III/LBC/9). Lizabeth is my friend. She is a beautiful girl, kind, cheerful, friendly, and very smart. Just like me he also has smooth and easy to comb hair (III/LBC/9). (3) Mataku bulat dan berwarna cokelat. Hidungku tidak terlalu mancung sih, tapi sangat sesuai dengan bentuk wajahku cantik kata banyak orang (V/MDV/11). (3) Mataku bulat dan berwarna cokelat. Hidungku tidak terlalu mancung sih, tapi sangat sesuai dengan bentuk wajahku cantik kata banyak orang (V/MDV/11). My eyes are round and brown. My nose is not too high, but it fits my face shape, many people say (V/MDV/11). Data (1) describes the facial condition of the main character, the author describes the main character as a girl with facial features that have brown eyes and curly eyelashes. In data (2) as well, the author describes the main female character with beautiful facial features and has smooth hair and is easy to comb. Data (3) describes the facial characteristics of the main character, namely round brown eyes, a proportional nose and a beautiful face. The depiction of facial features by female writers in the novel, proves that the author is very interested in physically beautiful and beautiful women. Physical Aspects in terms of Facial Characteristics Based on these data, it can be concluded that female writers will provide characteristics according to their understanding of women. The female characters are always depicted as beautiful and near perfect in the story. Girls writers strive to make girls more prominent in the story than boys. Physical Aspects in terms of Body Conditions (senses) Physical aspects in terms of body condition (pancaindra) are found in five children's novels by children. The female characters described by the author are also shown through the behavior of their bodies. The following is an excerpt from the data in the novel. Physical aspects in terms of body condition (pancaindra) are found in five children's novels by children. The female characters described by the author are also shown through the behavior of their bodies. The following is an excerpt from the data in the novel. (1) Langkah Farah dan temannya terdengar ramai saat menuju kantin. Suara sepatu Farah yang sudah usang beradu dengan sepatu lincah dan mengkilap milik perempuan di sebelahnya (I/LF/20). The steps of Farah and her friends sounded crowded when they headed to the canteen. The sound of Farah's worn-out shoes colliding with the bright and shiny shoes of the woman next to her (I/LF/20). (2) Elsie mengukir senyumnya. Lalu mengangguk-anggukkan kepala seraya memasang wajah memelas (II/TWBL/32). Elsie cracked a smile. Then nodded his head while putting on a pitiful face (II/TWBL/32). (3) Elsie mengacungkan jempol tanda mengerti (II/TWBL/57). Elsie gives a thumbs up sign of understanding (II/TWBL/57). (4) Aku berjalan dengan langkah gontai sampai di luar (IV/TPS/45). I walked with unsteady steps until outside (IV/TPS/45). (5) Aku melambaikan tangan (V/MDV/19). I waved (V/MDV/19). (5) Aku melambaikan tangan (V/MDV/19). I waved (V/MDV/19). Data (1) describes the steps of the main character named Farah as a woman while walking, the child is described as a female character who walks with fast steps. In data (2), it can be seen how the main female child character uses head movements as a signal for the actions to be taken. The depiction of the character's actions suggests that the female character doesn't talk much, so she chooses to use body language. Data (3) describes how the female character uses her thumb to express an attitude of understanding or understanding, the actions taken by the main female character also indicate that the female character does not like to say a lot of words so she chooses to use sign language through her limbs. Data (4) describes the body condition of the main female character who always walks slowly. In general, these actions are always done by women to reflect the feminine side. Data (5) describes the main female character who always waves as a sign of meeting or parting with other people. Physical Aspect in terms of Clothing Used The physical aspect of the clothes used are found in four children's novels by children. The author describes the main character as a girl also through the depiction of the clothes worn by the characters. The clothes that are generally used by girls are described by the author in the novel. The following is an excerpt from the data in the novel. (1) Aku mandi dan mengenakan baju seragam merah putih berlengan panjang dan mengenakan jilbab putih (I/LF/76). I took a shower and wore a long-sleeved red and white uniform and wore a white headscarf (I/LF/76). (2) Baju berwarna merah muda dengan ribuan glitters yang berkilauan. Itu gaun favoritku, aku langsung menyambar baju itu dan berganti baju. Saat keluar, aku berkaca di cermin. Gaun itu tampak indah di tubuhku (III/LBC/28). A pink dress with thousands of glittering glitters. That's my favorite dress, I immediately grabbed it and changed. When I came out, I looked in the mirror. The dress looks beautiful on me (III/LBC/28). (3) Ketika aku dan Diva berjalan kaki untuk sampai ke sekolah, aku dan Diva terkena cipratan air kubangan! Rokku dan Diva pun basah dan kotor (IV/TPS/43). When Diva and I walked to school, Diva and I were splashed by puddles! My and Diva's skirts were wet and dirty (IV/TPS/43). (3) Ketika aku dan Diva berjalan kaki untuk sampai ke sekolah, aku dan Diva terkena cipratan air kubangan! Rokku dan Diva pun basah dan kotor (IV/TPS/43). When Diva and I walked to school, Diva and I were splashed by puddles! My and Diva's skirts were wet and dirty (IV/TPS/43). 70 DIKSI, Vol. 30, No. 1 DIKSI, Vol. 30, No. 1 Data (1) describes the main character as a child who wears a long-sleeved shirt and a headscarf which is identical to women's clothing in general. The use of hijab is usually used by all Muslim women, both children and adults. Data (2) also describes how the main character is a girl who wears pink clothes with glittering glitters. The depiction illustrated by the author regarding the clothes of the female characters is not much different from the real situation. Where girls prefer pink and something shiny, to emphasize their feminine identity. Likewise with data (3) when it is described that the main character wears a skirt to school, in real life skirts are only worn by female students while boys do not. Physical Aspect in terms of Clothing Used y Based on these data, it can be concluded that children's writers also provide an overview of female characters through the clothes used, not only from their actions and facial features. The characters in the novel are described in their entirety to get a complete depiction of women. Physical Aspects in terms of Body Conditions (senses) This body condition is usually also owned by men, but in children's novels, these actions are more dominantly carried out by women, especially the main character. Women have a special physical character (Prismanisa, 2021). Based on these data, it can be concluded that the main female character is fully described by the author. The female character is also depicted through the state of her limbs as a sign in carrying out an action and behavior. 71 DIKSI, Vol. 30, No. 1 DIKSI, Vol. 30, No. 1 CONCLUSIONS Literary works have an important role in various aspects of human life for all people. 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https://openalex.org/W3122373505	https://revistas.ucm.es/index.php/RECI/article/download/66326/4564456554398	Spanish; Castilian	null	The Jake Mandell Test as a measure of individual differences in pitch discrimination: validity and reliability properties.	Revista electrónica complutense de investigación en educación musical	2,020	cc-by	7,211	Recibido: 4 de diciembre de 2019 / Aceptado: 27 de Abril de 2020 Resumen. El test de Jake Mandell (JMT) es un test online diseñado para evaluar la discriminación tonal en la población normal. El test está disponible online desde 2006, pero no se han publicado datos psicométricos. La presente investigación fue diseñada para proporcionar datos de validez y fiabilidad para este test. El estudio 1 se realizó en estudiantes universitarios, y el rendimiento en el JMT se comparó con las pruebas de detección online de AMUSIA. Las correlaciones fueron altas con los subtests de AMUSIA, pero especialmente con el de discriminación de tonos. El estudio 2 se realizó en niños, y el JMT se comparó con diferentes pruebas de habilidades musicales. El JMT mostró una buena relación con el subtest de discriminación de tono e imitación de ritmo, pero especialmente con la puntuación global de las habilidades musicales. Además, el test mostró una buena fiabilidad test-retest al cabo de un año. Finalmente, la validación externa del JMT se obtuvo al señalar que los músicos obtuvieron puntuaciones más altas que los no músicos. La discusión se centra en la posibilidad de utilizar el JMT como una medida de detección rápida de las diferencias individuales en la discriminación de tono en la población normal. ras clave: Discriminación de tono; Test de Jake Mandell (JMT); habilidades musicales; neuroimagen; cereb El test Jake Mandell como instrumento para medir las diferencias individuales en la discriminación tonal: propiedades de validez y fiabilidad1 María-Ángeles Palomar-García2, Gustau Olcina-Sempere3, Mireia Hernández4, María-Antonia Parcet5, Jacob C. Mandell6, César Ávila7 1 Este trabajo ha recibido financiación por parte del Ministerio de Economía y Competitividad de España (PSI-2016-78805-R) a C. Ávila. Además, este trabajo fue posible por la subvención del programa de posgrado postdoctoral (postdoc-UJI a M-Á. Palomar- García) y el Programa de Investi gación Ramón y Cajal del Ministerio de Ciencia, Innovación y Universidades de España (RYC-2016- 19477 a M. Hernández. 2 Universitat Jaume I TRABAJOS BILINGÜES Revista Electrónica Complutense de Investigación en Educación Musical ISSN-e 1698-7454 http://dx.doi.org/10.5209/reciem.66326 Revista Electrónica Complutense de Investigación en Educación Musical ISSN-e 1698-7454 http://dx.doi.org/10.5209/reciem.66326 Revista Electrónica Complutense de Investigación en Educación Musical ISSN-e 1698-7454 http://dx.doi.org/10.5209/reciem.66326 1. Introducción El diagnóstico de amusia requiere una puntuación baja en las tres tareas de discri minación de tono, mientras que se conservan las habilidades en la percepción del ritmo (Vuvan, Paquette, Goulet, Royal, Felezeu y Peretz, 2018). El MBEA ha demostrado su capacidad para detectar amusia congénita en estudios con poblaciones de individuos con lesiones cerebrales y con poblaciones sanas (Ayotte y cols., 2002; Peretz y cols., 2002). Las propiedades psicométricas del MBEA fueron aceptables. La fiabilidad test-retest a los 4 meses fue de 0,75, y la validez concurrente fue buena cuando se correlacionó con las puntuaciones en los tests de Aptitud Musical de Gordon [r = 0.53; (Peretz y cols., 2002)]. Sin embargo, las diversas pruebas del MBEA fueron fáciles para la mayoría de la población, ya que la distribución de las puntuaciones fue asimétrica, con una tendencia hacia puntuaciones altas (Peretz, Champod y Hyde, 2003). El Jake Mandell Tone Deaf Test (JMT) es otro test creado en 2006 que se ha centrado en medir la capacidad para la discriminación tonal, realizado por Jake Mandell y que ha estado disponible online durante más de 10 años. Ini cialmente se desarrolló para evaluar la sordera de tono (amusia congénita), pero al mismo tiempo fue diseñado para ser un desafio, incluso para sujetos con entrenamiento musical. Este nivel de dificultad se logró utilizando melodías complejas y más largas con múltiples timbres musicales donde una sola nota es la que cambia. Por lo tanto, el test fue diseñado para reducir cualquier sesgo potencial debido al entrenamiento específico del instrumento musical y evitar la agrupación de puntuaciones altas por parte de individuos entrenados. En la página web se presentan datos de 61.036 participantes que se han recogido de manera online y que muestran que este instrumento puede ser útil para evaluar la capacidad de percepción tonal. La distribución de las puntuaciones presenta una curva normal, con un porcentaje medio del 73,8% (DE = 9,99), donde muy pocos encuestados alcanzan puntuaciones superiores al 90%, lo que hace que el JMT sea un test interesante para medir las habilidades musicales. La presencia de melodías complejas y más largas aumenta los requisitos de la memoria tonal (Gosselin, Jolicoeur y Peretz, 2009; Tillmann, Schulze y Foxton, 2009). Este proceso se encuentra alterado en la amusia congénita y se ha relacionado con la corteza auditiva y las áreas frontales operculares (Albouy y cols., 2013). 1. Introducción La amusia congénita (también conocida como sordera del tono) es la falta de capacidad para reconocer diferencias re lativas en frecuencia que no pueden explicarse por pérdida auditiva, daño cerebral o un déficit cognitivo (Ayotte, Pe retz y Hude, 2002; Peretz y Hyde, 2003; Sihvonen, Sarkamo, Rodríguez-Fornells, Ripollés, Munte y Soinila, 2019). Esta presencia permanente de insensibilidad al tono musical es la característica principal de la amusia congénita, que también requiere preservar la capacidad de discriminar diferentes ritmos. Se estima que la amusia congénita está pre sente en aproximadamente el 4% de la población (Peretz y Vuvan, 2017). Los estudios de neuroimagen han asociado este trastorno con modificaciones en el volumen de sustancia gris en el giro temporal superior y/o en el giro frontal inferior, interpretada como una alteración en los sistemas cerebrales relacionados con el procesamiento auditivo y la memoria (Albouy y cols., 2013; Sihvonen, Ripollés, Leo, Rodríguez-Fornells, Soinila y Särkämö, 2016). Además, la amusia congénita está relacionada con una conectividad anormal en la red frontal-temporal, que se configura como un síndrome de desconexión. La evaluación de la sordera de tono y el diagnóstico de amusia congénita se han llevado a cabo principalmente con la Batería de Montreal para la Evaluación de Amusia (MBEA; 2). El desarrollo de esta batería se basó en el modelo cognitivo-neuropsicológico desarrollado por Pérez y Coltheart (Peretz y Hyde, 2003). Este modelo predice la existencia de módulos auditivos separados para el procesamiento del lenguaje y la música. El módulo para el pro cesamiento musical está formado por dos sistemas independientes y paralelos, uno para el procesamiento melódico (más relacionado con la discriminación de tono) y otro para la organización temporal (más asociado con el proce samiento del ritmo). Estos dos sistemas fueron descritos basándose en los datos obtenidos en pacientes con lesiones en el giro temporal superior que mostraron una alteración de la discriminación tonal en presencia de una capacidad preservada para las variaciones en el ritmo (Peretz, Kolinsky, Tramo, Labrecque, Hublet, Demeurisse y Belleville, 1994; Piccirilli, Sciarma y Luzzi, 2000). El MBEA se compone de seis pruebas diferentes, tres dedicadas a diferentes aspectos de la discriminación de tono, una para la percepción del ritmo, una para discriminar si la melodía era una marcha o un vals, y la última para el reconocimiento de la melodía. [en] The Jake Mandell Test as a Measure of Individual Differences in Pitch Discrimination: Validity and Reliability Properties. 5. Discusión. 6. Conclusiones. 7. Referencias bibliográficas. Sumario. 1. Introducción. 2. Estudio 1. 2.1 Materiales y métodos. 2.1.1. Participantes. 2.1.2. Materiales. 2.2 Resultados. 2.3. Análisis cualitativos. 3. Estudio 2. 3.1. Materiales y métodos. 3.1.1. Participantes. 3.1.2. Materiales. 3.2. Resultados. 4. Estudio 3. 4.1. Materiales y métodos. 4.1.1. Participantes. 4.1.2. Materiales. 4.2. Resultados. 5. Discusión. 6. Conclusiones. 7. Referencias bibliográficas. Cómo citar: Palomar-García, M. A.; Olcina-Sempere, G.; Hernández, M.; Parcet, M. A.; Mandell, J. C. y Ávila, C. (2020). El test Jake Mandell como instrumento para medir las diferencias individuales en la discriminación tonal: propiedades de validez y fiabilidad. Revista Electrónica Complutense de Investigación en Educación Musical, 17, 133-141. [en] The Jake Mandell Test as a Measure of Individual Differences in Pitch Discrimination: Validity and Reliability Properties. [en] The Jake Mandell Test as a Measure of Individual Differences in Pitch Discrimination: Validity and Reliability Properties. Abstract. The Jake Mandell Tone Deaf Test (JMT) is an online measure designed to evaluate pitch discrimination in the normal population. The test has been available online since 2006, but no psychometric data have been published. The present research was designed to provide validity and reliability data for this test. Study 1 was conducted in university students, and the performance on the JMT was compared to the AMUSIA online screening tests. Correlations were high with the subtests of the AMUSIA online screening tests, but especially with the Tone discrimination test. Study 2 was conducted in children, and the JMT was compared to different tests of musical abilities. The JMT showed a good relationship with the Tone Discrimination and Rhythm Imitation subtest, but especially with the global score of musical abilities. In addition, the test showed good one-year test-retest reliability. Finally, external validation of the JMT was obtained by noting that musicians obtained higher scores than non-musicians. Discussion is focused on the possibility of using the JMT as a rapid screening measure of individual differences in pitch discrimination in the normal population. K d Pit h di i i ti J k M d ll T D f T t (JMT) i l biliti i i b i eywords: Pitch discrimination; Jake Mandell Tone Deaf Test (JMT); musical abilities; neuroimaging; brain. Rev. electrón. complut. inves. educ. music. 17, 2020: 133-141 1 Este trabajo ha recibido financiación por parte del Ministerio de Economía y Competitividad de España (PSI-2016-78805-R) a C. Ávila. Ade este trabajo fue posible por la subvención del programa de posgrado postdoctoral (postdoc-UJI a M-Á. Palomar- García) y el Programa de In gación Ramón y Cajal del Ministerio de Ciencia, Innovación y Universidades de España (RYC-2016- 19477 a M. Hernández. 2 Universitat Jaume I mpalomar@uji.es 3 Universitat Jaume I golcina@uji.es 4 Universitat de Barcelona mireiahp@gmail.com 5 Universitat Jaume I parcet@psb.uji.es 6 Brigham and Women’s Hospital jmandell@partners.org 7 Universitat Jaume I avila@psb.uji.es 133 Palomar-García M. A. et alii. Rev. electrón. complut. inves. educ. music. 17, 2020: 133-141 134 Sumario. 1. Introducción. 2. Estudio 1. 2.1 Materiales y métodos. 2.1.1. Participantes. 2.1.2. Materiales. 2.2 Resultados. 2.3. Análisis cualitativos. 3. Estudio 2. 3.1. Materiales y métodos. 3.1.1. Participantes. 3.1.2. Materiales. 3.2. Resultados. 4. Estudio 3. 4.1. Materiales y métodos. 4.1.1. Participantes. 4.1.2. Materiales. 4.2. Resultados. 1. Introducción Por lo tanto, la literatura previa señala que tanto la percep ción del tono como la memoria tonal comparten una red cerebral común en áreas frontotemporales a nivel bilateral. 135 Palomar-García M. A. et alii. Rev. electrón. complut. inves. educ. music. 17, 2020: 133-141 La contribución del presente estudio es proporcionar datos de fiabilidad y validez concurrente para el JMT, con el fin de demostrar su viabilidad como una medida para conocer las diferencias individuales en la discriminación de tono y memoria tonal. Los datos online obtenidos para el test sugieren que el uso de estímulos complejos hace que el test sea más difícil que otras pruebas tonales, pero ningún estudio ha investigado esta posibilidad. Por lo tanto, nuestros objetivos específicos son: 1) estudiar la consistencia interna y la estabilidad de JMT; 2) comparar el JMT con otros tests que miden habilidades musicales; y 3) mostrar el redimiento en el JMT en participantes con entrenamiento musical. La elaboración de este instrumento permitirá disponer de una medida rápida y psicométricamente fiable para la evaluación de las capacidades de discriminación tonal que podría ser útil en el ámbito educativo y en la evaluación de las capacidades musicales en el ámbito clínico. Se realizaron tres estudios diferentes. El primer estudio se realizó con estudiantes universitarios y se diseñó para investigar la validez concurrente con el test online de AMUSIA (versión en línea; Peretz y Vuvan, 2017; Vuvan y cols., 2018). Esta herramienta de detección se compone de tres pruebas: la prueba de escala, la prueba de ritmo y la prueba de tono; que miden la discriminación tonal, la sordera basada en el tiempo y la detección de notas fuera de tono, respectivamente. En comparación con las pruebas de detección online de AMUSIA, esperábamos encontrar que el JMT presentara: i) correlación con las pruebas de detección online de AMUSIA (especialmente con la prueba de escala, que es muy similar), ii) una distribución más simétrica de las puntuaciones, y iii) puntuaciones más bajas debido a su mayor dificultad. El estudio 2 se realizó en niños y se hizo con la intención de obtener una distribución similar a la de los adultos y correlaciones específicas con las pruebas de discriminación de tono. Finalmente, en el ter cer estudio comparamos el rendimiento del JMT en músicos y no músicos. 2.1.1. Participantes Nuestra muestra estuvo compuesta por 61 estudiantes universitarios (34 mujeres). La edad de los participantes osciló entre 18 y 38 años (media 22.41; DE = 3.64). Ninguno de los participantes había tocado nunca un instrumento musi cal, y no habían recibido enseñanzas musicales más allá de la educación escolar normal. Se obtuvo el consentimiento informado por escrito de todos los participantes, siguiendo un protocolo aprobado por la Universidad Jaume I, y recibieron una compensación económica. 1. Introducción Esperábamos encontrar una distribución simétrica de las puntuaciones en ambos grupos, pero puntuaciones más altas en la prueba para el grupo de expertos. 1. Jake Mandell Tone Deaf Test (JMT) Este test consta de 36 ensayos basados en frases musicales complejas que utilizan diferentes sonoridades, utilizando instrumentos como el órgano, el piano, la percusión o la cuerda. Estas frases también fueron heterogéneas en dife rentes características, como la duración, la cantidad de tonos, la cantidad de sonidos cortos y largos, los cambios en la intensidad y el uso de sonidos sintetizados o naturales. Cada frase combina una variedad de timbres y estilos musicales, y sus dos presentaciones seguidas comparten el mismo contorno melódico, ritmo y timbre. La mitad de los pares (18/36) difieren solamente en el tono de una nota, con 9/18 de las diferentes notas que están fuera de la escala de la melodía y 9/18 propias de la escala. La diferencia de tono de una nota modificada de la frase inicial y repetida puede variar hasta 11 semitonos, pero no se utilizaron variaciones mayores de una octava. Estos cambios siempre ocurrieron en uno de los últimos 10 tonos de la melodía. En cada ensayo, el participante escucha dos melodías sucesivas cortas y tenía que indicar si eran iguales (botón verde “igual”) o diferentes (botón rojo “diferente”). En la mitad de los ensayos los pares de melodías eran iguales y en la otra mitad presentaban diferencias en uno o más tonos. Después de recibir las instrucciones, se ajustaba el volumen, y se presentaban cuatro ensayos de prueba. A continuación, aparecían en el mismo orden 36 melodías em parejadas para todos los participantes, sin que las melodías se presentaran en un orden de dificultad creciente. Este test se encuentra disponible en http://jakemandell.com/tonedeaf. Usamos el porcentaje de respuestas correctas como medida de la capacidad de percepción tonal. Se consideró un posible déficit en la discriminación tonal cuando las puntuaciones eran inferiores al 55%. 2. Pruebas de detección online de AMUSIA (Peretz y Vuvan, 2017; Vuvan y cols., 2018) 2. Pruebas de detección online de AMUSIA (Peretz y Vuvan, 2017; Vuvan y cols., 2018) Esta prueba se realiza con un ordenador a través de Internet (http://www.brams.org/amusia-public/) e incluye diferen tes subtests: el de escala extraído de la batería de evaluación de Montreal de Amusia (MBEA; Peretz y cols., 2002), y los dos subtests basados en las escalas MBEA: ritmo y tono (Peretz y cols., 2008). 136 Palomar-García M. A. et alii. Rev. electrón. complut. inves. educ. music. 17, 2020: 133-141 En el subtest de escala, los participantes realizaron 31ensayos. En cada ensayo se presentan dos melodías su cesivas cortas, y los participantes deben indicar si las melodías son iguales o diferentes haciendo clic en el botón “diferente” o “igual”. Las melodías que son diferentes tienen un tono que cambia. El subtest de ritmo contiene 24 ensayos que consisten en escuchar una melodía corta. Los sujetos deben identificar si aparece o no un retraso inusual en la melodía: si es así, hacen clic en “incorrecto”; si no es así, hacen clic en “correcto”. Este retraso es un silencio de 5/7 de la duración del tiempo (es decir, 357 ms) antes de la pulsación coincidente. El subtest de tono contiene 24 ensayos que consisten en escuchar una melodía corta e identificar si contiene una nota que está fuera de la escala de la melodía haciendo clic en “incongruente” o “correcto”. Todos los participantes realizan los subtests en el mismo orden (escala, ritmo y tono). Antes de comenzar, cada test se explicó verbalmente a los participantes, quienes realizaron dos o cuatro ensayos de práctica y ajustaron el volumen de los auriculares para escuchar claramente las melodías. En cada subtest, la mitad de los ensayos pertenecen a una condición y la otra mitad a la otra condición. Los ensayos se presentaban en un orden aleatorio. p En nuestro análisis utilizamos los porcentajes de las respuestas correctas como las puntuaciones de cada prueba. En base a estas puntuaciones, se consideró el posible déficit en la discriminación del tono (es decir, amusia) para las puntuaciones por debajo del 70%, en escala y tono, y una puntuación por encima del 70% en la prueba de ritmo (Vuvan y cols., 2018). 2.2. Resultados Los resultados del porcentaje de respuestas correctas, las desviaciones estándar y el rango para cada prueba pueden encontrarse en la Tabla 1. Se realizó una prueba t para muestras pareadas con el objetivo de investigar la diferencia entre las cuatro pruebas. Los resultados mostraron que el JMT fue más difícil que las tres pruebas de detección online de AMUSIA. No hubo diferencias de género en ninguna de las pruebas (P> 0,10). Las correlaciones de Pearson con la edad no fueron significativas (P> 0,10). Tabla 1. Porcentaje medio de respuestas correctas, desviaciones estándar y rango para las pruebas de detección online de AMUSIA y JMT, así como pruebas t pareadas entre ellas. Test Media (SD) Rango Pruebas t-pareadas 1. JMT 65.43 (8.70) 47-86.1 1 < 2; t (59) =8.66, p < .001 1 < 3; t (59) =8.25, p < .001 1 < 4; t (59) =3.16, p = .002 2. Escala 76.93 (10.53) 57-100 2 > 4; t (59) =3.01, p = .003 3. Ritmo 76.02 (8.85) 54-92 3 > 4; t (59) =2.32, p = .02 4. Tono 71.58 (15.05) 46-96 Tabla 1. Porcentaje medio de respuestas correctas, desviaciones estándar y rango para las pruebas de detección online de AMUSIA y JMT, así como pruebas t pareadas entre ellas. Tabla 1. Porcentaje medio de respuestas correctas, desviaciones estándar y rango para las pruebas de detección online de AMUSIA y JMT, así como pruebas t pareadas entre ellas. La consistencia interna del JMT se midió utilizando el coeficiente de fiabilidad ώH de McDonald ya que es mejor que el coeficiente alfa de Cronbach (Zinbarg, Revelle, Yovel y Li, 2005). El coeficiente de fiabilidad ώH describe la varianza de la puntuación total que puede atribuirse al factor general (Hermsen, Leone, Smalbrugge, Knol, van der Horst y Dekker, 2013). El coeficiente ώH obtenido para la presente muestra fue de 0,91, que debería interpretarse como un soporte para la unidimensionalidad de la medida. También ejecutamos correlaciones de Pearson entre las diferentes pruebas (Tabla 2). Como se esperaba, el JMT correlacionó significativamente con la prueba de escala en el MBEA, así como con las otras dos pruebas de MBEA. as correlaciones de Pearson entre las puntuaciones obtenidas en el JMT y las pruebas online de AMUSIA Table 2. Las correlaciones de Pearson entre las puntuaciones obtenidas en el JMT y las pruebas online de AMUSIA Table 2. 3.1.2. Materiales Los participantes completaron el JMT más otras pruebas desarrolladas para evaluar específicamente cinco aptitudes musicales diferentes: discriminación tonal, memoria rítmica, memoria tonal, imitación rítmica e imitación melódica. Estas aptitudes se inspiraron en la Prueba de Aptitud Musical de Bentley (Young, 1973), pero decidimos usar formas más cortas que las pruebas de Bentley por dos razones: 1) estas versiones más cortas se usan con frecuencia para seleccionar a los niños que empezarán los estudios musicales en los conservatorios de España; y 2) los participantes formaron parte en un estudio de neuroimagen más global, y no tuvimos tiempo suficiente para usar pruebas más lar gas. Los ítems de cada una de las pruebas fueron grabados, interpretados, administrados y evaluados por un músico profesional. Se puede solicitar información más detallada a los autores. 1. La prueba de discriminación tonal requiere que los participantes escuchen 10 ítems con dos notas cada uno in terpretados con una flauta. En 5 ítems, el segundo sonido es ascendente, mientras que, en los otros 5, el segundo sonido es descendente. En esta tarea, el niño debe reconocer si la segunda nota sube o baja en relación con la primera. La puntuación para esta prueba fue la suma de todas las respuestas correctas (puntuación máxima = 10). 2. La prueba de memoria rítmica consta de 10 ritmos musicales diferentes con cuatro pulsaciones. Los participantes escucharon dos repeticiones consecutivas de cada uno de estos ritmos. En el 50% de los ritmos, las pulsaciones en la primera y segunda repetición de ritmo fueron los mismos, mientras que una pulsación se modificó en el otro 50%. Por lo tanto, los participantes tenían que indicar si las pulsaciones del segundo ritmo eran iguales o difer entes respecto del primero. Si el participante notó algún cambio, se le pidió que identificara qué pulsación había cambiado. La puntuación para esta prueba fue la suma de todas las respuestas correctas (puntuación máxima = 10). ) 3. La prueba de memoria tonal consta de 10 melodías musicales diferentes con cinco ritmos cada una. Los partici pantes escucharon dos repeticiones consecutivas de cada una de estas melodías. Los investigadores alteraron una nota en 5 de las 10 melodías musicales originales en la segunda repetición. Por lo tanto, los participantes tenían que identificar si cada melodía era igual o diferente de la primera. 2.2. Resultados Las correlaciones de Pearson entre las puntuaciones obtenidas en el JMT y las pruebas online de AMUSIA Escala Ritmo Tono 1. Jake Mandell Test .44 .33 .38** 2. Escala .28* .51** 3. Fuera de ritmo .35* 4. Fuera de tono *P <0.01; P <0.05 Palomar-García M. A. et alii. Rev. electrón. complut. inves. educ. music. 17, 2020: 133-141 137 3.1.1. Participantes Nuestra muestra estaba compuesta por 33 niños en edad escolar provenientes de diversas escuelas de la ciudad de Castellón sin formación musical previa (más allá de la obligatoria contemplada en los estudios reglados de la edu cación primaria) que estaban interesados en acceder a estudios formales de música en el conservatorio. El grupo estaba compuesto por 15 niños y 18 niñas. La edad de los participantes varió de 6 a 12 años (M = 8’47; DE = 1’50). Los participantes nunca habían tocado un instrumento musical y no habían recibido formación musical más allá de la educación escolar. Los padres dieron su consentimiento por escrito para que sus hijos participaran en este estudio, siguiendo un protocolo aprobado por la Universidad Jaume I, y recibieron una compensación económica por su participación. 2.3. Análisis cualitativos Según los criterios de JMT, se puede considerar amusia cuando la precisión en la prueba es inferior al 55%, y el 11,4% de nuestra muestra cumplió con este criterio. Del mismo modo, las pruebas de detección online de AMUSIA requirieron puntuaciones de menos del 70% en las pruebas de escala y tono y puntuaciones mayores del 70% en la prueba ritmo, y el 11.5% de nuestra muestra cumplió con este criterio. El acuerdo entre las dos medidas alcanzó el 85.2% (Kappa de Cohen = 2.48, p = .013), con 3 casos de 7 diagnosticados como amúsicos por ambas medidas. 3. Estudio 2 3.1.2. Materiales La puntuación para esta prueba fue la suma de todas las respuestas correctas (puntuación máxima = 10). 4. La prueba de imitación rítmica consta de 5 ritmos diferentes de aplausos. Primero, el examinador mostraba a los participantes cómo realizar un ritmo de aplausos sin darles una guía visual. Luego, los participantes escucharon los 5 ritmos de aplausos (sin guía visual) y tuvieron que imitarlos. La puntuación para esta prueba fue la suma de todas las respuestas correctas (puntuación máxima = 10). 5. La prueba de imitación melódica se compone de 5 frases musicales diferentes. El examinador canta cada una de las 5 frases musicales con la sílaba “la”, y los niños tienen que volver a cantar las melodías. La puntuación para esta prueba fue la suma de todas las respuestas correctas (puntuación máxima = 10). 5. La prueba de imitación melódica se compone de 5 frases musicales diferentes. El examinador canta cada una de las 5 frases musicales con la sílaba “la”, y los niños tienen que volver a cantar las melodías. La puntuación para esta prueba fue la suma de todas las respuestas correctas (puntuación máxima = 10). 6. Puntuación global. Calculamos una puntuación global para las habilidades musicales utilizando puntuaciones Z estándar (media = 0; DE = 1) obtenidas de las puntuaciones totales en cada una de las pruebas anteriores. 6. Puntuación global. Calculamos una puntuación global para las habilidades musicales utilizando puntuaciones Z estándar (media = 0; DE = 1) obtenidas de las puntuaciones totales en cada una de las pruebas anteriores. Palomar-García M. A. et alii. Rev. electrón. complut. inves. educ. music. 17, 2020: 133-141 138 3.2. Resultados Las medias y las desviaciones estándar para todas las pruebas se presentan en la Tabla 3. Las puntuaciones de JMT fueron menores en los niños que las obtenidas por los adultos en el estudio 1 (diferencia = 4.07), pero la diferencia no fue significativa (t = 1.91; P = 0.058). Las puntuaciones en el JMT siguieron una distribución normal (prueba K-S Z = 0.73, P> 0.10). Las correlaciones parciales (controlando por la edad) entre las diferentes medidas se presentan en la Tabla 3. Como se esperaba, el JMT correlacionaba con las otras variables, especialmente con las pruebas de discrimi nación tonal y discriminación de ritmo. Es importante destacar que la correlación parcial del JMT con la puntuación global en las pruebas musicales fue muy significativa. Tabla 3. Correlaciones parciales entre las puntuaciones obtenidas en el JMT y las diferentes pruebas musicales, así como la media de las respuestas correctas, las desviaciones estándar, el rango y las correlaciones de Pearson con la edad JMT Discrimina ción Tonal Memoria Rítmica Memoria Tonal Imitación Rítmica Imitación Melódica JMT 1 Test Discriminación Tonal .38* 1 Test Memoria Rítmica .33 .45* 1 Test Memoria Tonal .24 .27 .40* 1 Test Imitación Rítmica .40* .21 .44* .51 1 Test Imitación Melódica .30 .18 .29 .20 .30 1 Puntuación Total .56 Media (SD) 61.36 (9.3) 8.32 (1.70) 8.59 (1.40) 8.71 (1.19) 6.35 (3.25) 2.94 (2.44) Rango 38-86 3-10 6-10 6-10 0-10 0-10 Correlación de Pearson con la edad .28 .42* .22 .26 .18 .28 P <0.05; *P <0.01 La fiabilidad test-retest al cabo de un año se calculó con toda la muestra. Los resultados mostraron que las pun t i j d é d ñ (M 66 25 SD 9 73) l fi i t d l ió i t l (ICC) t Tabla 3. Correlaciones parciales entre las puntuaciones obtenidas en el JMT y las diferentes pruebas m como la media de las respuestas correctas, las desviaciones estándar, el rango y las correlaciones de Pea Tabla 3. 4.1. Materiales y métodos 4.1. Materiales y métodos 4.1.2. Materiales Los participantes completaron el JMT (ver Estudio 1 para los detalles metodológicos). 4.1.1. Participantes La muestra estuvo compuesta por 61 participantes, 33 músicos y 28 no músicos que respondieron a anuncios para participar en un estudio de neuroimagen. Los músicos habían completado estudios formales de música (conservato rio, escuelas privadas) durante al menos 9 años, y eran músicos activos (11 mujeres; edad media = 20.09 años; DE = 2.01; rango: 18-26 años). Los no músicos solo habían recibido instrucción musical obligatoria en la escuela (12 mujeres; edad media = 20.68 años; DE = 2.21; rango: 18-27 años). Los dos grupos no diferían en la distribución por edad o género. Se obtuvo el consentimiento informado por escrito de todos los participantes, siguiendo un protocolo aprobado por la Universidad Jaume I, y recibieron una compensación económica. 4.2. Resultados El porcentaje medio de respuestas correctas y las desviaciones estándar de ambos grupos se presentan en la Tabla 4. Realizamos un ANOVA utilizando el JMT como variable dependiente, género y grupo como factores entre sujetos, y la edad como covariable. Solamente se encontró un efecto significativo para el grupo [F (1, 36) = 12.68, P = .002, η2 = .40). Como se esperaba, los músicos obtuvieron mejores puntuaciones que los no músicos. Tabla 4. Media de las respuestas correctas y desviaciones estándar en el JMT en los grupos de músicos y no músicos Grupo N Media DE Error típico de la media No-músicos 28 67.85 10.43 1.97 Músicos 33 76.84 9.43 1.64 Tabla 4. Media de las respuestas correctas y desviaciones estándar en el JMT en los grupos de músico a de las respuestas correctas y desviaciones estándar en el JMT en los grupos de músicos y no músicos 3.2. Resultados Correlaciones parciales entre las puntuaciones obtenidas en el JMT y las diferentes pruebas musicales, así como la media de las respuestas correctas, las desviaciones estándar, el rango y las correlaciones de Pearson con la eda elaciones parciales entre las puntuaciones obtenidas en el JMT y las diferentes pruebas musicales, así e las respuestas correctas, las desviaciones estándar, el rango y las correlaciones de Pearson con la edad La fiabilidad test-retest al cabo de un año se calculó con toda la muestra. Los resultados mostraron que las pun tuaciones mejoraron después de un año (M = 66.25; SD = 9.73), y el coeficiente de correlación intraclase (ICC) entre las dos medidas fue alto [ICC = 0.59 (IC 95% 0.172-0.80); ver Fig. 1]. La fiabilidad test-retest al cabo de un año se calculó con toda la muestra. Los resultados mostraron que las pun tuaciones mejoraron después de un año (M = 66.25; SD = 9.73), y el coeficiente de correlación intraclase (ICC) entre las dos medidas fue alto [ICC = 0.59 (IC 95% 0.172-0.80); ver Fig. 1]. Fig. 1. El diagrama de dispersión muestra la correlación intraclase (ICC) entre las puntuaciones obtenidas en la Prueba de Jake Mandell con las puntuaciones obtenidas en el Retest Fig. 1. El diagrama de dispersión muestra la correlación intraclase (ICC) entre las puntuaciones obtenidas en la Prueba de Jake Mandell con las puntuaciones obtenidas en el Retest 139 Palomar-García M. A. et alii. Rev. electrón. complut. inves. educ. music. 17, 2020: 133-141 Además, el coeficiente de fiabilidad ώH de McDonald obtenido para la presente muestra fue de 0,81, lo que debe interpretarse como un soporte para la unidimensionalidad de la medida. 4. Estudio 3 4.1. Materiales y métodos 5. Discusión y conclusiones inves. educ. music. 17, 2020: 133-141 140 (Sihvonen y cols., 2016; Peretz y Vuvan, 2017). Además, el JMT mostró correlaciones significativas pero menores con las otras dos pruebas de AMUSIA: las pruebas de tono y ritmo. Estos resultados fueron consistentes con estudios anteriores que muestran correlaciones significativas entre todas las pruebas de MBEA (Pfeifer y Hamann, 2015), incluso el requisito de que los amúsicos tengan una percepción preservada del ritmo. De hecho, cuando se realizó el análisis factorial de las diferentes pruebas MBEA, se obtuvo un modelo de un factor, que indica que las diferentes habilidades musicales estaban todas correlacionadas. Por lo tanto, el JMT también puede considerarse una medida de las habilidades musicales generales. El JMT se administró a niños de 6 a 12 años en el estudio 2. La puntuación media fue aproximadamente 4 puntos más baja que en los adultos, con un rango entre 38 y 86%. Las diferencias entre niños y adultos y las correlaciones con la edad dentro de la muestra de niños no fueron significativas. Esto sugiere que el desarrollo de la discrimina ción tonal en los no músicos, medido con el JMT, no varió con la edad y puede establecerse durante la infancia a los 7 años. De hecho, investigaciones previas mostraron que estas habilidades se desarrollan principalmente hasta la edad de 7 años, sin que se encuentren diferencias entre la infancia y la adolescencia (Thompson, Cranford y Hoyer, 1999). Nuestros resultados fueron consistentes con estos resultados anteriores. Además, el JMT también mostró un buen índice de fiabilidad test-retest al año, lo que sugiere que el rendimiento de la prueba es estable, a pesar de que la segunda administración del test arrojó una mejora en el rendimiento (de menos del 5%). g j j ( ) También estudiamos la validez concurrente en niños al correlacionar las puntuaciones de JMT con los resultados de cinco pruebas musicales diferentes. Como se esperaba, la correlación fue positiva con todas las pruebas, pero solo fue significativa con la prueba de discriminación tonal y la prueba de imitación rítmica. Al igual que en el Estudio 1, la prueba de discriminación de tono fue una versión similar y más fácil del JMT, que requería que los participantes indicaran si dos melodías consecutivas eran iguales o diferentes. 5. Discusión y conclusiones La prueba de imitación rítmica requirió que los participantes aplaudieran diversos patrones rítmicos presentados, por lo que también se requirió una buena memoria tonal. Las correlaciones con las otros tres subtests fueron positivas, pero no fueron significativas, lo que indica que se requerían algunas habilidades de percepción musical. Es importante destacar que la correlación más fuerte se observó con un factor compuesto de todas las pruebas, lo que indica que el JMT se asoció con todas las habilidades musicales. Por lo tanto, el JMT podría reflejar una medida global para evaluar todas las habilidades musicales relacionadas con la percepción y la memoria tonal. En este sentido, el JMT ha mostrado una buena asociación con un aumento en el volumen de sustancia gris en las áreas fronto-temporales (giro temporal superior y giro frontal inferior) en niños y adultos (Palomar-García y cols., 2020). El tercer estudio fue diseñado para evaluar la validez predictiva mediante el estudio del rendimiento en el JMT en un grupo de músicos. Los músicos generalmente muestran una mayor capacidad de discriminación de tono en comparación con los no músicos, lo que es consistente con el hallazgo de que los músicos son más sensibles a algunas características acústicas que son críticas para el procesamiento musical (Spiegel y Watson, 1984; Micheyl, Delhom meau, Perrot y Oxenham, 2006). Consistentemente, la experiencia en el procesamiento y el desempeño de la música se asoció con puntuaciones más altas en el JMT, en comparación con los no músicos. Las puntuaciones fueron casi un 10% más altas que la de los no músicos. Sin embargo, las puntuaciones de los músicos también siguieron una distribución normal, lo que indica la falta de un efecto techo. Es importante destacar que las puntuaciones en el JMT no correlacionaron con el entrenamiento musical temprano o con años de entrenamiento musical, lo que sugiere una posible independencia del entrenamiento. En general, los estudios actuales sugieren que las habilidades de discriminación tonal se establecen a los 6-7 años de edad y apenas mejoran en el período de la infancia a la edad adulta sin entrenamiento específico. La falta de correlación con la edad y la falta de diferencias significativas entre niños y adultos sugieren la ausencia de efectos de maduración durante el desarrollo. 5. Discusión y conclusiones Por lo tanto, no podemos concluir que el mejor desempeño de los músicos en el JMT se deba al entrenamiento porque un proceso de autoselección puede hacer que las personas con buenas habili dades de discriminación tonal se conviertan en músicos. El instrumento actual tiene varios usos potenciales. Primero, debería ayudar a medir rápidamente las habilidades de discriminación tonal y las habilidades musicales generales en adultos sanos, y establecer diferencias individuales entre los músicos. En segundo lugar, el JMT podría ser útil como herramienta para investigar el desarrollo de la per cepción musical en los niños. Sus aplicaciones pueden centrarse en el campo educativo como medida del potencial de la capacidad musical, así como en el campo clínico como medida de los déficits de procesamiento musical asociados a lesiones cerebrales. 5. Discusión y conclusiones En este trabajo, se han presentado los datos psicométricos del JMT en adultos y niños para validar este instrumento como una medida de las diferencias individuales en la discriminación tonal. Los estudios 1 y 2 mostraron una buena consistencia interna de la escala y una buena validez convergente cuando las puntuaciones del JMT se correlacio naron con otras variables que medían capacidades musicales similares. Es importante destacar que las puntuaciones medias en el JMT fueron más bajas que las obtenidas para las pruebas de detección en línea de AMUSIA, mostrando una distribución más simétrica y normal. El estudio 2 también mostró que el JMT tenía una buena fiabilidad test-re test y estaba relacionado con las habilidades musicales globales. Finalmente, el estudio 3 aportó validez de contenido a la prueba al mostrar que los músicos tuvieron un mejor desempeño que los no músicos, pero sin encontrar un efecto techo. En general, los datos actuales son consistentes con la idea de que el JMT podría ser un buen instrumento psi cométrico y rápido para medir las diferencias individuales en la percepción y la memoria tonal. El estudio 1 se realizó en adultos no músicos y se encontró una puntuación media en el JMT del 65.4%, un 10% menos que la puntuación media encontrada en la muestra online. Las puntuaciones obtenidas se distribuyeron simé tricamente entre los participantes en un rango de 47 a 86%. En este grupo de participantes, el 11.5% fueron conside rados amúsicos de acuerdo con los criterios propuestos por la prueba (puntuaciones inferiores al 55%). Por otro lado, el JMT también fue sensible para la identificación de participantes con buenas habilidades en la discriminación tonal porque no hubo efecto techo, es decir, ninguno de los participantes evaluados alcanzó más del 87% de precisión. La validez concurrente del JMT se estudió utilizando tres pruebas diferentes incluidas en las pruebas de detec ción de AMUSIA. Como se esperaba, la media de JMT fue menor que la de las tres pruebas de detección online de AMUSIA. Los análisis de correlación revelaron una fuerte asociación entre el JMT y el subtest de escala de la prueba AMUSIA. La prueba de escala tiene un formato similar al JMT, pero es más fácil y, por lo tanto, produce efectos de techo. Algunos estudios sólo usaron esta prueba de escala para estimar posibles déficits en la percepción musical Palomar-García M. A. et alii. Rev. electrón. complut. Albouy, P., Mattout, J., Bouet, R., Maby, E., Sanchez, G., Aguera, P. 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https://openalex.org/W2896004276	https://europepmc.org/articles/pmc6262914?pdf=render	English	null	Pronounced genetic differentiation in <i>Fokienia hodginsii</i> revealed by simple sequence repeat markers	Ecology and evolution	2,018	cc-by	11,053	Received: 29 October 2017 \| Revised: 16 August 2018 \| Accepted: 29 August 2018 Received: 29 October 2017 \| Revised: 16 August 2018 \| Accepted: 29 August 2018 Received: 29 October 2017 \| Revised: 16 August 2018 \| Accepted: 29 August 2018 DOI: 10.1002/ece3.4560 Qianyi Yin1 \| Sufang Chen1 \| Wei Guo2 \| Yanshuang Huang1 \| Yelin Huang1 \| Renchao Zhou1 \| Qiang Fan1 \| Wenbo Liao1 1State Key Laboratory of Biocontrol and Guangdong Provincial Key Laboratory of Plant Resources, Sun Yat‐sen University, Guangzhou, China Correspondence Correspondence Wenbo Liao and Qiang Fan, School of Life Sciences, Sun Yat‐Sen University, Guangzhou, China. Correspondence Wenbo Liao and Qiang Fan, School of Life Sciences, Sun Yat‐Sen University, Guangzhou, China. Email: lsslwb@mail.sysu.edu.cn (WL); fanqiang@mail.sysu.edu.cn (QF) Email: lsslwb@mail.sysu.edu.cn (WL); fanqiang@mail.sysu.edu.cn (QF) Pronounced genetic differentiation in Fokienia hodginsii revealed by simple sequence repeat markers Qianyi Yin1 \| Sufang Chen1 \| Wei Guo2 \| Yanshuang Huang1 \| Yelin Huang1 \| Renchao Zhou1 \| Qiang Fan1 \| Wenbo Liao1 10938 \| Ecology and Evolution. 2018;8:10938–10951. www.ecolevol.org 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. © 2018 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. Abstract Abstract Fokienia hodginsii is a Tertiary relict conifer of the monotypic genus Fokienia (Cupressaceae s.l.). Currently, the species is distributed in southern China, northern Vietnam, and northern Laos and listed as a “near threatened” species by the IUCN. In this study, a total of 427 individuals of F. hodginsii were sampled from China and Vietnam to characterize its genetic diversity and population differentiation. Based on the profiles of 12 simple sequence repeat (SSR) markers, we observed a high level of genetic diversity in F. hodginsii at the species level (He =0.635), albeit slightly lower than that of its sister species Chamaecyparis obtusa. Signals of bottleneck events were detected in the populations GXDMS, GXHJ, V‐PXB, and V‐HB, probably due to Pleistocene glaciations or overexploitation in recent years. Pronounced genetic dif‐ ferentiation (Fst = 0.157) was found in this species. The inbreeding index (Fis = 0.176 ± 0.024) indicated that F. hodginsii has a mixed mating system. Significant correlation was found between the pairwise genetic differentiation and geographic distance (r = 0.882, p = 0.01), suggesting that genetic differentiation among the pop‐ ulations follows the model of isolation by distance (IBD). STRUCTURE analysis and principal coordinate analysis revealed that these populations were divided into four groups: the western China group located mainly in the Yunnan–Guizhou Plateau, the central China group located mostly in the Luoxiao Mountains and Nanling Mountains, the eastern China group located in the Wuyi Mountains and the Vietnam group con‐ taining two populations in Vietnam. The different terrains and elevations of popula‐ tions may be the most likely factors leading to the differentiation between the western China group and the central China group, while the geographic isolation caused by the lack of appropriate habitats may greatly contribute to the differentia‐ tion between the central China group and the eastern China group. Based on the results, some conservation suggestions for this species are provided, such as estab‐ lishing seed orchards and multiple nature reserves. 2Department of Horticulture and Landscape Architecture, Zhongkai University of Agriculture and Engineering, Guangzhou, China 1 \| INTRODUCTION 1 the International Union for Conservation of Nature Red List (IUCN 2004) and the National Secondary Protected Plants by Order of the Forestry Bureau and Ministry of Agriculture of China (https://www. gov.cn/gongbao/content/2000/content_60072.htm), the vulnerable species by the Information System of Chinese Rare and Endangered Plants (https://rep.iplant.cn/protlist), National Secondary Protected Plants in China and a K‐class protected plant species in Vietnam (Vuong, 2009). the International Union for Conservation of Nature Red List (IUCN 2004) and the National Secondary Protected Plants by Order of the Forestry Bureau and Ministry of Agriculture of China (https://www. gov.cn/gongbao/content/2000/content_60072.htm), the vulnerable species by the Information System of Chinese Rare and Endangered Plants (https://rep.iplant.cn/protlist), National Secondary Protected Plants in China and a K‐class protected plant species in Vietnam (Vuong, 2009). Under current rapid global climate change, many endemic species are facing a high risk of extinction due to limited natural ranges re‐ sulting from genetic stochasticity or demographic, environmental, or other factors (Caughley, 1994; Gitzendanner & Soltis, 2000; Lande, 1993). It is vital to understand the genetic characteristics of these species, such as genetic diversity and population structure, for their management and the development of effective conservation strat‐ egies (Eckert, Samis, & Lougheed, 2008; Lesica & Allendorf, 2010). Most recent studies on F. hodginsii mainly focused on seed breeding, nursery technology, plantation cultivation, essential oil ex‐ traction and development and utilization of other resources (Huang et al., 2013; Zhao, 2005). Only one paper mentioned the progress in genetics of F. hodginsii, according to Tam, Trang, and Hoa (2011), who investigated the genetic diversity and population structure of F. hodginsii in Vietnam by applying ISSR markers and showed that F. hodginsii maintained a low level of genetic variability and a high level of genetic differentiation. They supposed that human distur‐ bance may play a key role in the present status of F. hodginsii by lead‐ ing to the degradation and fragmentation of its habitats. The gymnosperm family Cupressaceae Bartling comprises ap‐ proximately 22 genera and 150 species. Most of these species are Tertiary relict species that arose in the Jurassic (possibly as early as the Triassic), thrived in the Jurassic, and decreased in members con‐ tinuously up to the present. It is also the only family of gymnosperms that is present on all continents except Antarctica (Yang, Ran, & Wang, 2012). 1 \| INTRODUCTION However, except for Juniperus, Sabina, and Cupressus, most species in this family are locally endemic, and ensuring their survival under future climate change will require public and scientific attention. Simple sequence repeat (SSR; microsatellite) markers, codomi‐ nant markers with good reproducibility and high variability, are one of the best tools to understand species genetic diversity and popula‐ tion structure (Wang, Huang, & Long, 2013). Based on transcriptome sequencing, we synthesized 108 SSR primers that were successfully amplified in F. hodginsii (Ding et al., 2017). Applying these SSR mark‐ ers, we aimed to investigate the levels of genetic diversity and pop‐ ulation structure of this species, which could provide some reliable information for the protection of this endangered species. The genus Fokienia Henry et Thomas (Cupressaceae s.l.) contains only one extant species, Fokienia hodginsii (Dunn) Henry et Thomas (Farjon, 2005; Figure 1). Fossil records show that Fokienia was widely distributed in the Northern Hemisphere in ancient periods: fossils in forms with foliage and attached seed cones of Fokienia were re‐ ported from the Paleocene in Saskatchewan, central Canada (McIver & Basinger, 1990); the Oligocene in Jilin, northeastern China (Guo & Zhang, 2002); and the Miocene in Zhejiang, eastern China (He, Sun, & Liu, 2012). However, this genus is currently distributed in only south‐ ern China, northern Vietnam, and northern Laos (Zheng & Fu, 1978). In China, it occurs at elevations between approximately 1,000 and 1,800 m as a minor constituent of the subtropical evergreen (mixed) forest (Zheng & Fu, 1978). This conifer is a good landscape tree spe‐ cies with a beautiful shape and straight trunk (Huang et al., 2013) and is commonly cut down for building materials because of its light texture and material stability (Huang, Huang, Guo, & Zheng, 2015). Currently, this conifer is listed as “near threatened (NT)” as part of Funding information g the Fourth National Survey on Chinese Material Medical Resources Program of State Administration of Traditional Chinese Medicine of the People's Republic of China, Grant/Award Number: 2017-152- 003; the Special Program for Key Basic Research of the Ministry of Science and Technology,China, Grant/Award Number: 2013FY111500; Chang Hungta Science Foundation of Sun Yat‐sen University.; National Natural Science Foundation of China, Grant/Award Number: 31570195 and 31670189; Natural Science Foundation of Guangdong Province, China, Grant/Award Number: 2016A030313326; Foundation of Jinggangshan Administration of Jiangxi Province, Grant/Award Number: 33000- 7102993; Fundamental Research Funds for the Central Universities, Grant/Award Number: 16lgjc38 conservation, endangered species, Fokienia hodginsii, genetic differentiation, microsatellite, southern China Ecology and Evolution. 2018;8:10938–10951. 10939 Yin et al. 2.1 \| Sample collection and DNA extraction A total of 427 individuals of F. hodginsii were sampled from 24 lo‐ cations across twelve provinces of China and Vietnam (Table 1; Figure 2). A Garmin GPS unit (GPSMAP 62sc, Taiwan) was used to record the sample geographic locations with a margin of 10 m. For each population, fresh leaves were collected from 5 to 23 randomly selected fully grown individuals, which were at least 30 m apart from each other. Then, the leaf tissues were dried by silica gel and stored in zip‐lock plastic bags for DNA extraction. Voucher specimens for each population were all deposited in the Herbarium of Sun Yat‐sen University (SYS). FI G U R E 1 Photograph of Fokienia hodginsii Total DNA was extracted from dried leaf tissue using the modi‐ fied CTAB method (Doyle & Doyle, 1987). For each population, two individuals were randomly selected for PCR amplifications with all 108 primers designed by Ding et al. (2017). Fluorescence was added to the 3′ end of the 12 SSR markers (Table 2) with the highest poly‐ morphism levels, and PCR amplifications were performed for all 427 individuals, in which the annealing temperature for each primer was set to 52°C. The PCR products were first inspected in 1% agarose gel and then electrophoresed on an Applied Biosystems 3730xl DNA Analyzer (Applied Biosystems, Foster City, California, USA). FI G U R E 1 Photograph of Fokienia hodginsii Yin et al. Yin et al. \| TA B LE 1 Groups based on the result from SAMOVA and geographic information for populations of Fokienia hodginsii Pop. 2.1 \| Sample collection and DNA extraction ID Geographic locality Geographic coordinates Altitude (m) Sample size The eastern China group ZJJD Jiande, Zhejiang, China 119°33′19.98″E, 29°34′40.56″N 877 20 ZJFYS Longquan, Zhejiang, China 119°10′11.05″E, 27°52′49.63″N 1,471 20 FJHBL Nanjing, Fujian, China 117°15′38.83″E, 24°31′13.57″N 762 15 FJDYS Dehua, Fujian, China 118°13′2.34″E, 25°38′27.1″N 1,095 20 FJFHS Shaxian, Fujian, China 117°47′29.86″E, 26°23′32.6″N 369 20 FJMHS Longyan, Fujian, China 116°51′17.78″E, 25°16′0.61″N 830 20 JXSQS Shangrao, Jiangxi, China 118°3′50″E, 28°54′10.5″N 1,354 20 JXMTS Zixi, Jiangxi, China 117°8′11.81″E, 27°50′6.31″N 805 11 The central China group GDQXD Zhaoqing, Guangdong, China 111°57′56.82″E, 23°33′29.25″N 1,068 20 JXJGS Jinggangshan, Jiangxi, China 114°09′16.36″E, 26°30′32.82″N 1,311 20 JXWZF Shangyou, Jiangxi, China 114°19′12″E, 25°28′47.99″N 1,488 20 HNMS Yizhang, Hunan, China 112°57′19.63″E, 24°57′49.43″N 1,103 20 HNYY Daoxian, Hunan, China 111°20′45.39″E, 25°33′38.92″N 1,247 23 The western China group GXCWLS Baise, Guangxi, China 106°22′36.07″E, 24°25′9.19″N 1671 20 GXDMS Nanning, Guangxi, China 108°26′17.47″E, 23°29′46.39″N 1,203 5 GXHP Longsheng, Guangxi, China 109°54′51.55″E, 25°36′14.52″N 1,290 20 GXHJ Dongxing, Guangxi, China 108°38′23.94″E, 25°12′9.82″N 1,139 7 GXJX Jinxiu, Guangxi, China 110°19′15.11″E, 24°12′40.19″N 989 20 YNLFZ Mengzi, Yunnan, China 103°49′6.11″E, 22°52′12.27″N 1503 19 GZYC Yuchong, Guizhou, China 105°58′50.32″E, 27°22′2.01″N 1,323 20 CQSMS Jiangjin, Chongqing, China 106°20′55.27″E, 28°34′38.61″N 1,170 20 SCHGX Xuyong, Sichuan, China 105°33′7.84″E, 28°14′40.64″N 1,122 20 The Vietnam group V‐PXB Fansipan, Sapa, Vietnam 103°46′22.34″E,22°21′03.54″N 1823 11 V‐HB Mai Châu, Hòa Binh, Vietnam 104°53′25.10″E,20°44′19.48″N 1,366 16 TA B LE 1 Groups based on the result from SAMOVA and geographic information for pop ed on the result from SAMOVA and geographic information for populations of Fokienia hodginsii (Wright, 1969). Four abiotic‐climate variables, namely, minimum temperature, maximum temperature, average temperature, and precipitation, from the sampled locations were obtained from the WorldClim database (Version 1.4; https://www.worldclim.org/) and used to calculate the differentiation matrix. Mantel tests (Mantel, 1967) between the matrix of the pairwise population differentiation in terms of Fst/(1 − Fst) and the differentiation matrix of geographic distances or abiotic‐climate variables were performed with GenAlEx with 1,000 random permutations (Rousset, 1997). TA B LE 2 The information for the 12 microsatellites TA B LE 2 The information for the 12 microsatellites TA B LE 2 The information for the 12 microsatellites TA B LE 2 The information for the 12 microsatellites Locus Primer sequences (5′–3′) Repeat Expected size (bp) Putative function F015 F: TGTAATAACTCTGTCCCTTCC (TA)7 200–210 Arabidopsis thaliana SIT4 phosphatase‐ associated family protein R: CTCTGTGCTCCTCTCCAA F017 F: AAGACAAGATGCTCAGATCA (AG)7 192–196 Picea glauca clone GQ03325_I06 mRNA R: GTGGTAGCCTAGAACTTCAT F020 F: TTCCTGCTTGAATGAATCCA (CT)7 232–238 Arabidopsis thaliana armadillo/ beta‐catenin repeat family protein R: GCGGAGGAGAAGGAGATT F036 F: GCCGAGACAGAGATAGAGA (AG)6 260–268 Oryza sativa (japonica cultivar‐group) U1 small nuclear ribonucleoprotein 70 K R: ATAGCATAACAGCACCTCAT F042 F: TGGAAGAAGATATGGTCAAGG (GA)6 264–270 Arabidopsis thaliana auxilin‐like protein R: TCAATAGCTGCTCTGTCAC F049 F: CAATGTTCCTTCTGTGTCTG (CAG)7 221–245 Picea sitchensis clone WS02761_D24 unknown mRNA R: TTGATACTGAGGTGCTTGAA F089 F: TACGGATGAGCAGTCCAT (TGG)5 276–291 Cryptomeria japonica putative glycine‐ rich RNA binding protein R: CACCTCCACCACCATTAC F127 F: CCTTCAACTCATCATAGAATGG (TTC)6 230–242 Not found R: TGAGCCTTCACTGCTAATG F173 F: TTATTCTACAGGCGAAGCAT (AAC)5 194–206 Arabidopsis thaliana zinc‐binding family protein R: TATTCTGGATAAGACGGTGAG F204 F: TCTGGGAATGTTTGGGAAG (CAG)5 201–210 Pisum sativum ultraviolet‐B‐repressible dehydrin‐related protein R: CTGCGTCTATAAAGCCTAATC F210 F: TGGAAGGAAGAAGGAAGATG (GTG)5 291–306 Not found R: CGGACCTCATGTAAGAACTT F217 F: GCATATAAGGTGGCGACTC (CAT)5 200–212 Pinus radiata PrLTP1 R: GCAGGAAGTGGTGAGAAG 2.2 \| Data analyses Linkage disequilibrium (LD) between pairs of loci and deviation from Hardy–Weinberg equilibrium (HWE) for each locus/population combination were tested using ARLEQUIN version 3.1 (Schneider, Roessli, & Excoffier, 2000). Parameters of genetic variation were calculated using GenAlEx v6.41 (Peakall & Smouse, 2006), includ‐ ing the total number of alleles (Na), the effective number of alleles (Ne), the expected and observed heterozygosities (He and Ho, respec‐ tively), the Shannon information index (I) and the fixation (inbreed‐ ing) index (Fis). Additionally, FSTAT version 2.9.3.2 (Goudet, 2002) was used to calculate the allelic richness (AR), the unbiased estimate of Wright’s F‐statistic (including total‐population inbreeding coef‐ ficients (Fit), the overall intrapopulation inbreeding coefficient (Fis) and the interpopulation genetic differentiation coefficient (Fst), Weir & Cockerham, 1984), and pairwise Fst between paired popu‐ lations. Based on pairwise Fst, gene flow between populations (Nm) was further estimated with the following formula: Nm = (1 − Fst)/4Fst Taking into account the geographic location of each population and the genetic differentiation within and among populations, Spatial Analysis of Molecular Variance (SAMOVA) software (Dupanloup, Schneider, & Excoffier, 2002) was used to define the best number of groups; then, ARLEQUIN version 3.11 was used for the analysis of molecular variance (AMOVA; Excoffier, Smouse, & Quattro, 1992), in which three levels of genetic differentiation were calculated: genetic differentiation within populations, genetic differentiation among populations within groups, and genetic differentiation among groups. Yin et al. 10941 \| 10941 Yin et al. FI G U R E 2 Geographic locations of the 24 populations of Fokienia hodginsii \| 10941 Yin et al. FI G U R E 2 Geographic locations of the 24 populations of Fokienia hodginsii Notes. AR: allelic richness; Fis: coefficient of inbreeding; He: expected frequency of heterozygotes; Ho: observed frequency of heterozygotes; I: Shannon index; N: number of alleles; Na: observed number of alleles; Ne: effective number of alleles. Notes. AR: allelic richness, i.e. the average number of alleles per locus; Fis: inbreeding coefficient; Fit: total‐population inbreeding coefficient; Fst: among‐ population genetic differentiation coefficient; He: unbiased expected heterozygosity; Ho: observed heterozygosity; Na observed number of alleles; Ne: effective number of alleles; Nm: gene flow; NT: number of alleles per locus. BOTTLENECK 1.2.02 (Piry, Luikart, & Cornuet, 1999) was used In addition, a Bayesian clustering approach implemented in STRUCTURE v2.3.4 (Evanno, Regnaut, & Goudet, 2005) was used to investigate population structure, in which a 100,000 burn‐in pe‐ riod was followed by 10 iterations of 100,000 Markov chain Monte Carlo replicates per K (1–10). Then, STRUCTURE HARVESTER (Earl & Vonholdt, 2012) was used to determine the optimum K. Further, a principal coordinate analysis (PCoA) was conducted based on BOTTLENECK 1.2.02 (Piry, Luikart, & Cornuet, 1999) was used to detect signals of recent bottleneck effects, in which one‐tailed Wilcoxon signed‐rank tests (10,000 replications) based on the “in‐ finite allele model of mutation” (I.A.M.), the “stepwise mutation model’’ (S.M.M.), and the “two‐phased model of mutation” (T.P.M.; 70% of alleles under S.M.M.) were performed, and Bonferroni cor‐ rections for multiple tests were made. Yin et al. Yin et al. 10942 TA B LE 3 Genetic variability for the 12 SSR markers within populations Pop N AR Na Ne Ho He Fis I ZJJD 42 3.181 3.5 2.877 0.533 0.639 0.161 1.117 ZJFYS 45 3.365 3.75 3.089 0.496 0.659 0.241 1.178 FJHBL 44 3.348 3.667 3.024 0.544 0.666 0.18 1.178 FJDYS 43 3.318 3.583 3.106 0.521 0.669 0.22 1.179 FJFHS 43 3.273 3.583 3.031 0.542 0.658 0.166 1.158 FJMHS 44 3.283 3.667 2.989 0.517 0.658 0.21 1.161 JXSQS 43 3.253 3.583 3.062 0.567 0.656 0.124 1.152 JXMTS 39 3.078 3.25 2.804 0.583 0.628 0.062 1.069 GDQXD 41 3.2 3.417 2.956 0.517 0.637 0.172 1.114 JXJGS 40 3.083 3.333 2.775 0.563 0.624 0.09 1.077 JXWZF 41 3.114 3.417 2.804 0.521 0.63 0.175 1.091 HNMS 42 3.251 3.5 3.023 0.563 0.662 0.147 1.156 HNYY 42 3.147 3.5 2.826 0.496 0.634 0.219 1.106 GXCWLS 44 3.15 3.667 2.624 0.496 0.604 0.174 1.076 GXDMS 39 3.25 3.25 2.517 0.533 0.59 0.286 1.011 GXHP 43 3.201 3.583 2.775 0.496 0.633 0.22 1.112 GXHJ 39 3.147 3.25 2.662 0.524 0.606 0.262 1.04 GXJX 44 3.244 3.667 3.048 0.475 0.661 0.084 1.159 YNLFZ 44 3.217 3.667 2.908 0.518 0.65 0.207 1.139 GZYC 42 3.2 3.5 2.923 0.479 0.651 0.129 1.134 CQSMS 41 3.135 3.417 2.87 0.488 0.645 0.242 1.109 SCHGX 42 3.244 3.5 3.034 0.542 0.662 0.181 1.155 V‐PXB 32 2.967 3 2.47 0.508 0.573 0.111 0.93 V‐HB 34 2.988 2.917 2.461 0.51 0.551 0.066 0.908 Mean 3.193 ± 0.067 3.465 ± 0.044 2.861 ± 0.034 0.522 ± 0.007 0.635 ± 0.005 0.172 ± 0.011 1.105 ± 0.012 Notes. AR: allelic richness; Fis: coefficient of inbreeding; He: expected frequency of heterozygotes; Ho: observed frequency of heterozygotes; I: Shannon 3.1 \| Genetic diversity According to the LD analysis for these 12 polymorphic loci, no pairs of loci showed linkage disequilibrium after a sequential Bonferroni cor‐ rection for multiple tests, indicating that the 12 markers can be con‐ sidered independent markers for population genetics studies. The genetic variation across the 24 natural populations is summarized in Table 3. According to Table 3, a total of 78 alleles were detected from these 12 SSR loci, ranging from 4 to 8 per locus. The average allelic richness (AR) for each population ranged from 2.967 to 3.365 (average: 3.193 ± 0.067). The value of Na ranged from 2.917 to 3.750 (average: 3.465 ± 0.044), Ne ranged from 2.461 to 3.106 (average: 2.861 ± 0.034), and He and Ho ranged from 0.551 to 0.669 (average: 0.635 ± 0.005) and 0.475 to 0.583 (average: 0.523 ± 0.007), respec‐ tively. After Bonferroni corrections, no loci showed deviations from Hardy–Weinberg equilibrium (Supporting Information Table S1). The Fis (inbreeding coefficient) averaged across all loci ranged from 0.048 to 0.326 (average: 0.176 ± 0.024, Table 4). In the results of the STRUCTURE analysis, ΔK showed the highest value at K = 3 (Figure 4). Assignment results for K = 3 showed that all individuals could be roughly divided into three gene pools: the east‐ ern China and Vietnam gene pool (mainly in green), the central China gene pool (mainly in red), and the western China gene pool (mainly in blue; Figure 5). When K = 4, the eastern China and Vietnam gene pool were divided into the eastern China gene pool (mainly in green) and the Vietnam gene pool (mainly in yellow; Figure 5), which agreed with the four groups divided by the SAMOVA (Figure 6). Populations V‐PXB and V‐HB, located in Vietnam, had the low‐ est genetic diversity (V‐PXB: He = 0.573 and Ho = 0.508; V‐HB: He = 0.551 and Ho = 0.510). Among the 22 populations in China, GXDMS and GXHJ harbored the lowest genetic diversity (He = 0.590 and 0.606 and Ho = 0.533 and 0.524, respectively). In contrast, the populations FJDYS, FJHBL, HNMS and SCHGX showed the highest genetic diversity (He = 0.662–0.669 and Ho = 0.521 – 0.563). 3.1 \| Genetic diversity Principal coordinate analysis showed that most populations of the western China group were located on the lower left side; popula‐ tions of the central China group, on the middle left side; populations of the eastern China group, on the upper left side; and populations of the Vietnam group, on the right side (Figure 7). the Jaccard distance between populations using MVSP software (Kovach, 1999). including the remaining populations, mostly in the Wuyi Mountains; and the last group including two populations in Vietnam. Based on this division, the AMOVA showed that genetic differentiation among groups accounted for 13.14% of the variation, genetic differentiation among populations within groups accounted for 2.20%, and genetic differentiation within populations accounted for 84.66% (Table 7). The gene flow among populations within groups and between dif‐ ferent groups was also calculated. The results showed that the gene flow in the eastern China group had the maximum value (11.486) and that the Vietnam group had the minimum value (4.527) compared to the central China group (10.584) and the western China group (8.448). The gene flow between the eastern China group and the central China group was 2.960, and the gene flow between the west‐ ern China group and the central China group was 3.892. 3.2 \| Genetic structure The results from F‐statistics showed that the overall intrapopulation inbreeding coefficient (Fis) was 0.176 ± 0.024, the total‐population inbreeding coefficient (Fit) was 0.308 ± 0.019, the interpopula‐ tion genetic differentiation coefficient (Fst) was 0.157 ± 0.019, and the gene flow (Nm) was estimated to be 2.013 ± 0.698 (Table 4). All pairwise Fst values were highly significant (p < 0.001), ranging from 0.009 (between FJDYS and FJFHS) to 0.234 (between V‐HB and ZJJD; Table 5). Correlation analyses showed that the genetic differ‐ entiation was most correlated with geographic distance (r = 0.882, p = 0.01, Figure 3), longitudinal changes (r = 0.466, p = 0.01), lati‐ tudinal changes (r = 0.432, p = 0.01), precipitation differentiation (r = 0.256, p = 0.01), elevational changes (r = 0.205, p = 0.01), and average temperature changes (r = 0.178, p = 0.04; Table 6). 3.3 \| Genetic bottleneck assessments The Wilcoxon test and sign test indicated that bottleneck events may have occurred in the populations GXDMS, GXHJ, V‐PXB, and V‐ HB via the infinite allele model and the two‐phased mutation model (Table 8). TA B LE 3 Genetic variability for the 12 SSR markers within populations Notes. AR: allelic richness; Fis: coefficient of inbreeding; He: expected frequency of heterozygotes; Ho: observed frequency of heterozygotes; I: Shannon index; N: number of alleles; Na: observed number of alleles; Ne: effective number of alleles. TA B LE 4 Genetic diversity at the 12 microsatellite loci TA B LE 4 Genetic diversity at the 12 microsatellite loci Loci NT AR Na Ne Ho He Fis Fit Fst Nm F015 8 4.233 4.000 3.405 0.583 0.700 0.167 0.284 0.140 1.533 F017 6 2.769 2.958 2.609 0.541 0.607 0.109 0.227 0.132 1.647 F020 5 4.071 3.250 2.846 0.429 0.636 0.326 0.411 0.126 1.730 F036 9 3.323 4.292 3.294 0.522 0.688 0.241 0.342 0.133 1.634 F042 4 3.520 3.917 3.213 0.552 0.686 0.195 0.216 0.025 9.610 F049 7 3.214 2.875 2.415 0.546 0.574 0.048 0.334 0.300 0.582 F089 5 3.339 3.000 2.415 0.531 0.568 0.065 0.250 0.198 1.012 F127 7 4.546 4.375 3.433 0.574 0.699 0.178 0.283 0.127 1.712 F173 7 3.926 3.125 2.452 0.407 0.589 0.308 0.431 0.178 1.158 F204 6 3.279 2.958 2.637 0.518 0.616 0.158 0.304 0.173 1.194 F210 8 3.947 3.625 2.721 0.520 0.617 0.156 0.323 0.198 1.016 F217 6 4.171 3.208 2.890 0.541 0.644 0.161 0.293 0.158 1.330 Mean 3.695 ± 0.044 3.465 ± 0.044 2.861 ± 0.034 0.522 ± 0.007 0.635 ± 0.005 0.176 ± 0.024 0.308 ± 0.019 0.157 ± 0.019 2.013 ± 0.698 Notes. AR: allelic richness, i.e. the average number of alleles per locus; Fis: inbreeding coefficient; Fit: total‐population inbreeding coefficient; Fst: among‐ population genetic differentiation coefficient; He: unbiased expected heterozygosity; Ho: observed heterozygosity; Na observed number of alleles; Ne: effective number of alleles; Nm: gene flow; NT: number of alleles per locus. 10943 Yin et al. hodginsii habitat in Vietnam has been degraded and fragmented, which may also serve as a good explanation for the low genetic vari‐ ability in Vietnam, as signals of bottleneck events were also detected FI G U R E 3 Relationship between pairwise Fst/(1 − Fst) and the geographic distance among the populations of Fokienia hodginsii (r = 0.882, p = 0.01) y = 0.0003x + 0.013 R² = 0.816 0 0.05 0.1 0.15 0.2 0.25 0.3 0 100 200 300 400 500 600 700 800 900 Fst (1-Fst) Geographic distance (km) POP ZJJD ZJFYS FJHBL FJDYS FJFHS FJMHS JXMTS JXSQS JXJGS JXWZF GDQXD ZJJD 0.000 15.728 16.894 8.984 7.049 9.803 8.197 4.429 2.827 2.753 3.140 ZJFYS 0.016 0.000 13.977 10.007 8.882 10.419 6.643 3.796 2.555 2.571 2.627 FJHBL 0.015 0.018 0.000 10.589 6.818 10.312 10.169 5.290 3.454 3.430 3.901 FJDYS 0.027 0.024 0.023 0.000 29.065 22.317 5.358 4.917 3.143 3.288 3.337 FJFHS 0.034 0.027 0.035 0.009 0.000 20.908 4.115 4.006 2.506 2.724 2.681 FJMHS 0.025 0.023 0.024 0.011 0.012 0.000 4.969 4.514 2.763 2.887 3.113 JXMTS 0.030 0.036 0.024 0.045 0.057 0.048 0.000 4.346 2.849 2.733 2.863 JXSQS 0.053 0.062 0.045 0.048 0.059 0.052 0.054 0.000 7.558 8.447 8.834 JXJGS 0.081 0.089 0.067 0.074 0.091 0.083 0.081 0.032 0.000 18.349 15.456 JXWZF 0.083 0.089 0.068 0.071 0.084 0.080 0.084 0.029 0.013 0.000 15.694 GDQXD 0.074 0.087 0.060 0.070 0.085 0.074 0.080 0.028 0.016 0.016 0.000 HNMS 0.070 0.075 0.066 0.065 0.076 0.073 0.095 0.037 0.028 0.025 0.028 HNYY 0.078 0.083 0.072 0.081 0.092 0.083 0.098 0.045 0.026 0.025 0.025 GXJX 0.084 0.087 0.071 0.076 0.086 0.080 0.092 0.044 0.044 0.034 0.042 GXHP 0.094 0.097 0.082 0.078 0.087 0.077 0.117 0.063 0.063 0.048 0.052 GXHJ 0.115 0.123 0.109 0.105 0.114 0.106 0.129 0.089 0.088 0.072 0.072 GXDMS 0.095 0.100 0.084 0.079 0.087 0.076 0.116 0.083 0.084 0.072 0.067 GXCWLS 0.101 0.112 0.094 0.087 0.095 0.084 0.124 0.070 0.075 0.056 0.057 GZYC 0.093 0.098 0.082 0.085 0.097 0.087 0.099 0.078 0.075 0.067 0.072 CQSMS 0.093 0.099 0.085 0.087 0.099 0.089 0.107 0.082 0.073 0.069 0.076 SCHGX 0.093 0.096 0.085 0.091 0.100 0.094 0.101 0.080 0.069 0.066 0.073 YNLFZ 0.099 0.104 0.093 0.092 0.102 0.095 0.113 0.085 0.083 0.073 0.078 V‐PXB 0.196 0.178 0.179 0.183 0.187 0.191 0.185 0.165 0.189 0.183 0.186 V‐HB 0.234 0.211 0.203 0.211 0.217 0.216 0.213 0.198 0.209 0.204 0.213 FI G U R E 3 Relationship between pairwise Fst/(1 − Fst) and the geographic distance among the populations of Fokienia hodginsii (r = 0.882, p = 0.01) y = 0.0003x + 0.013 R² = 0.816 0 0.05 0.1 0.15 0.2 0.25 0.3 0 100 200 300 400 500 600 700 800 900 Fst (1-Fst) Geographic distance (km) FI G U R E 3 Relationship between pairwise Fst/(1 − Fst) and the geographic distance among the populations of Fokienia hodginsii (r = 0.882, p = 0.01) FI G U R E 3 Relationship between pairwise Fst/(1 − Fst) and the geographic distance among the populations of Fokienia hodginsii (r = 0.882, p = 0.01) Kirk, & Petersen, 2011; Hamilton, 2009). 4.1 \| Genetic diversity Genetic diversity is crucial for species, as it may influence the ability of species to cope with environmental change (Frankham, Ballou, & Briscoe, 2002; Frankham, 1995a, 1995b). In this study, microsatel‐ lite markers were used to estimate population genetic diversity and to investigate the genetic structure of F. hodginsii. Slightly lower ge‐ netic diversity was found in F. hodginsii (He = 0.635 ± 0.005) than in Chamaecyparis obtusa (He = 0.780), the sister species of F. hodginsii (Matsumoto, Uchida, Taguchi, Tani, & Tsumura, 2010). Compared to other species (Nybom, 2004), the expected heterozygosities (He) of F. hodginsii are similar to those of regional species (He = 0.65) and long‐lived woody perennial species (He = 0.68). Allelic diver‐ sity (Na) and expected heterozygosity (He) are also commonly used to estimate the genetic diversity in natural populations (Freeland, The SAMOVA demonstrated the highest value of FCT (FCT = 0.25346, p < 0.05; Supporting Information Figure S1) when it divided all 24 populations into four groups as follows: the west‐ ern China group including the populations located in western China (mostly the Yunnan–Guizhou Plateau); the central China group in‐ cluding the populations located in central China (Luoxiao Mountains, Nanling Mountains, and adjacent areas); the eastern China group 10944 \| Yin et al. Kirk, & Petersen, 2011; Hamilton, 2009). The He and Na values of F. hodginsii (He = 0.635, Na = 3.465) are slightly lower than those of C. obtusa (He = 0.780, Na = 7.038), albeit higher than those of other conifer species, such as Cryptomeria japonica (He = 0.277, Na = 2.000, Tsumura & Tomaru, 1999). In this study, the lowest genetic diversity was found in the two populations in Vietnam (V‐PXB: He = 0.573; V‐HB: He = 0.551). This phenomenon agreed with previous reports that most populations in Vietnam harbor low genetic diversity (HT = 0.0970 ± 0.0101, ISSR markers used by Tam et al., 2011). It is possible that China serves as the central distributional area of F. hodginsii, such that its genetic diversity decreased as it dispersed from its central area to its mar‐ ginal areas, such as Vietnam (Wei, Sork, Meng, & Jiang, 2016). Tam et al. (2011) also indicated that, as a result of human disturbance, the F. 4.1 \| Genetic diversity hodginsii habitat in Vietnam has been degraded and fragmented, which may also serve as a good explanation for the low genetic vari‐ ability in Vietnam, as signals of bottleneck events were also detected in these two populations. TA B LE 5 Pairwise population matrix of gene flow (upper triangle) and Fst values (lower triangle) for all populations POP ZJJD ZJFYS FJHBL FJDYS FJFHS FJMHS JXMTS JXSQS JXJGS JXWZF GDQXD ZJJD 0.000 15.728 16.894 8.984 7.049 9.803 8.197 4.429 2.827 2.753 3.140 ZJFYS 0.016 0.000 13.977 10.007 8.882 10.419 6.643 3.796 2.555 2.571 2.627 FJHBL 0.015 0.018 0.000 10.589 6.818 10.312 10.169 5.290 3.454 3.430 3.901 FJDYS 0.027 0.024 0.023 0.000 29.065 22.317 5.358 4.917 3.143 3.288 3.337 FJFHS 0.034 0.027 0.035 0.009 0.000 20.908 4.115 4.006 2.506 2.724 2.681 FJMHS 0.025 0.023 0.024 0.011 0.012 0.000 4.969 4.514 2.763 2.887 3.113 JXMTS 0.030 0.036 0.024 0.045 0.057 0.048 0.000 4.346 2.849 2.733 2.863 JXSQS 0.053 0.062 0.045 0.048 0.059 0.052 0.054 0.000 7.558 8.447 8.834 JXJGS 0.081 0.089 0.067 0.074 0.091 0.083 0.081 0.032 0.000 18.349 15.456 JXWZF 0.083 0.089 0.068 0.071 0.084 0.080 0.084 0.029 0.013 0.000 15.694 GDQXD 0.074 0.087 0.060 0.070 0.085 0.074 0.080 0.028 0.016 0.016 0.000 HNMS 0.070 0.075 0.066 0.065 0.076 0.073 0.095 0.037 0.028 0.025 0.028 HNYY 0.078 0.083 0.072 0.081 0.092 0.083 0.098 0.045 0.026 0.025 0.025 GXJX 0.084 0.087 0.071 0.076 0.086 0.080 0.092 0.044 0.044 0.034 0.042 GXHP 0.094 0.097 0.082 0.078 0.087 0.077 0.117 0.063 0.063 0.048 0.052 GXHJ 0.115 0.123 0.109 0.105 0.114 0.106 0.129 0.089 0.088 0.072 0.072 GXDMS 0.095 0.100 0.084 0.079 0.087 0.076 0.116 0.083 0.084 0.072 0.067 GXCWLS 0.101 0.112 0.094 0.087 0.095 0.084 0.124 0.070 0.075 0.056 0.057 GZYC 0.093 0.098 0.082 0.085 0.097 0.087 0.099 0.078 0.075 0.067 0.072 CQSMS 0.093 0.099 0.085 0.087 0.099 0.089 0.107 0.082 0.073 0.069 0.076 SCHGX 0.093 0.096 0.085 0.091 0.100 0.094 0.101 0.080 0.069 0.066 0.073 YNLFZ 0.099 0.104 0.093 0.092 0.102 0.095 0.113 0.085 0.083 0.073 0.078 V‐PXB 0.196 0.178 0.179 0.183 0.187 0.191 0.185 0.165 0.189 0.183 0.186 V‐HB 0.234 0.211 0.203 0.211 0.217 0.216 0.213 0.198 0.209 0.204 0.213 5 Pairwise population matrix of gene flow (upper triangle) and Fst values (lower triangle) for all populations Kirk, & Petersen, 2011; Hamilton, 2009). The He and Na values of F. hodginsii (He = 0.635, Na = 3.465) are slightly lower than those of C. obtusa (He = 0.780, Na = 7.038), albeit higher than those of other conifer species, such as Cryptomeria japonica (He = 0.277, Na = 2.000, Tsumura & Tomaru, 1999). In this study, the lowest genetic diversity was found in the two populations in Vietnam (V‐PXB: He = 0.573; V‐HB: He = 0.551). This phenomenon agreed with previous reports that most populations in markers used by Tam et al., 2011). It is possible that China serves as the central distributional area of F. hodginsii, such that its genetic diversity decreased as it dispersed from its central area to its mar‐ ginal areas, such as Vietnam (Wei, Sork, Meng, & Jiang, 2016). Tam et al. (2011) also indicated that, as a result of human disturbance, the F. 4.1 \| Genetic diversity FI G U R E 3 Relationship between pairwise Fst/(1 − Fst) and the geographic distance among the populations of Fokienia hodginsii (r = 0.882, p = 0.01) y = 0.0003x + 0.013 R² = 0.816 0 0.05 0.1 0.15 0.2 0.25 0.3 0 100 200 300 400 500 600 700 800 900 Fst (1-Fst) Geographic distance (km) TA B LE 5 Pairwise population matrix of gene flow (upper triangle) and Fst values (lower triangle) for all populations POP ZJJD ZJFYS FJHBL FJDYS FJFHS FJMHS JXMTS JXSQS JXJGS JXWZF GDQXD ZJJD 0.000 15.728 16.894 8.984 7.049 9.803 8.197 4.429 2.827 2.753 3.140 ZJFYS 0.016 0.000 13.977 10.007 8.882 10.419 6.643 3.796 2.555 2.571 2.627 FJHBL 0.015 0.018 0.000 10.589 6.818 10.312 10.169 5.290 3.454 3.430 3.901 FJDYS 0.027 0.024 0.023 0.000 29.065 22.317 5.358 4.917 3.143 3.288 3.337 FJFHS 0.034 0.027 0.035 0.009 0.000 20.908 4.115 4.006 2.506 2.724 2.681 FJMHS 0.025 0.023 0.024 0.011 0.012 0.000 4.969 4.514 2.763 2.887 3.113 JXMTS 0.030 0.036 0.024 0.045 0.057 0.048 0.000 4.346 2.849 2.733 2.863 JXSQS 0.053 0.062 0.045 0.048 0.059 0.052 0.054 0.000 7.558 8.447 8.834 JXJGS 0.081 0.089 0.067 0.074 0.091 0.083 0.081 0.032 0.000 18.349 15.456 JXWZF 0.083 0.089 0.068 0.071 0.084 0.080 0.084 0.029 0.013 0.000 15.694 GDQXD 0.074 0.087 0.060 0.070 0.085 0.074 0.080 0.028 0.016 0.016 0.000 HNMS 0.070 0.075 0.066 0.065 0.076 0.073 0.095 0.037 0.028 0.025 0.028 HNYY 0.078 0.083 0.072 0.081 0.092 0.083 0.098 0.045 0.026 0.025 0.025 GXJX 0.084 0.087 0.071 0.076 0.086 0.080 0.092 0.044 0.044 0.034 0.042 GXHP 0.094 0.097 0.082 0.078 0.087 0.077 0.117 0.063 0.063 0.048 0.052 GXHJ 0.115 0.123 0.109 0.105 0.114 0.106 0.129 0.089 0.088 0.072 0.072 GXDMS 0.095 0.100 0.084 0.079 0.087 0.076 0.116 0.083 0.084 0.072 0.067 GXCWLS 0.101 0.112 0.094 0.087 0.095 0.084 0.124 0.070 0.075 0.056 0.057 GZYC 0.093 0.098 0.082 0.085 0.097 0.087 0.099 0.078 0.075 0.067 0.072 CQSMS 0.093 0.099 0.085 0.087 0.099 0.089 0.107 0.082 0.073 0.069 0.076 SCHGX 0.093 0.096 0.085 0.091 0.100 0.094 0.101 0.080 0.069 0.066 0.073 YNLFZ 0.099 0.104 0.093 0.092 0.102 0.095 0.113 0.085 0.083 0.073 0.078 V‐PXB 0.196 0.178 0.179 0.183 0.187 0.191 0.185 0.165 0.189 0.183 0.186 V‐HB 0.234 0.211 0.203 0.211 0.217 0.216 0.213 0.198 0.209 0.204 0.213 10944 Yin et al. Yin et al. The He and Na values of F. hodginsii (He = 0.635, Na = 3.465) are slightly lower than those of C. obtusa (He = 0.780, Na = 7.038), albeit higher than those of other conifer species, such as Cryptomeria japonica (He = 0.277, Na = 2.000, Tsumura & Tomaru, 1999). markers used by Tam et al., 2011). It is possible that China serves as the central distributional area of F. hodginsii, such that its genetic diversity decreased as it dispersed from its central area to its mar‐ ginal areas, such as Vietnam (Wei, Sork, Meng, & Jiang, 2016). Tam et al. (2011) also indicated that, as a result of human disturbance, the F. hodginsii habitat in Vietnam has been degraded and fragmented, which may also serve as a good explanation for the low genetic vari‐ ability in Vietnam, as signals of bottleneck events were also detected in these two populations. markers used by Tam et al., 2011). It is possible that China serves as the central distributional area of F. hodginsii, such that its genetic diversity decreased as it dispersed from its central area to its mar‐ ginal areas, such as Vietnam (Wei, Sork, Meng, & Jiang, 2016). Tam et al. (2011) also indicated that, as a result of human disturbance, the F. hodginsii habitat in Vietnam has been degraded and fragmented, which may also serve as a good explanation for the low genetic vari‐ ability in Vietnam, as signals of bottleneck events were also detected in these two populations. In this study, the lowest genetic diversity was found in the two populations in Vietnam (V‐PXB: He = 0.573; V‐HB: He = 0.551). This phenomenon agreed with previous reports that most populations in Vietnam harbor low genetic diversity (HT = 0.0970 ± 0.0101, ISSR 10945 \| 1 Yin et al. 10945 In China, the populations GXDMS and GXHJ, where only 5–7 in‐ dividuals were collected, had the lowest genetic diversity (He = 0.590 and 0.606, respectively), and signals of bottleneck events were also detected in these two populations (Table 8). These phenomena may be explained by insufficient sampling. However, as a Tertiary relict species, this conifer was strongly influenced by the Pleistocene gla‐ ciations, resulting in the populations contracting sharply. In China, it has been more than 2,600 years since this conifer was used to build boats and houses, and due to extensive deforestation, the lower dis‐ tribution limit of this conifer has moved up by 500 m since the 1980s (Hou, Cheng, Lin, & Yu, 2004). During our field investigations, we also observed substantial evidence of deforestation near the F. hodginsii populations, and in many places where ample specimens were re‐ corded, few or no individual were found, especially in the populations of GXDMS and GXHJ. Further, the geographic locations of these two populations were near Vietnam, indicating that the low genetic diver‐ sity observed in GXDMS and GXHJ may be caused by the same fac‐ tors that account for the low genetic diversity observed in Vietnam. These phenomena may be explained by insufficient sampling. However, as a Tertiary relict species, this conifer was strongly influenced by the Pleistocene gla‐ ciations, resulting in the populations contracting sharply. In China, it has been more than 2,600 years since this conifer was used to build boats and houses, and due to extensive deforestation, the lower dis‐ tribution limit of this conifer has moved up by 500 m since the 1980s TA B LE 7 Analysis of molecular variance (AMOVA) for the 24 populations TA B LE 6 The relationship between genetic differentiation (Fst / (1 ‐ Fst)) and the differences in environmental factors HNMS HNYY GXJX GXHP GXHJ GXDMS GXCWLS GZYC CQSMS SCHGX YNLFZ V‐PXB V‐HB 3.343 2.949 2.733 2.423 1.929 2.384 2.214 2.446 2.433 2.446 2.272 1.028 0.834 3.095 2.769 2.621 2.330 1.781 2.256 1.990 2.294 2.268 2.356 2.146 1.153 0.937 3.567 3.211 3.264 2.805 2.053 2.739 2.400 2.809 2.690 2.708 2.450 1.145 0.982 3.569 2.824 3.055 2.937 2.140 2.918 2.628 2.682 2.610 2.505 2.457 1.114 0.937 3.046 2.475 2.655 2.627 1.944 2.626 2.393 2.330 2.275 2.253 2.200 1.083 0.901 3.194 2.757 2.885 3.008 2.105 3.054 2.733 2.615 2.565 2.405 2.374 1.059 0.906 2.369 2.290 2.461 1.890 1.694 1.902 1.768 2.282 2.093 2.232 1.971 1.098 0.924 6.432 5.252 5.487 3.726 2.559 2.765 3.342 2.966 2.785 2.879 2.690 1.268 1.013 8.783 9.286 5.492 3.714 2.576 2.736 3.083 3.103 3.152 3.375 2.772 1.071 0.948 9.949 9.895 7.042 4.949 3.204 3.234 4.229 3.456 3.382 3.542 3.168 1.114 0.978 8.729 9.654 5.705 4.548 3.210 3.481 4.142 3.200 3.051 3.178 2.943 1.092 0.924 0.000 16.444 8.039 7.419 3.663 4.156 4.843 4.550 4.413 4.601 4.243 1.224 1.078 0.015 0.000 5.557 5.149 3.316 2.930 3.795 3.485 3.530 3.752 3.908 1.150 1.006 0.030 0.043 0.000 11.990 4.966 6.925 8.235 8.724 7.952 9.582 5.730 1.096 0.995 0.033 0.046 0.020 0.000 5.076 10.578 16.548 5.910 6.292 6.249 5.671 1.021 0.999 0.064 0.070 0.048 0.047 0.000 3.609 4.202 4.178 3.508 3.323 3.564 0.954 0.854 0.057 0.079 0.035 0.023 0.065 0.000 10.311 4.494 5.002 4.432 3.287 0.881 0.889 0.049 0.062 0.029 0.015 0.056 0.024 0.000 4.992 5.596 4.800 4.649 0.899 0.857 0.052 0.067 0.028 0.041 0.056 0.053 0.048 0.000 24.807 19.394 14.473 1.043 0.981 0.054 0.066 0.030 0.038 0.067 0.048 0.043 0.010 0.000 24.201 14.370 1.033 1.007 0.052 0.062 0.025 0.038 0.070 0.053 0.050 0.013 0.010 0.000 16.536 1.099 1.065 0.056 0.060 0.042 0.042 0.066 0.071 0.051 0.017 0.017 0.015 0.000 1.115 1.087 0.170 0.179 0.186 0.197 0.208 0.221 0.218 0.193 0.195 0.185 0.183 0.000 4.527 0.188 0.199 0.201 0.200 0.226 0.220 0.226 0.203 0.199 0.190 0.187 0.052 0.000 TA B LE 6 The relationship between genetic differentiation (Fst / (1 ‐ Fst)) and the differences in environmental factors Influencing factors Formula r p Δmin temperature y = 0.0015x + 0.0808 0.067 0.27 Δaverage temperature y = 0.0017x + 0.0798 0.178 0.04 Δmax temperature y = 0.0019x + 0.0786 0.092 0.21 Δ precipitation y = 4E−05x + 0.0676 0.256 0.01 Δ elevation y = 3E−05x + 0.00707 0.205 0.1 Δ latitude y = 0.0094x + 0.052 0.432 0.01 Δ longitude y = 0 0043x + 0 0478 0 466 0 01 TA B LE 7 Analysis of molecular variance (AMOVA) for the 24 populations Source of variation Sum of squares Variance components Percentage of variation F‐statistics Among groups 394.651 0.61683 13.14 Fst:0.21430 Among populations within groups 169.975 0.10347 2.20 Fsc:0.02538 Within populations 3277.343 3.97323 84.66 FCT:0.13142 Total 3841 969 4 69353 100 00 HNMS HNYY GXJX GXHP GXHJ GXDMS GXCWLS GZYC CQSMS SCHGX YNLFZ V‐PXB V‐HB 3.343 2.949 2.733 2.423 1.929 2.384 2.214 2.446 2.433 2.446 2.272 1.028 0.834 3.095 2.769 2.621 2.330 1.781 2.256 1.990 2.294 2.268 2.356 2.146 1.153 0.937 3.567 3.211 3.264 2.805 2.053 2.739 2.400 2.809 2.690 2.708 2.450 1.145 0.982 3.569 2.824 3.055 2.937 2.140 2.918 2.628 2.682 2.610 2.505 2.457 1.114 0.937 3.046 2.475 2.655 2.627 1.944 2.626 2.393 2.330 2.275 2.253 2.200 1.083 0.901 3.194 2.757 2.885 3.008 2.105 3.054 2.733 2.615 2.565 2.405 2.374 1.059 0.906 2.369 2.290 2.461 1.890 1.694 1.902 1.768 2.282 2.093 2.232 1.971 1.098 0.924 6.432 5.252 5.487 3.726 2.559 2.765 3.342 2.966 2.785 2.879 2.690 1.268 1.013 8.783 9.286 5.492 3.714 2.576 2.736 3.083 3.103 3.152 3.375 2.772 1.071 0.948 9.949 9.895 7.042 4.949 3.204 3.234 4.229 3.456 3.382 3.542 3.168 1.114 0.978 8.729 9.654 5.705 4.548 3.210 3.481 4.142 3.200 3.051 3.178 2.943 1.092 0.924 0.000 16.444 8.039 7.419 3.663 4.156 4.843 4.550 4.413 4.601 4.243 1.224 1.078 0.015 0.000 5.557 5.149 3.316 2.930 3.795 3.485 3.530 3.752 3.908 1.150 1.006 0.030 0.043 0.000 11.990 4.966 6.925 8.235 8.724 7.952 9.582 5.730 1.096 0.995 0.033 0.046 0.020 0.000 5.076 10.578 16.548 5.910 6.292 6.249 5.671 1.021 0.999 0.064 0.070 0.048 0.047 0.000 3.609 4.202 4.178 3.508 3.323 3.564 0.954 0.854 0.057 0.079 0.035 0.023 0.065 0.000 10.311 4.494 5.002 4.432 3.287 0.881 0.889 0.049 0.062 0.029 0.015 0.056 0.024 0.000 4.992 5.596 4.800 4.649 0.899 0.857 0.052 0.067 0.028 0.041 0.056 0.053 0.048 0.000 24.807 19.394 14.473 1.043 0.981 0.054 0.066 0.030 0.038 0.067 0.048 0.043 0.010 0.000 24.201 14.370 1.033 1.007 0.052 0.062 0.025 0.038 0.070 0.053 0.050 0.013 0.010 0.000 16.536 1.099 1.065 0.056 0.060 0.042 0.042 0.066 0.071 0.051 0.017 0.017 0.015 0.000 1.115 1.087 0.170 0.179 0.186 0.197 0.208 0.221 0.218 0.193 0.195 0.185 0.183 0.000 4.527 0.188 0.199 0.201 0.200 0.226 0.220 0.226 0.203 0.199 0.190 0.187 0.052 0.000 In China, the populations GXDMS and GXHJ, where only 5–7 in‐ dividuals were collected, had the lowest genetic diversity (He = 0.590 and 0.606, respectively), and signals of bottleneck events were also detected in these two populations (Table 8). TA B LE 6 The relationship between genetic differentiation (Fst / (1 ‐ Fst)) and the differences in environmental factors populations Source of variation Sum of squares Variance components Percentage of variation F‐statistics Among groups 394.651 0.61683 13.14 Fst:0.21430 Among populations within groups 169.975 0.10347 2.20 Fsc:0.02538 Within populations 3277.343 3.97323 84.66 FCT:0.13142 Total 3841.969 4.69353 100.00 Influencing factors Formula r p Δmin temperature y = 0.0015x + 0.0808 0.067 0.27 Δaverage temperature y = 0.0017x + 0.0798 0.178 0.04 Δmax temperature y = 0.0019x + 0.0786 0.092 0.21 Δ precipitation y = 4E−05x + 0.0676 0.256 0.01 Δ elevation y = 3E−05x + 0.00707 0.205 0.1 Δ latitude y = 0.0094x + 0.052 0.432 0.01 Δ longitude y = 0.0043x + 0.0478 0.466 0.01 Yin et al. Yin et al. 10946 10946 \| FI G U R E 4 The best K value based on the result from STRUCTURE HARVESTER (a: ΔK; b: mean L(k)) –18,000 –16,000 –14,000 –12,000 –10,000 –8,000 –6,000 –4,000 –2,000 0 0 2 4 6 8 10 12 Mean (K) K MeanL (K) (a) (b) (a) in this study, the genetic diversity of F. hodginsii is primarily main‐ tained within populations (84.66%, p < 0.01), while the genetic dif‐ ferentiation among populations of F. hodginsii (Fst = 0.157 ± 0.019) is weak; however, the value of Fis was 0.176 ± 0.024, indicating a mixed mating system in which inbreeding occurred frequently. The genetic differentiation among populations of F. hodginsii (Fst = 0.157 ± 0.019) is also in accordance with that of other mixed‐breeding species of seed plants (79.2%, Nybom & Bartish, 2000), slightly higher than that of wind‐dispersed species (Fst = 0.13), and much lower than that of entomophilous species (Fst = 0.21) (Nybom, 2004). This pattern is also in accordance with previous observations that the dispersal of Fokienia is mainly through the wind, though sometimes also through insects (Jin et al., 2012; Lu et al., 2011; Wang & Ran, 2014). Such pat‐ terns were also observed in Cupressus funebris, for which the genetic diversity within populations is 88.15%, Fst = 0.1580 and Fis = 0.1579 (Lu et al., 2014). For the species C. obtusa, much higher genetic di‐ versity was maintained within populations (91.7%), and genetic dif‐ ferentiation among populations was lower (Fst = 0.039). The Fis value estimated for C. obtusa was only 0.034, indicating a random mating system. Therefore, the different levels of genetic differentiation among the three species may be caused primarily by the differentia‐ tion of mating systems. TA B LE 6 The relationship between genetic differentiation (Fst / (1 ‐ Fst)) and the differences in environmental factors –18,000 –16,000 –14,000 –12,000 –10,000 –8,000 –6,000 –4,000 –2,000 0 0 2 4 6 8 10 12 Mean (K) K MeanL (K) (b) (b) Mean (K) In this study, a significant correlation was found between ge‐ netic differentiation (Fst/(1 − Fst)) and geographic distance (r = 0.882, p = 0.01), suggesting that the genetic differentiation among pop‐ ulations follows the model of isolation by distance (IBD), that is, the differentiation among populations is strongly associated with geo‐ graphic distance. Such a phenomenon was also observed in C. obtusa (r2 = 0.3997 and p = 0.001, Matsumoto et al., 2010). It is also known that the dispersal of Fokienia is mainly through the wind (Jin et al., 2012; Lu et al., 2011; Wang & Ran, 2014); thus, its capability for long‐distance dispersal could be limited as the geographic distance increases. FI G U R E 4 The best K value based on the result from STRUCTURE HARVESTER (a: ΔK; b: mean L(k)) FI G U R E 6 Grouping of populations according to STRUCTURE (K = 3 or K = 4) and their geographic locations 4.2 \| Genetic differentiation Most conifers have high levels of genetic diversity within popula‐ tions and low levels of differentiation among populations (Hamrick, Godt, & Sherman‐Broyles, 1992). According to the AMOVA results Although significant correlations were also found between ge‐ netic differentiation and climatic variables in the sampled locations, FI G U R E 5 STRUCTURE individual assignment results for K = 3 and K = 4, based on simple sequence repeat data. Different colors represent different gene pools. K is the number of gene pools FI G U R E 5 STRUCTURE individual assignment results for K = 3 and K = 4, based on simple sequence repeat data. Different colors represent different gene pools. K is the number of gene pools FI G U R E 5 STRUCTURE individual assignment results for K = 3 and K = 4, based on simple sequence repeat data. Different colors represent different gene pools. K is the number of gene pools Yin et al. 10947 FI G U R E 6 Grouping of populations according to STRUCTURE (K = 3 or K = 4) and their geographic locations FI G U R E 7 Principal coordinate analysis of individual genotypes obtained from four groups GDQXD JXSQS JXJGS JXWZF JXMTS HNMS HNYY ZJJD ZJFYS FJHBL FJDYS FJFHS FJMHS GXCWLS GXDMS GXHP GXHJ GXJX YNLFZ GZYC CQSMS SCHGX V-PXB V-HB Coord. 2 (41.41%) Coord. 1 (44.11%) Principal coordinates (PCoA) E 6 Grouping of populations g to STRUCTURE (K = 3 or K = 4) geographic locations Principal coordinates (PCoA) FI G U R E 6 Grouping of populations according to STRUCTURE (K = 3 or K = 4) and their geographic locations FI G U R E 6 Grouping of populations according to STRUCTURE (K = 3 or K = 4) and their geographic locations Principal coordinates (PCoA) GDQXD JXSQS JXJGS JXWZF JXMTS HNMS HNYY ZJJD ZJFYS FJHBL FJDYS FJFHS FJMHS GXCWLS GXDMS GXHP GXHJ GXJX YNLFZ GZYC CQSMS SCHGX V-PXB V-HB Coord. 2 (41.41%) Coord. 1 (44.11%) Principal coordinates (PCoA) Coord. 2 (41.41%) FI G U R E 7 Principal coordinate analysis of individual genotypes obtained from four groups Yin et al. Yin et al. 10948 \| POP ID Wilcoxon test Sign test Model shift test I.A.M. T.P.M. I.A.M. T.P.M. 0948 \| TA B LE 8 Results of bottleneck analyses for each population Note. I.A.M.: infinite allele model of mutation; T.P.M.: two‐phased model of mutation. The bold values represent the significance values lower than 0.05 (p < 0.05). such as average temperature (r = 0.178, p = 0.04) and precipitation (r = 0.256, p = 0.01), their correlations were rather weak compared to those with geographic distance (r = 0.882, p = 0.01). It was ob‐ served that the flowering period of F. hodginsii is delayed with a de‐ crease in temperature and precipitation (Hou et al., 2006); therefore, climatic factors may also actively increase the genetic differentiation among populations to a lesser extent. of geographic distance. More molecular data need to be analyzed to understand this pattern. In this study, the assignment results for K = 4 were the same as the results from SAMOVA and PCoA. Therefore, it is reasonable to divide all populations into four groups: the eastern China group, the central China group, the western China group, and the Vietnam group. The terrain of China from west to east forms a flight of three steps, commonly called the “Three Steps”. The first step located in southwestern China mainly includes the Qinghai‐Tibetan Plateau, which has an elevation above 4,000 m. The second step lies in cen‐ tral and western China with an elevation of 1,000–3,000 m and includes the Xuefeng Mountains, Qinling Mountains, and Yunnan– Guizhou Plateau. The third step spans all remaining regions, covering eastern and southern China with an elevation of 500 m (Huang et al., 2012). The western China group is located on the second step, which mainly contains plateau and basin, while the central China group and the eastern China group are located on the third step, which mainly contains plain and hills. Additionally, the elevation of the sampled populations in the western China group is generally higher than that of populations in the central China group and eastern China group (Table 1). According to Hou et al. (2006), the flowering period of 4.2 \| Genetic differentiation ZJJD 0.0744 0.1618 0.2645 0.0623 L‐shaped ZJFYS 0.0853 0.1543 0.4768 0.1857 L‐shaped FJHBL 0.1034 0.1764 0.3783 0.2879 L‐shaped FJDYS 0.0953 0.1665 0.0624 0.2645 L‐shaped FJFHS 0.0847 0.1555 0.1742 0.6829 L‐shaped FJMHS 0.0963 0.1685 0.5305 0.1198 L‐shaped JXSQS 0.0748 0.133 0.3195 0.0456 L‐shaped JXMTS 0.0764 0.1319 0.381 0.2663 L‐shaped GDQXD 0.0608 0.1338 0.5969 0.6244 L‐shaped JXJGS 0.0608 0.1219 0.3142 0.2091 L‐shaped JXWZF 0.0543 0.1256 0.3201 0.3694 L‐shaped HNMS 0.0814 0.1706 0.3142 0.2377 L‐shaped HNYY 0.0764 0.1391 0.12 0.1542 L‐shaped GXCWLS 0.0975 0.625 0.1857 0.2645 L‐shaped GXDMS 0.0159 0.0312 0.0288 0.048 L‐shaped GXHP 0.1019 0.1497 0.6829 0.6238 L‐shaped GXHJ 0.0102 0.0096 0.0268 0.0379 L‐shaped GXJX 0.0858 0.1531 0.1238 0.1742 L‐shaped YNLFZ 0.0921 0.16 0.4487 0.5305 L‐shaped GZYC 0.0715 0.1479 0.2397 0.3192 L‐shaped CQSMS 0.091 0.1624 0.0803 0.3711 L‐shaped SCHGX 0.0784 0.1574 0.3169 0.4143 L‐shaped V‐PXB 0.0472 0.0264 0.0278 0.0326 L‐shaped V‐HB 0.0376 0.0473 0.0154 0.0471 L‐shaped Note. I.A.M.: infinite allele model of mutation; T.P.M.: two‐phased model of mutation. The bold values represent the significance values lower than 0.05 (p < 0.05). TA B LE 8 Results of bottlen analyses for each population 10948 AUTHORS’ CONTRIBUTIONS Liao, W.B. and Fan, Q. designed the research. Guo, W. and Huang, Y.SH. collected the samples. Yin, Q.Y., Huang, Y.L. and Zhou, R.CH. generated the data. Yin, Q.Y., Chen, S.F. and Zhou, R.CH. analyzed and interpreted the data. Yin, Q.Y. wrote the manuscript, and Chen, S.F. and Zhou, R.CH. edited the manuscript. 4.4 Genetic diversity plays an important role in determining the sur‐ vival and adaptability of a species (Liao et al., 2015). The high genetic diversity maintained within F. hodginsii and the initial sig‐ nificant genetic differentiation among its populations found in this study are encouraging. However, we found recent bottleneck events in the populations GXDMS, GXHJ, V‐PXB, and V‐HB, sug‐ gesting that individual populations may suffer from a dramatic de‐ cline in population size. As a Tertiary relict species, the range of this conifer contracted sharply during the Pleistocene glaciations, and our field investigations also showed that the F. hodginsii popula‐ tions have been overexploited since the 1980s, especially in the last ten years. For the conservation of this species, measures should be taken to increase the number of individuals and avoid the destruc‐ tion caused by human activities. Ex situ conservation and breed‐ ing can also be considered to maintain the greatest within‐species genetic variation, especially for the populations GXHJ and GXDMS, with higher inbreeding coefficients. Establishing seed orchards is also a good method, which could preserve favorable genes and prepare for breeding in the future. According to the results from STRUCTURE, the optimum number of groups is 4; thus, we also should establish seed orchards for these four groups to preserve their genotypes. In addition, establishing multiple F. hodginsii na‐ ture reserves, such as the Daiyunshan National Nature Reserve and Nanling National Nature Reserve, is needed, and the communities containing F. hodginsii should be classified as absolute protection areas to avoid human destruction. ACKNOWLEDGMENTS We thank the Daiyunshan National Nature Reserve, Mangshan National Nature Reserve, and Vietnam National Museum of Nature for allowing us to collect samples. This work was supported by the National Natural Science Foundation of China (31670189 and 31570195), the Natural Science Foundation of Guangdong Province, China(2016A030313326), the Special Program for Science and Technology Basic Research of the Ministry of Science and Technology of China (2013FY111500), the Foundation of Jinggangshan Administration of Jiangxi Province (33000‐7102993), the Fourth National Survey on Chinese Material Medical Resources Program for State Administration of Traditional Chinese Medicine of the People’s Republic of China (2017‐152‐003), the Fundamental Research Funds for the Central Universities (16lgjc38), and the Chang Hungta Science Foundation of Sun Yat‐sen University. Population differentiation was also found between the central China group and the eastern China group even though both of them are located on the third step. It was found that the central China group belongs to the Guangdong and Guangxi Hills while the eastern China group belongs to the Zhejiang and Fujian Hills, and between them, most areas are plains with a low elevation where no specimen records of F. hodginsii were found. Therefore, the plain area between the cen‐ tral and eastern China groups may have limited the gene flow between them and led to genetic differentiation, as we have found that iso‐ lation by distance was the main reason for genetic differentiation of F. hodginsii. However, it was surprisingly that the population JXSQS, located in the eastern China group, was closer to the central China group genetically (Figure 5). It is possible that some of the individuals could be later generations of ancient transplants from the central area, considering that F. hodginsii was often planted around the tombs and temples in China. DATA ACCESSIBILITY The primers used in this study are shown in Table 2, and all other data supporting the findings are available within the article and sup‐ plementary information file. 4.3 \| Population structure The STRUCTURE model based on 12 loci identified three as the most likely number of genetic clusters, as the highest ΔK value was at K = 3. The assignment results for K = 3 showed that the two populations in Vietnam were clustered with the eastern China group. In contrast, the results for K = 4 showed that the Vietnam populations were sepa‐ rated from the eastern China group and clustered as a fourth group. However, the populations located in Vietnam are located far away from those in eastern China, and the climatic conditions are much dif‐ ferent between the two regions. It is surprising that the two popula‐ tions in Vietnam were clustered with the eastern China group and not the western China group, which is much closer to Vietnam in terms Yin et al. Yin et al. 10949 \| F. hodginsii is delayed with an increase in elevation. Therefore, the change in topography may be the main reason for the population dif‐ ferentiation between the western China group and the central China group. Based on the specimen records and our field collections, the distribution of F. hodginsii is continuous between the western China group and the central China group; thus, populations located near the border, such as GXJX and GXHP, may receive gene flow from both groups and ultimately harbor mixed gene pools. CONFLICT OF INTEREST None declared. Qiang Fan http://orcid.org/0000-0003-4254-6936 implementing the Evanno method. Conservation Genetics Resources, 4(2), 359–361. https://doi.org/10.1007/s12686-011-9548-7 implementing the Evanno method. Conservation Genetics Resources, 4(2), 359–361. https://doi.org/10.1007/s12686-011-9548-7 Kovach, W. L. (1999). MVSP – A MultiVariate statistical package for win‐ dows, ver. 3.1 (p. 133). 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https://openalex.org/W3159742764	https://www.researchsquare.com/article/rs-449758/latest.pdf	English	null	ERIL: An Algorithm for Emotion Recognition From Indian Languages Using Machine Learning	Research Square (Research Square)	2,021	cc-by	708	ERIL: An Algorithm for Emotion Recognition From Indian Languages Using Machine Learning RAMOD MEHRA (  pramodmehra11@gmail.com ) entral Institute of Petrochemicals Engineering & Technology - Lucknow Roorkee Institute of Technology, Roorkee, Uttarakhand, India ERIL: An Algorithm for Emotion Recognition From Indian Languages Using Machine Learning PRAMOD MEHRA (  pramodmehra11@gmail.com ) CIPET - Lucknow: Central Institute of Petrochemicals Engineering & Technology - Lucknow Parag Jain Roorkee Institute of Technology, Roorkee, Uttarakhand, India Research Article Keywords: MFCC, LPC, Pitch, Indian Speech, Emotion recognition, emotion classification, Catboost Posted Date: April 23rd, 2021 DOI: https://doi.org/10.21203/rs.3.rs-449758/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License ERIL: An Algorithm for Emotion Recognition From Indian Languages Using Machine Learning PRAMOD MEHRA (  pramodmehra11@gmail.com ) CIPET - Lucknow: Central Institute of Petrochemicals Engineering & Technology - Lucknow Parag Jain Roorkee Institute of Technology, Roorkee, Uttarakhand, India Research Article Keywords: MFCC, LPC, Pitch, Indian Speech, Emotion recognition, emotion classification, Catboost Posted Date: April 23rd, 2021 DOI: https://doi.org/10.21203/rs.3.rs-449758/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Research Article License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/6 Abstract For a human interaction with machine, it is important that it understand the mood of the speaker. Until now we train machines on neutral speeches or utterances. The mood of a person would affect their performances. Deciphering human mood is challenging for the machines, as human can create fourteen distinct sound in a second. For a machine to understand the human behaviour, it should understand the acoustic abilities of the human ear. Mel Frequency Cepstral Coefficients (MFCC) and Linear Prediction coefficients (LPC) can replicate human auditory system. The proposed model Emotion Recognition from Indian Languages (ERIL) extracts emotions like fear, anger, surprise, sadness, happiness, and neutral. ERIL first pre-processes the voice signal, extracts selective MFCC, LPC, pitch, and voice quality features, then classifies the speech using Catboost. ERIL is a multilingual emotion classifier, it is independent of any language. We checked it on Hindi, Gujarati, Marathi, Punjabi, Bangla, Tamil, Oriya, and Telugu. We recorded a speech dataset of various emotions in these languages. ERIL is compared to other benchmark classifiers. Full-text Due to technical limitations, full-text HTML conversion of this manuscript could not be completed. However, the manuscript can be downloaded and accessed as a PDF. Figures Figure 1 Proposed Model Page 2/6 Figure 2 Original voice Figure 3 Pitch estimation plot Page 3/6 Figure 2 Original voice Figure 3 Pitch estimation plot Figure 2 g Figure 3 Figure 3 Pitch estimation plot Page 3/6 Page 3/6 Figure 4 Filtered voice Figure 5 Effect of Windowing Figure 6 Plot of the filter points Figure 4 Filtered voice Page 4/6 Figure 5 Effect of Windowing Figure 6 Plot of the filter points Figure 5 Effect of Windowing Figure 6 Plot of the filter points Effect of Windowing Figure 6 Plot of the filter points Plot of the filter points Page 4/6 Figure 7 Plot of Mel bands calculated Figure 8 Spectrogram plot of the second derivative of MFCC Figure 7 Plot of Mel bands calculated Figure 8 Spectrogram plot of the second derivative of MFCC Figure 7 Plot of Mel bands calculated Figure 8 Spectrogram plot of the second derivative of MFCC Figure 8 Spectrogram plot of the second derivative of MFCC Spectrogram plot of the second derivative of MFCC Page 5/6 Page 5/6 Figure 9 LPC plot with filtered voice Figure 10 Figure 9 LPC plot with filtered voice Figure 10 Based on table 6 a plot is drawn of contribution of each language in the accuracy. Figure 10 Based on table 6 a plot is drawn of contribution of each language in the accuracy Figure 10 Based on table 6 a plot is drawn of contribution of each language in the accuracy. Based on table 6 a plot is drawn of contribution of each language in the accuracy. Based on table 6 a plot is drawn of contribution of each language in the accuracy. Page 6/6 Page 6/6
https://openalex.org/W2609767462	https://research.rug.nl/files/56241617/Interpretation_of_the_cosmic_ray_air_shower_signal.pdf	English	null	Interpretation of the cosmic-ray air shower signal in Askaryan radio detectors	EPJ web of conferences	2,017	cc-by	2,995	Interpretation of the cosmic-ray air shower signal in Askaryan radio detectors Interpretation of the cosmic-ray air shower signal in Askaryan radio detectors p y g y de Vries, Krijn D.; Buitink, Stijn; van Eijndhoven, Nick; Meures, Thomas; O'Murchadha, Aongus; Scholten, Olaf Published in: 7th International Conference on Acoustic and Radio EeV Neutrino Detection Activities Published in: 7th International Conference on Acoustic and Radio EeV Neutrino Detection Activities DOI: 10.1051/epjconf/201713505001 IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF also known as Version of record IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Document Version Publisher's PDF, also known as Version of record Document Version Publisher's PDF, also known as Version of record Publication date: 2017 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): de Vries, K. D., Buitink, S., van Eijndhoven, N., Meures, T., O'Murchadha, A., & Scholten, O. (2017). Interpretation of the cosmic-ray air shower signal in Askaryan radio detectors. In 7th International Conference on Acoustic and Radio EeV Neutrino Detection Activities: ARENA 2016 (Vol. 135). Article 5001 https://doi.org/10.1051/epjconf/201713505001 Citation for published version (APA): de Vries, K. D., Buitink, S., van Eijndhoven, N., Meures, T., O'Murchadha, A., & Scholten, O. (2017). Interpretation of the cosmic-ray air shower signal in Askaryan radio detectors. In 7th International Conference on Acoustic and Radio EeV Neutrino Detection Activities: ARENA 2016 (Vol. 135). Article 5001 https://doi.org/10.1051/epjconf/201713505001 Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Citation for published version (APA): de Vries, K. D., Buitink, S., van Eijndhoven, N., Meures, T., O'Murchadha, A., & Scholten, O. (2017). Interpretation of the cosmic-ray air shower signal in Askaryan radio detectors. In 7th International Conference on Acoustic and Radio EeV Neutrino Detection Activities: ARENA 2016 (Vol. 135). Article 5001 https://doi.org/10.1051/epjconf/201713505001 1 Introduction In [1], we presented a calculation for the radio emission from a cosmic-ray air shower hitting an ice surface. It is shown that this signal should be observable by the currently existing Askaryan radio detectors [2–4]. The in-air emission, the in-ice emission, as well as the coherent transition radiation from the boundary were considered. In this article we focus on the interpretation of the observed signal and discuss the properties of the coherent transition radiation in view of the sudden appearance and sudden death signals. The sudden appearance signal is for example seen during beam- test experiments, for which the beam is hidden to the observer (within the observing frequency band) while inside the accelerator, after which it ’suddenly’ becomes visible while exiting the accelerator. An example of the sudden death signal is the signal originating from a beam-dump process. The importance of the cosmic-ray air shower signal in Askaryan radio detectors, if identiﬁed cor- rectly, lies in its possibility to calibrate the currently existing Askaryan radio detectors. Furthermore, if detected, this signal immediately shows the on-site feasibility of the Askaryan radio detection tech- nique. If misidentiﬁed, however, this signal might pose a background in the search for the signal from a high-energy neutrino-induced particle cascade in ice. ⋆e-mail: krijndevries@gmail.com Interpretation of the cosmic-ray air shower signal in Askaryan radio detectors Krijn D. de Vries1,⋆, Stijn Buitink2, Nick van Eijndhoven1, Thomas Meures3, Aongus O’Murchadha3, and Olaf Scholten1,4 Krijn D. de Vries1,⋆, Stijn Buitink2, Nick van Eijndhoven1, Thomas Meures3, Aongus O’Murchadha3, and Olaf Scholten1,4 1Vrije Universiteit Brussel, Dienst ELEM, IIHE, Pleinlaan 2, 1050 Brussels, Belgium 2Vrije Universiteit Brussel, Astrophysical Institute, Pleinlaan 2, 1050 Brussels, Belgium 1Vrije Universiteit Brussel, Dienst ELEM, IIHE, Pleinlaan 2, 1050 Brussels, Belgium 2Vrije Universiteit Brussel, Astrophysical Institute, Pleinlaan 2, 1050 Brussels, Belgium 3Dept. of Physics and Wisconsin IceCube Particle Astrophysics Center, University of Wisconsin, Madison, WI 53706,USA of Groningen, KVI-Center for Advanced Radiation Technology, 9747 AA Groningen, The Nethe 4University of Groningen, KVI-Center for Advanced Radiation Technology, 9747 AA Groningen, The Nether- lands Abstract. We discuss the radio emission from a cosmic-ray air shower propagating in air before it hits an air-ice boundary after which it completes its propagation inside the ice. The in-air emission, the in-ice emission, as well as the transition radiation from the shower crossing the boundary is considered. We discuss the interpretation of the radio signal observed by an in-ice observer. Copyright Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license. More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne- amendment. 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Download date: 24-10-2024 , (2017) 135 EPJ Web of Conferences ARENA 2016 05001 DOI: 10.1051/ 7135 epjconf/201 05001 © 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/). 2 Macroscopic modeling (5) (5) The obtained ﬁelds agree well with the microscopic calculations presented in [8–11], and references therein. The obtained ﬁelds agree well with the microscopic calculations presented in [8–11], and references therein. The coherent transition radiation obtained within this formalism can be understood as the emission just above the air-ice boundary at tr −ϵ, and the emission just below the boundary at tr + ϵ. Linking Eqs. 3 and 4, the coherent transition radiation can be interpreted as the superposition of the sudden appearance signal and the sudden death signal. As will be shown in the following section, under the proper circumstances these signals will indeed be observed separately in the case of coherent transition radiation. It should be noted that these solutions are limiting solutions. The derivation of the vector potential from Maxwell’s equations has to be considered with great care, since this is done under the assumption of a continuous medium, where in our situation we consider a hard boundary. This is reﬂected by the fact that tr(t) is discontinuous at the boundary, and hence the retarded distance is ill deﬁned at this point. 2 Macroscopic modeling The calculations in [1] are based on a macroscopic calculation [5–7], starting from the Liénard- Wiechert potentials from classical electrodynamics. The electric ﬁeld is directly obtained from the © 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/). , (2017) 135 EPJ Web of Conferences ARENA 2016 05001 , (2017) 135 EPJ Web of Conferences ARENA 2016 05001 DOI: 10.1051/ 7135 epjconf/201 05001 charge and current distributions in the cascade front. The ﬁelds have to be evaluated at the negative retarded emission time, tr, which links to the observer time, t, through the optical path length L from the emission point to the observer, c(t −tr) = L. (1) (1) The optical path length inside a medium consisting out of m layers with diﬀerent index of refraction ni, can be deﬁned by L = m X i=1 nidi. (2) (2) Here the distance di, traversed by the emission in layer i, is obtained by using a ray-tracing procedure based on Snell’s law. The in-air, as well as the in-ice emission originates from the time-variation of the total number of particles in the cascade, dN(tr)/dtr, in combination with relativistic boosting eﬀects. In this article, however, we will focus on the coherent transition radiation and its link to the sudden appearance and sudden death signal. In [1], the coherent transition radiation signal was given by, ⃗Etr(t, ⃗x) = lim ϵ→0 Z d2⃗re d Ne(tr) w(⃗r, h) 4πϵ0c ×  1 \|D\|2 tr−ϵ − 1 \|D\|2 tr+ϵ ˆp h=c(tr−tb) . (3) (3) Following the same procedure the sudden appearance signal is given by, ⃗Esa(t, ⃗x) = −lim ϵ→0 Z d2⃗r e d Ne(tr) w(⃗r, h) 4πϵ0c \|D\|2 tr+ϵ ˆp h=c(tr−tb) . (4) (4) Here d denotes the impact distance from the observer to the shower axis, where w(⃗r, h) denotes the particle distribution in the cascade front at lateral position ⃗r and a distance h behind the cascade front. The retarded distance D is deﬁned by, d Here d denotes the impact distance from the observer to the shower axis, where w(⃗r, h) denotes the particle distribution in the cascade front at lateral position ⃗r and a distance h behind the cascade front. The retarded distance D is deﬁned by, d D = L dt dtr . 3 Results and interpretation How are these results reﬂected in our simulation? This is shown in Fig. (1), where we show the signal observed for a perpendicular incoming air shower which penetrates the ice at an elevation of 3 km, 2 2 , (2017) 135 EPJ Web of Conferences ARENA 2016 05001 , (2017) 135 EPJ Web of Conferences ARENA 2016 05001 DOI: 10.1051/ 7135 epjconf/201 05001 -1200 -800 -400 0 400 E( V/m) 262.5 280.0 297.5 315.0 t(ns) d=40 m In-air In-Ice TR 1 10 10 2 10 3 10 4 (z-90)[m] 262.5 280.0 297.5 315.0 t(ns) 0.0 0.875 1.75 2.625 3.5 Ne(z) [107] Ne air ice air ice -4200 -2800 -1400 0 1400 E( V/m) 400 600 800 1000 1200 1400 t(ns) d=240 m In-air In-Ice TR 1 10 102 10 3 10 4 (z-90)[m] 287.5 575.0 862.5 1150.0 t(ns) 0.0 14.375 28.75 43.125 57.5 Ne(z) [107] Ne air ice air ice Figure 1. The electric ﬁeld at diﬀerent observer distances equal to, a) d = 40 m, b) d = 240 m. The ﬁgures on the right show the emission height, plotted as function of the observer time. The full red line gives the emission in air, the dotted purple line gives the transition radiation, and the dashed blue line gives the in-ice emission. For the ﬁgures on the right, the total number of particles is given by the full green line. Figure and caption taken from [1]. 1 10 10 2 10 3 10 4 (z-90)[m] 262.5 280.0 297.5 315.0 t(ns) 0.0 0.875 1.75 2.625 3.5 Ne(z) [107] Ne air ice air ice -1200 -800 -400 0 400 E( V/m) 262.5 280.0 297.5 315.0 t(ns) d=40 m In-air In-Ice TR E( V/m) 1 10 102 10 3 10 4 (z-90)[m] 287.5 575.0 862.5 1150.0 t(ns) 0.0 14.375 28.75 43.125 57.5 Ne(z) [107] Ne air ice air ice -4200 -2800 -1400 0 1400 E( V/m) 400 600 800 1000 1200 1400 t(ns) d=240 m In-air In-Ice TR E( V/m) Figure 1. The electric ﬁeld at diﬀerent observer distances equal to, a) d = 40 m, b) d = 240 m. The ﬁgures on the right show the emission height, plotted as function of the observer time. The full red line gives the emission in air, the dotted purple line gives the transition radiation, and the dashed blue line gives the in-ice emission. 3 Results and interpretation For the ﬁgures on the right, the total number of particles is given by the full green line. Figure and caption taken from [1]. after which the cascade completes its propagation inside the ice. The observer is positioned in the ice, 100 m below the air-ice boundary. The detailed particle distributions are given in [1]. The top ﬁgure shows the signal observed by an observer located inside the in-ice Cherenkov cone, but outside the in-air Cherenkov cone. The interpretation of this signal can be seen in the top-right ﬁgure, where we show the emission height, linked to the emission time by z = −ctr, as function of the observer time. For the in-air emission, since the observer is positioned outside the Cherenkov cone, early emission from large heights is seen before late emission, where the emission from close to the boundary is observed latest. The in-ice emission, since seen from inside the Cherenkov cone, is seen diﬀerently. Late emission is seen before early emission. It also follows that the in-ice emission is observed at the same time as the in-air emission. Lastly the emission from just above and just below the boundary superimpose to give the coherent transition radiation component. The bottom ﬁgure shows a very interesting situation. Here the point where the cascade hits the boundary is both outside the in-air, as well as outside the in-ice Cherenkov cone. Since the in-ice Cherenkov angle also denotes the critical angle, the in-air emission only enters the ice at this angle. 3 3 DOI: 10.1051/ 7135 epjconf/201 05001 DOI: 10.1051/ 7135 epjconf/201 05001 , (2017) 135 EPJ Web of Conferences ARENA 2016 05001 Hence, the emission from just above the boundary ﬁrst travels through the air, before it breaks into the ice at the critical angle. Even though this is the longer path, since the signal is not delayed by the medium for its in-air travel, it will arrive before the signal emitted from just below the boundary which traverses its full path to the observer inside the ice. It follows that in these geometries, the in-air and the in-ice emission are completely separated in time. Hence, the observer will ﬁrst observe the emission from the in-air cascade, after which it appears that the cascade suddenly dies out. The dying out of the cascade is given by the sudden death signal. 3 Results and interpretation After some delay, from the observer point of view, the cascade suddenly appears after which the in-ice emission is seen. Hence in this situation, the emission just above the boundary is completely separated from the emission just below the boundary. Therefore, the transition radiation is not seen at a single time, but becomes separated in time. Within this framework, therefore the more fundamental description is given by the sudden appearance and sudden death signals. The observation of such a signal separated in time would con- ﬁrm the interpretation of coherent transition radiation as the superposition of a the more fundamental sudden appearance and sudden death signals. 4 Summary We discussed the interpretation of the coherent transition radiation signal, which originates from a cosmic-ray-induced air shower traversing between air and ice. It follows that within the given for- malism, this can be interpreted as the superposition of two more fundamental signals given by the cascade sudden appearance and sudden death signals. This interpretation should be taken with care, since the derived potentials on which the calculations are based implicitly rely on the assumption of a continuous medium. This is reﬂected in the fact that the retarded distance is ill deﬁned at the air-ice boundary. To solve for the ﬁelds, the limiting situation of the emission just above and just below the air-ice boundary is considered. The superposition of these two components gives rise to the coherent transition radiation signal. It is shown that in certain geometries, where the observer is positioned outside the in-ice Cherenkov cone for emission directly below the air-ice boundary, the coherent transition radiation is split in two signal separated in time. These signals, the sudden appear- ance signal and the sudden death signal, can therefore within the given formalism be interpreted as more fundamental. References [1] K.D. de Vries et al., Astropart. Phys. 74, 96-104 (2016) [2] P.W. Gorham et al., Phys. Rev. Lett. 103, 051103 (2009) [3] P. Allison et al., ARA Collaboration, Astropart. Phys. 35, 457-477 (2012) [4] A. Nelles et al., ARIANNA Collaboration, these proceedings [5] O. Scholten, K. Werner, F. Rusydi, Astropart. Phys. 29, 94-103 (2008) [6] K.D. de Vries, A.M. van den Berg, O. Scholten, K. Werner, Phys. Rev. Lett. 107, 061101 (2011) [7] K. Werner, K.D. de Vries, O. Scholten, Astropart. Phys. 37, 5-16 (2012) [8] V.L. Ginzburg, V.N. Tsytovich, Transition Radiation and Transition Scattering, Adam Hilger Press, New York, 1990 [9] S. ter Veen et al., Phys. Rev. D 82, 103014 (2010) [10] C.W. James, H. Falcke, T. Huege, M. Ludwig, Phys. Rev. E 84, 056602 (2011) [11] P. Motloch, J. Alvarez-Muñiz, P. Privitera, E. Zas, Phys. Rev. D 93, 043010 (2016) [1] K.D. de Vries et al., Astropart. Phys. 74, 96-104 (2016) [3] P. Allison et al., ARA Collaboration, Astropart. Phys. 35, 457-477 (2012) [4] A. Nelles et al., ARIANNA Collaboration, these proceedings [5] O. Scholten, K. Werner, F. Rusydi, Astropart. Phys. 29, 94-103 (2008) [6] K.D. de Vries, A.M. van den Berg, O. Scholten, K. Werner, Phys. Rev. Lett. 107, 061101 (2011) [8] V.L. Ginzburg, V.N. Tsytovich, Transition Radiation and Transition Scattering, Adam Hilger Press, New York, 1990 [8] V.L. Ginzburg, V.N. Tsytovich, Transition Radiation and Transition Scattering, Adam Hilger Press, New York, 1990 [9] S. ter Veen et al., Phys. Rev. D 82, 103014 (2010) C.W. James, H. Falcke, T. Huege, M. Ludwig, Phys. Rev. E 84, 056602 (2011) [10] C.W. James, H. Falcke, T. Huege, M. Ludwig, Phys. Rev. E 84, 056602 (2011) [11] P. Motloch, J. Alvarez-Muñiz, P. Privitera, E. Zas, Phys. Rev. D 93, 043010 (2016) 4 4
https://openalex.org/W4214706658	https://dergipark.org.tr/tr/download/article-file/2029685	English	null	Koronavirüs 2019 Geçirmiş Vertigosu Olan ve Olmayan Hastaların Subjektif Değerlendirilmesi	İnönü üniversitesi sağlık hizmetleri meslek yüksekokulu dergisi :	2,022	cc-by	4,233	SUBJECTIVE EVALUATION OF CORONAVIRUS 2019 PATIENTS WITH AND WITHOUT VERTIGO WITHOUT VERTIGO Koronavirüs 2019 Geçirmiş Vertigosu Olan ve Olmayan Hastaların Subjektif Değerlendirilmesi Tuğba EMEKCİ1 Fatmanur UYSAL2 Serpil DEMİR3 Mehmet Akif DÜNDAR4 1,4Necmettin Erbakan University, Faculty of Medicine ENT Clinic, Konya 2Doğuş University, İstanbul 3Başkent University, Ankara Subjektif Değerlendirilmesi İ1 Fatmanur UYSAL2 Serpil DEMİR3 Mehmet Akif DÜNDAR4 1,4Necmettin Erbakan University, Faculty of Medicine ENT Clinic, Konya 2Doğuş University, İstanbul 3Başkent University, Ankara j ğ Tuğba EMEKCİ1 Fatmanur UYSAL2 Serpil DEMİR3 Mehmet Akif DÜNDAR4 1,4Necmettin Erbakan University, Faculty of Medicine ENT Clinic, Konya 2Doğuş University, İstanbul 3Başkent University, Ankara Tuğba EMEKCİ1 1 4 Geliş Tarihi / Received: 15.10.2021 Kabul Tarihi / Accepted: 01.03.2022 Journal of Inonu University Health Services Vocational School ISSN: 2147-7892, Volume 10, Issue 2 (2022) 521-529 doi: 10.33715/inonusaglik.1010406 Journal of Inonu University Health Services Vocational School ISSN: 2147-7892, Volume 10, Issue 2 (2022) 521-529 doi: 10.33715/inonusaglik.1010406 İnönü Üniversitesi Sağlık Hizmetleri Meslek Yüksekokulu Dergisi ISSN: 2147-7892, Cilt 10, Sayı 2 (2022) 521-529 doi: 10.33715/inonusaglik.1010406 İnönü Üniversitesi Sağlık Hizmetleri Meslek Yüksekokulu Dergisi ISSN: 2147-7892, Cilt 10, Sayı 2 (2022) 521-529 doi: 10.33715/inonusaglik.1010406 Original Article / Araştırma Makalesi Original Article / Araştırma Makalesi Original Article / Araştırma Makalesi Keywords: COVID-19, SARS-CoV-2, Vertigo. Keywords: COVID-19, SARS-CoV-2, Vertigo. Keywords: COVID-19, SARS-CoV-2, Vertigo. Keywords: COVID-19, SARS-CoV-2, Vertigo. 5 Bu makaleye atıf yapmak için(How to cite this article): Emekci, T., Uysal, F., Demir, S. & Dündar, M. A. (2022). Subjective evaluation of coronavirus 2019 patients with and without vertigo. İnönü Üniversitesi Sağlık Hizmetleri Meslek Yüksekokulu Dergisi, 10(2), 521-529. doi: 10.33715/inonusaglik.1010406 ğ Necmettin Erbakan University, Faculty of Medicine ENT Clinic, Konya ABSTRACT The purpose of the present study is to compare individuals with and without vertigo who have experienced COVID-19 in terms of their psychological, emotional, sleep quality, and concerns about the possibility of falls. A total of 30 individuals were included in the study, including the case group with 15 subjects who were diagnosed with vertigo with past COVID-19, and the control group with 15 subjects who had past COVID-19 and were not diagnosed with vertigo. The Falls Efficacy Scale-International (FES-I), Hospital Anxiety and Depression Scale (HADS), and Pittsburgh Sleep Quality Index (PSQI) were applied face-to-face to the individuals who were included in the research. Among the participants’, who were included in the study; statistically significant differences in anxiety, depression, falls, and PUKI scores have been detected between the experimental and control groups (p<0.05). Of those in the control group; anxiety, depression, falls and PUKI scale scores have been found to be lower than the experimental group. Patients with vertigo who apply to the clinic must be evaluated in this respect, and clinicians must be careful in terms of the patients to receive psychological support. Tuğba EMEKCİ, t.emekci@hotmail.com Tuğba EMEKCİ, t.emekci@hotmail.com INTRODUCTION The coronavirus 2019 (COVID-19) is a single-stranded RNA virus that can cause a wide spectrum of clinical manifestations, from the common cold, pneumonia, respiratory failure and death to the much more severe lower respiratory tract diseases (Batra et al., 2020). The first case was seen in Wuhan, China and then spread to the whole world (Alhazzani et al., 2021). The reference name of the virus causing the disease was determined as severe acute respiratory syndrome-coronavirus-2 (severe acute respiratory syndrome-coronavirus-2 [SARSCoV-2]) by the World Health Organization. In clinical studies, the most common symptoms of COVID-19 were reported as fever, cough, shortness of breath, myalgia, arthralgia, headache, diarrhea, rhinorrhea, and sore throat (Wan et al., 2020; Wong, Leo & Tan, 2020). There are studies conducted on whether the SARS-CoV-2 virus has indirect or direct neurotrophic effects on the nervous system (Niazkar, Zibaee, Nasimi & Bahri, 2020; Román et al., 2020). In a study conducted with people with and without a diagnosis of COVID-19, it was reported that both the auditory and vestibular systems were affected (Tan et al., 2022). On the other hand, various neurological symptoms such as loss of consciousness, headache, and vertigo were also reported in COVID-19 patients (Ahmad & Rathore, 2020; Korkmaz, Eğilmez, Özçelik & Güven, 2021; Mao et al., 2020; Moriguchi et al., 2020). Among otological symptoms, the cases of; facial paralysis, sudden hearing loss, and vertigo were associated with COVID-19 (Sriwijitalai & Wiwanitkit, 2020; Vaira, Salzano, Deiana & De Riu, 2020). Although it is not known how the COVID-19 virus affects both peripheral and central cochleovestibular pathways, objective findings were reported in many studies (Ahmad & Rathore, 2020; Korkmaz et al., 2021; Mao et al., 2020; Moriguchi et al., 2020; Niazkar et al., 2020; Román et al., 2020; Sriwijitalai & Wiwanitkit, 2020; Tan et al., 2022; Vaira et al., 2020; Wong et al., 2020). However, individuals who had vertigo with past COVID-19 must also be evaluated subjectively in addition to objective findings. The purpose of the present study was to compare individuals with and without vertigo with past COVID-19 in terms of psychological, emotional, sleep quality, and concerns about the possibility of falls. ÖZ Bu çalışmanın amacı; COVID 19 geçirmiş, vertigosu olan ve olmayan bireylerin psikolojik, emosyonel, uyku kalitesi ve düşme ihtimaline yönelik endişeleri açısından karşılaştırılmasıdır. Araştırmaya, COVID 19 geçirmiş vertigo tanısı almış 15 denek vaka grubu ve COVID 19 geçirmiş vertigo tanısı almamış 15 denek kontrol grubu olmak üzere 30 birey dâhil edildi. Araştırmaya dahil edilen bireylere, Uluslararası Düşme Etkinliği Ölçeği (Falls Efficacy Scale International- FES -I), Hastane Anksiyete ve Depresyon Ölçeği (Hospital Anxiety and Depression Scale-HADS) ve Pittsburgh Uyku Kalite İndeksi (Pittsburgh Sleep Quality Index-PSQI) yüz yüze uygulandı. Çalışmaya alınan katılımcıların; anksiyete, depresyon, düşme ve PUKİ ölçeğinden alınan puanlarda deney ve kontrol grupları arasında istatistiksel olarak anlamlı farklılık bulunmuştur (p<0,05). Kontrol grubunda yer alanların; anksiyete, depresyon, düşme ve PUKİ ölçek puanlarının deney grubuna göre düşük olduğu tespit edilmiştir. Kliniğe başvuran vertigolu hastalar bu açıdan değerlendirilmeli ve klinisyenler hastaların psikolojik destek almaları açısından dikkatli olmalıdır. Anahtar kelimeler: COVID-19, SARS-CoV-2, Vertigo. 521 ISSN: 2147-7892, Cilt 10 Sayı 2 (2022) 521-529 doi: 10.33715/inonusaglik.1010406 ISSN: 2147 7892, Cilt 10 Sayı 2 (2022) 521 529 Subjective Evaluation of Coronavirus 2019 Patients with and without Vertigo. Tuğba EMEKCİ, Fatmanur UYSAL, Serpil DEMİR, Mehmet Akif DÜNDAR MATERIAL AND METHOD Approval was obtained from Necmettin Erbakan University Health Sciences Institute Non-Interventional Clinical Research Ethics Committee (Decisions Number: 2021/3420), and “informed consent” was taken from all individuals participating in the study. 522 ISSN: 2147-7892, Cilt 10 Sayı 2 (2022) 521-529 doi: 10.33715/inonusaglik.1010406 y Subjective Evaluation of Coronavirus 2019 Patients with and without Vertigo. Tuğba EMEKCİ, Fatmanur UYSAL, Serpil DEMİR, Mehmet Akif DÜNDAR y Subjective Evaluation of Coronavirus 2019 Patients with and without Vertigo. Tuğba EMEKCİ, Fatmanur UYSAL, Serpil DEMİR, Mehmet Akif DÜNDAR The study was conducted prospectively in the Audiology Unit of the Ear Nose and Throat Clinic of Necmettin Erbakan University Hospital between October 2021 and December 2021. A total of 30 individuals were included in the study, the case group with 15 subjects who were diagnosed with vertigo with past COVID-19, and the control group with 15 subjects who have past COVID-19 and were not diagnosed with vertigo. Exclusion criteria were the presence of communication barrier, chronic disease, history of previously diagnosed balance problems, and other otological-neurorootological diseases. The International Falls Efficacy Scale (FES-I), Hospital Anxiety and Depression Scale (HADS), and Pittsburgh Sleep Quality Index (PSQI) were applied face-to-face to the individuals who were included in the study. International Falls Effectiveness Scale is a feedback scale on the level of anxiety about falls during activities of daily living (Yardley et al., 2005). The Turkish validity and reliability study was conducted by Ulus et al. in 2012. The scale consists of 16 questions, and the total score varies between 16 and 64 (Ulus et al., 2012). Hospital Anxiety and Depression Scale was developed by Zigmond and Snaith in 1983 to evaluate the anxiety and depression of patients (Zigmond & Snaith, 1983). Aydemir et al. (1997) conducted the Turkish validity and reliability of the scale, which is not used to diagnose but to define anxiety and depression in a short time in patients who have physical illnesses and in those applying to primary healthcare services. The scale includes; 7 questions on anxiety (odd-numbered questions), 7 questions that evaluate depression (even-numbered questions), and consists of 14 questions in total. The responses are scored between 0 and 3. The lowest score that patients can receive from both subscales is 0 and the highest score is 21. Pittsburgh Sleep Quality Index was developed by Buysse et al. MATERIAL AND METHOD to evaluate sleep quality and disorder in the last month (Buysse, Reynolds III, Monk, Berman & Kupfer, 1989). It was adapted into Turkish by Agargun et al. in 1996. The scale consists of 24 questions, 10 of which are answered by the individual himself, and 5 questions are answered based on the observations of his spouse or roommate. The total score ranges between 0 and 21. A total score greater than 5 indicates “poor sleep quality” (Agargun, Kara & Anlar, 1996). Demographic Data A total of 30 participants were included in the study, of which 15 were in the experimental group, and 15 were in the control group. The mean age of the participants was 42.13 ± 9.05 in the experimental group, and the age range varied between 28 and 60. The mean age of the participants in the Control Group was 41.40 ± 10.45, and the age range varied between 24 and 63. Statistical Analysis The analysis of the data of the study was made with the SPSS (Statistical Program in Social Sciences) 25 program. The Kolmogorov Smirnov Test was used to check whether the data fit the Normal distribution. Since the data were distributed normally, comparisons between the case and control (Covid (+), Covid (-)) group were made with the significance test (t-test) of the difference between the two mean values. The homogeneity of variance was 523 doi: 10.33715/inonusaglik.1010406 ISSN: 2147-7892, Cilt 10 Sayı 2 (2022) 521-529 checked with the Levine’s Test to decide which test result would be used in the comparison (p>0.05). The values of the variables are given as number, percentage, mean, and standard deviation. The Cronbach α Coefficient was used to determine the reliability analysis of the scales. The Cronbach α Coefficient of the participants was calculated as 0.89 for anxiety, 0.83 for depression, 0.92 for falls, and 0.91 for PUKI in the experimental group. The Cronbach α Coefficient of the participants was calculated as 0.91 for anxiety, 0.81 for depression, 0.94 for falls, and 0.87 for PUKI in the control group. The reliability of the scales was detected to be adequate for both groups. The correlation coefficients are the criteria that provide information on the strength (degree) and direction of the relations between variables. Values used frequently in the evaluation of the findings were interpreted as 0.40 - 0.69 moderate relation, 0.70 - 0.89 strong relation, and 0.90 - 1.00 very strong relation (Alpar, 2020). The Pearson Relation Coefficient was used as the variables included in the study showed normal distribution. In the Experimental Group Participants A high-level, positive (r=0.760), and statistically significant relation was detected between anxiety and depression (p<0.05). Statistically significant positive relations were detected (r=0.705) between anxiety and PUKI (p<0.05, Table 2). No statistically significant relations were detected between anxiety and falls (p>0.05). Positive correlation was found (r=0.766) between depression and PUKI (p<0.05). No statistically significant relations were detected between depression and falls (p>0.05). No statistically significant relations were detected between PUKI and falls (p>0.05, Table 2). Comparison of Groups According to Scale Scores It was tested whether the participants who were included in the study showed differences between the control and case groups in the scores of anxiety, depression, falls, and the PUKI scale, and the results of the analysis are given in the table below. Table 1. Comparison of Groups According to Scale Scores Variable Group Mean ± sd Test Value p Value Anxiety Control 6.73 ± 2.31 -3.845 0.001* Case 11.27 ± 3.94 Depression Control 7.27 ± 2.28 -2.486 0.019* Case 10.27 ± 4.08 Falls Total Score Control 10.67 ± 3.92 -4.502 0.001* Case 23.2 ± 10.04 PUKI score Control 6.87 ± 2.45 -3.109 0.004* Case 10.8 ± 4.25 Mean; Mean, sd; standard deviation, Test value; significance test t value of the difference between the two means; p; statistical significance, p<0.05; there is a statistically significant difference between the groups. Table 1. Comparison of Groups According to Scale Scores Table 1. Comparison of Groups According to Scale Scores 524 ISSN: 2147-7892, Cilt 10 Sayı 2 (2022) 521-529 ISSN: 2147-7892, Cilt 10 Sayı 2 (2022) 521-529 doi: 10.33715/inonusaglik.1010406 Subjective Evaluation of Coronavirus 2019 Patients with and without Vertigo. Tuğba EMEKCİ, Fatmanur UYSAL, Serpil DEMİR, Mehmet Akif DÜNDAR Statistically significant differences were detected between the experimental and control groups in the anxiety, depression, falls, and PUKI scores of the participants who were included in the study (p<0.05, Table 1). It was found that the scores of the control group were lower in the anxiety, depression, falls, and PUKI scales than the experimental group. Comparison of the Relations of the Scale Scores Between Groups The participants of the experimental and control groups were tested whether there were relations between anxiety, depression, falls, and the scores obtained in the PSQI scale, and the results are given in the table below. Table 2. Comparison of the Relations of Scale Scores between Groups Table 2. Comparison of the Relations of Scale Scores between Groups First variable Second Variable Control Experimental r Value p Value r Value p Value Anxiety Depression 0.731 0.002 0.760 0.001* Falls 0.297 0.283 0.409 0.130 PUKI 0.422 0.117 0.705 0.003* Depression Falls 0.529 0.042* 0.375 0.168 PUKI 0.340 0.216 0.766 0.001* Falls PUKI 0.218 0.434 -0.016 0.956 r; Pearson correlation coefficient, p; statistical significance, *p<0.05; there is a statistically significant relation between scores. DISCUSSION Vertigo, or dizziness, has recently been identified as a clinical manifestation of COVID- 19, according to studies conducted around the world (Baig, Khaleeq, Ali & Syeda, 2020; Mao et al., 2020; Wu et al., 2020). In a study conducted in China, researchers stated that the most common symptom of COVID-19 is dizziness (Mao et al., 2020). Another study by Baig et al. suggests that the virus enters neural tissue from the circulation and binds to angiotensin- converting enzyme 2 receptors located in the capillary endothelium (Baig et al., 2020). Apart from this, it is assumed that mechanisms such as direct invasion, neuronal invasion, hypoxia, and hypercoagulopathy cause dizziness (Wu et al., 2020). Epidemic/pandemics affect both physical and mental health negatively (Xiao, Zhang, Kong, Li & Yang, 2020; Xue et al., 2020). During the SARS (severe acute respiratory syndrome) epidemic, stress, anxiety, and depression increased, and sleep quality was affected in the general population (Altena et al., 2020; Wu, Chan & Ma, 2005). Decreased sleep duration and quality increase the risk of viral infections (Gamaldo, Shaikh & McArthur, 2012; Xiao et al., 2020), and stress impairs sleep quality (Van Reeth et al., 2000). In our study which was conducted to investigate the psychological, emotional, sleep quality, and the possibility of falls in individuals with vertigo and past COVID-19, it was found that the patient group had higher anxiety, depression, falls, and sleep quality scores than the control group. It was observed in general that there were positive relations between anxiety and depression in the patient and control groups in line with the literature, and anxiety and depression negatively affected sleep in the patient group. In the literature, there is no study evaluating mental status and falling in patients with vertigo diagnosed with COVID-19. Although delirium, depression, insomnia, anxiety, and post-traumatic stress disorder have been reported in the acute phase of COVID-19 infection, few studies are investigating long-term psychiatric symptoms after infection (Rogers et al., 2020). Studies investigating psychiatric findings in patients who recovered from COVID-19 infection reported a high rate of insomnia, post-traumatic stress disorder, depression, and anxiety symptoms (Liu et al., 2020; Mazza et al., 2020; Tomasoni et al., 2021). In a study, it was reported that more than half of those who had COVID-19 infection experienced anxiety, depression, post-traumatic stress disorder, and/or obsessive-compulsive symptoms in a month after treatment (Mazza et al., 2020). In the Control Group Participants Positive correlation was found (r=0.731) between anxiety and depression (p<0.05). No statistically significant relations were detected between anxiety and PUKI (p>0.05). No statistically significant relations were detected between anxiety and falls (p>0.05). Positive correlation was found (r=0.529) between depression and falls (p<0.05). No statistically significant relations were detected between depression and PUKI (p>0.05). No statistically significant relations were detected between PUKI and falls (p>0.05, Table 2). 525 ISSN: 2147-7892, Cilt 10 Sayı 2 (2022) 521-529 doi: 10.33715/inonusaglik.1010406 DISCUSSION In another study, “moderate-severe” depression was reported by 10%, anxiety by 20%, and post-traumatic stress disorder by 12% in patients with COVID-19 infection approximately one month after discharge from the hospital (Liu et al., 526 doi: 10.33715/inonusaglik.1010406 ISSN: 2147-7892, Cilt 10 Sayı 2 (2022) 521-529 2020). In a study by Tomasoni et al., a statistically significant rate of anxiety and/or depression was reported in one-third of patients with COVID-19 infection, 46 days after recovery (Tomasoni et al., 2021). A study by Poyraz et al. showed that a large proportion of patients with COVID-19 infection continue to experience psychological symptoms for approximately 50 days after recovery. Moderate and severe post-traumatic stress disorder was observed in approximately one-quarter of these patients, and depression was reported in more than 40% of the patients. These study findings prove that the majority of patients with COVID-19 infection may experience psychiatric symptoms up to a few months after the illness (Poyraz et al., 2021). The findings of this study are similar to the findings of Lee et al.'s study after the SARS and MERS (Middle East respiratory syndrome) epidemics. They reported a psychiatric illness between 10% and 35% after recovery from the infection (Lee et al., 2019). Our study is important because it is the first study to evaluate mental status and falling in patients with vertigo diagnosed with COVID-19. Patients with vertigo who apply to the clinic must be evaluated in this respect, and clinicians must be careful in terms of the patients to receive psychological support. REFERENCES Agargun, M. Y., Kara, H. & Anlar, Ö. (1996). 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https://openalex.org/W4246672502	http://www.odermatol.com/odermatology/20182/20.Poroqueratosis-PincayL.pdf	Spanish; Castilian	null	Poroqueratosis punctata: reporte de un caso y revisión de la literatura	Nasza Dermatologia Online	2,018	cc-by	4,851	Letty Pincay-Cedeño1, Leonardo Espinoza-Benavides2, Isabel Maldonado3, Roberto Cullen4, Susana Ruiz-Tagle1, Jorge Chamorro5, Daniel Pasmanik1,2 1Dermatology Department, Hospital Militar de Santiago, Chile, 2Universidad de los Andes Medical School, Santiago, Chile, 3Pediatric Resident, Pontiﬁ cia Universidad Católica de Chile, Chile, 4Dermatology Resident, Universidad de Chile, Chile, 5Anatomical-Pathology Department, Hospital Militar de Santiago, Chile Corresponding author: Dr. Leonardo Espinoza-Benavides, E-mail: leoespinoza@hotmail.cl Our Dermatology Online Our Dermatology Online Case Report How to cite this article: Pincay-Cedeño L, Espinoza-Benavides L, Maldonado I, Cullen R, Ruiz-Tagle S, Chamorro J, Pasmanik D. Punctate porokeratosis: Case report and review of the literature. Our Dermatol Online. 2018;9(2):180-186. Submission: 09.11.2017; Acceptance: 28.02.2018 DOI: 10.7241/ourd.20182.20 Punctate porokeratosis: Case report and review of the Punctate porokeratosis: Case report and review of the literature literature Letty Pincay-Cedeño1, Leonardo Espinoza-Benavides2, Isabel Maldonado3, Roberto Cullen4, Susana Ruiz-Tagle1, Jorge Chamorro5, Daniel Pasmanik1,2 How to cite this article: Pincay-Cedeño L, Espinoza-Benavides L, Maldonado I, Cullen R, Ruiz-Tagle S, Chamorro J, Pasmanik D. Poroqueratosis punctata: reporte de un caso y revisión de la literatura. Our Dermatol Online. 2018;9(2):180-186. Submission: 09.11.2017; Acceptance: 28.02.2018 DOI: 10.7241/ourd.20182.20 ABSTRACT We present the case of a 63-year-old woman with a one-year history of punctiform, hyperkeratotic lesions on the palms of both hands. The histologic examination showed the presence of a cornoid lamella and allowed the establishment of the diagnosis of punctate porkeratosis. Porokeratosis is a clonal disorder of keratinization that exhibits multiple forms of presentation, with punctate porokeratosis being an infrequent form. The differential diagnosis of palmo-plantar punctiform, hyperkeratotic lesions is composed of porokeratosis punctata, the spiny keratodermas, other punctate keratodermas, and other pathologies with distinctive characteristics. In the review of literature of this article we have focused on explaining and clarifying the historical problem concerning the terminology used for these pathologies. okeratosis; Cornoid lamella; Disorders of keratinization; Punctate keratoderma; Spiny keratoderma Key words: Porokeratosis; Cornoid lamella; Disorders of keratinization; Punctate keratoderma; Spin Submission: 09.11.2017; Acceptance: 28.02.2018 DOI: 10.7241/ourd.20182.20 180 180 © Our Dermatol Online 2.2018 Our Dermatology Online Case Report Case Report Corresponding author: Dr. Leonardo Espinoza-Benavides, E-mail: leoespinoza@hotmail.cl Corresponding author: Dr. Leonardo Espinoza-Benavides, E-mail: leoespinoza@hotmail.cl INTRODUCTION caracterizado por lesiones puntiformes en palmas de ambas manos, generalmente asintomáticas, con episodios de dolor ocasionales. Sin antecedentes de exposición a arsénico ni relato de casos familiares. Al examen físico se constataron lesiones de 1-2 milímetros de diámetro en ambas superficies palmares y en zonas flexoras de todos los dedos a excepción de ambos pulgares (Fig. 1). Las lesiones eran espiculadas, hiperqueratósicas, algunas de color marrón y otras de coloración similar a la piel, levemente sensibles a la palpación. La poroqueratosis es un desorden clonal de la queratinización que exhibe múltiples formas de presentación. La característica común a este grupo heterogéneo de manifestaciones clínicas es el hallazgo histológico denominado lámina o laminilla cornoide [1]. Reportamos un caso de poroqueratosis punctata, variante inusual caracterizada por lesiones puntiformes hiperqueratósicas de ubicación palmo-plantar [2]. RESUMEN Presentamos el caso de una paciente de 63 años con lesiones puntiformes hiperqueratósicas de un 1 año de evolución en palmas de ambas manos. El estudio histológico demostró la presencia de una lámina cornoide y permitió establecer el diagnóstico de poroqueratosis punctata. La poroqueratosis es un desorden clonal de la queratinización que exhibe múltiples formas de presentación, siendo la variante punctata una forma infrecuente. El diagnóstico diferencial de lesiones puntiformes hiperqueratósicas palmo-plantares está compuesto por la poroqueratosis punctata, las queratodermias espinosas, otras queratodermias punctatas y otras patologías de características distintivas. En la revisión del presente reporte nos hemos enfocado en exponer y clarificar la histórica problemática que ha existido en la terminología empleada para estas patologías. Palabras clave: Poroqueratosis; Laminilla cornoide; Desorden de la queratinización; Queratodermia punctata; Queratodermia espinosa. Letty Pincay-Cedeño1, Leonardo Espinoza-Benavides2, Isabel Maldonado3, Roberto Cullen4, Susana Ruiz-Tagle1, Jorge Chamorro5, Daniel Pasmanik1,2 Letty Pincay-Cedeño1, Leonardo Espinoza-Benavides2, Isabel Maldonado3, Roberto Cullen4, Susana Ruiz-Tagle1, Jorge Chamorro5, Daniel Pasmanik1,2 1Dermatology Department, Hospital Militar de Santiago, Chile, 2Universidad de los Andes Medical School, Santiago, Chile, 3Pediatric Resident, Pontiﬁ cia Universidad Católica de Chile, Chile, 4Dermatology Resident, Universidad de Chile, Chile, 5Anatomical-Pathology Department, Hospital Militar de Santiago, Chile y-Cedeño L, Espinoza-Benavides L, Maldonado I, Cullen R, Ruiz-Tagle S, Chamorro J, Pasmanik D. Poroqueratosis punctata: e la literatura. Our Dermatol Online. 2018;9(2):180-186. CASE REPORT (a) Histology reveals a compact parakeratotic column with an underlying decreased stratum granulosum. (b) The periphery of the parakeratotic column reveals an almost absent stratum granulosum along with the presence of some vacuolated keratinocytes]. Figure 2: Biopsia obtenida de lesión hiperqueratósica en palma izquierda. (a) Se distingue una columna paraqueratósica compacta con un estrato granular subyacente adelgazado. (b) Se observa en un borde de la columna paraqueratósica un adelgazamiento casi completo del estrato granular, con algunos queratinocitos vacuolados en el estrato espinoso adyacente, [Biopsy of a hyperkeratotic papule on the left hand. (a) Histology reveals a compact parakeratotic column with an underlying decreased stratum granulosum. (b) The periphery of the parakeratotic column reveals an almost absent stratum granulosum along with the presence of some vacuolated keratinocytes]. Figure 1: Lesiones puntiformes hiperqueratósicas en palmas y zonas ﬂ exoras de los dedos. [Hyperkeratotic punctiform papules involving the palms and the ventral surface of digits] contactar a la epidermis adyacente, se logró observar un adelgazamiento casi total del estrato granular, con algunos queratinocitos vacuolados en el estrato espinoso aledaño (Figs. 2a and 2b). Además de esto, se encontró una discreta acantosis y discreta papilomatosis de la epidermis. A nivel de la dermis papilar se observó un escaso infiltrado inflamatorio mononuclear con escasos linfocitos de ubicación perivascular. Los hallazgos fueron compatibles con la presencia de una lámina cornoide y fue establecido el diagnóstico de poroqueratosis. hasta constituir lesiones anulares, demarcadas, de centro atrófico y con una periferia hiperqueratósica solevantada, pudiendo ser asintomáticas o asociadas a prurito [2]. Al obtener una biopsia de la región periférica, la histología revela el hallazgo común a todo tipo de poroqueratosis: la lámina cornoide, una columna compacta paraqueratósica que deprime un área epidérmica caracterizada por un adelgazamiento o ausencia del estrato granular. Para completar el cuadro histológico, queratinocitos vacuolados y/o disqueratósicos deben observarse en el estrato espinoso subyacente [1]. Junto a esto se ha descrito también un compromiso inflamatorio de la dermis de tipo linfohistiocitario [1-3]. Se inició tratamiento con tretinoína crema al 0,1% y emulsión fluida queratorreguladora con lactato de amonio al 15%, citándose a control para luego de 1 mes. Sin embargo, la paciente acudió a la consulta dermatológica 7 meses después. Durante la segunda visita se constató que las lesiones seguían de igual aspecto. La paciente relató que controlaba sus lesiones utilizando una lima y retirándolas con pinza. CASE REPORT La lámina cornoide representa una forma anormal de queratinización. A pesar de ser el elemento unificador de la poroqueratosis, no constituye un hallazgo patognomónico; es posible observarla en otros trastornos cutáneos inflamatorios o hereditarios, siendo en ocasiones un hallazgo incidental [1]. Descrita por primera vez en 1893 por Mibelli [4], el nombre “poroqueratosis” surge ante la idea errónea de que las láminas cornoides pudiesen tener su origen en las glándulas ecrinas [1,5,6]. Wade y Ackerman fueron los que describieron por primera vez en 1980 la participación de esta lámina en múltiples patologías, considerándola más bien una “reacción tisular menor” en lo que corresponde a la aproximación diagnóstica de las patologías inflamatorias de la piel basada en patrones [7]. Patologías como psoriasis, queratosis liquenoide crónica, dermatomiositis, corresponden CASE REPORT Se realizó una biopsia de una lesión en palma izquierda. La muestra obtenida permitió evidenciar histológicamente la presencia de una columna paraqueratósica compacta, subyacente a la cual se evidenció un marcado adelgazamiento del estrato granular. Al observarse uno de los bordes basales de la columna paraqueratósica, en donde comenzaba a Paciente de sexo femenino, de 63 años de edad, con antecedentes de hipertensión arterial, hipotiroidismo, depresión, fibromialgia y cáncer de mama operado. Usuaria de ácido acetil salicílico, levotiroxina, escitalopram, carbamazepina, pregabalina y duloxetina. Consultó por un cuadro de 1 año de evolución p p p g y Consultó por un cuadro de 1 año de evolución 181 © Our Dermatol Online 2.2018 www.ode Figure 1: Lesiones puntiformes hiperqueratósicas en palmas y zonas ﬂ exoras de los dedos. [Hyperkeratotic punctiform papules involving the palms and the ventral surface of digits] matol.com Figure 2: Biopsia obtenida de lesión hiperqueratósica en palma izquierda. (a) Se distingue una columna paraqueratósica compacta con un estrato granular subyacente adelgazado. (b) Se observa en un borde de la columna paraqueratósica un adelgazamiento casi completo del estrato granular, con algunos queratinocitos vacuolados en el estrato espinoso adyacente, [Biopsy of a hyperkeratotic papule on the left hand. (a) Histology reveals a compact parakeratotic column with an underlying decreased stratum granulosum. (b) The periphery of the parakeratotic column reveals an almost absent stratum granulosum along with the presence of some vacuolated keratinocytes]. b a matol.com b a www.odermatol.com b a www.odermatol.com b a Figure 2: Biopsia obtenida de lesión hiperqueratósica en palma izquierda. (a) Se distingue una columna paraqueratósica compacta con un estrato granular subyacente adelgazado. (b) Se observa en un borde de la columna paraqueratósica un adelgazamiento casi completo del estrato granular, con algunos queratinocitos vacuolados en el estrato espinoso adyacente, [Biopsy of a hyperkeratotic papule on the left hand. (a) Histology reveals a compact parakeratotic column with an underlying decreased stratum granulosum. (b) The periphery of the parakeratotic column reveals an almost absent stratum granulosum along with the presence of some vacuolated keratinocytes]. Figure 2: Biopsia obtenida de lesión hiperqueratósica en palma izquierda. (a) Se distingue una columna paraqueratósica compacta con un estrato granular subyacente adelgazado. (b) Se observa en un borde de la columna paraqueratósica un adelgazamiento casi completo del estrato granular, con algunos queratinocitos vacuolados en el estrato espinoso adyacente, [Biopsy of a hyperkeratotic papule on the left hand. DISCUSSION Las variantes más comúnmente descritas de poroqueratosis son la forma clásica de Mibelli, la poroqueratosis diseminada superficial actínica (DSAP, de su nombre en inglés, disseminated superficial actinic porokeratosis), la poroqueratosis diseminada superficial, la poroqueratosis linear, la poroqueratosis palmaris et plantaris disseminata y la poroqueratosis punctata (PP) [3]. Las dos primeras mencionadas son las formas más frecuentes, observándose en ellas las lesiones típicas de este desorden: pápulas queratósicas que evolucionan de forma centrífuga © Our Dermatol Online 2.2018 182 www.odermatol.com puntiformes hiperqueratósicas localizadas en palmas y/o plantas [5]. En el caso de nuestro paciente, el compromiso fue exclusivamente palmar. Clásicamente las lesiones se describen en “forma de semilla” (seed- like), pudiendo ser solevantadas de forma espicular o más bien deprimidas en forma de pits [1,2]. Las lesiones suelen ser asintomáticas, pero pueden también ser sensibles a la presión [2]. Es notoria la existencia de una gran confusión respecto a la terminología a emplear para describir a la gama de patologías que cursan con estas lesiones palmo-plantares como manifestación clínica [3]. El diagnóstico diferencial ante este cuadro, utilizando la nomenclatura que nos parece actualizada y correspondiente, gira entorno a cuatro grandes grupos: la PP, las queratodermias espinosas, otras queratodermias punctatas y otras patologías de características distintivas [2,13,16,17]. Para comprender la confusión y los términos planteados, resulta imperiosa una revisión histórica de tales descripciones. De lo contrario resulta difícil tener una visión íntegra de la problemática. a algunos ejemplos en los que la histología puede también revelar una lámina cornoide; sin embargo, son otros los elementos clínico-patológicos los que guían el diagnóstico de aquellos cuadros [1]. En cuanto a la morfogénesis de la lámina cornoide, se han observado clones mutantes de queratinocitos predispuestos genéticamente con alteraciones en la ploidía del ADN. Una apoptosis prematura con alteración de la diferenciación terminal explicaría la queratinización desorganizada [1]. Se ha reportado también una queratinización rápida asociada a una descamación defectuosa y se ha demostrado con inmunohistoquímica la sobreexpresión del producto del gen P53 en los queratinocitos subyacentes a la lámina cornoide [1,8]. La asociación entre poroqueratosis y malignidades cutáneas ha sido descrita por múltiples autores [2,9,10]. Esta asociación puede comprenderse a través de la morfogénesis señalada: los queratinocitos comprometidos corresponderían a un estadío celular intermedio entre células normales y las células halladas en la enfermedad de Bowen. DISCUSSION La transformación maligna se estima en un 7.5% de los casos, con predominio de la variante linear y con el carcinoma espinocelular (CEC) siendo el tumor principalmente asociado [1,2]. En 1923, Sweitzer publica un artículo titulado “Keratoderma punctatum” [18]. En este, el autor describe un cuadro de hiperqueratosis punctata palmo-plantar y considera que ninguna de aquellas lesiones puede ser considerada una “poroqueratosis” hasta demostrar una relación con las glándulas ecrinas, característica considerada aún esencial en aquel entonces. En su revisión de la literatura, destaca como primera descripción de un cuadro similar un artículo de 1879, de Davies-Colley, donde se describe un cuadro clasificado como “disseminated clavus of hands and feet” (“clavos diseminados de manos y pies”) [19]. Besnier, comenta el autor, describe un caso con lesiones hiperqueratósicas punctata en palmas, a lo que denominó “queratodermia eritematosa simétrica de las extremidades: forma punctata” [20]. Sweitzer termina su análisis con un comentario personal: expresa haber recopilado 18 casos, ninguno de ellos idénticos entre sí, evidenciando una nomenclatura que alternaba entre poroqueratosis y queratodermias. Algunos de esos casos, considera, probablemente eran verrugas o incluso liquen plano. El artículo de Sweitzer corresponde al primer incentivo explícito hacia la comunidad científica para indagar en estas manifestaciones clínicas difusamente comprendidas en su época. La patogenia de la poroqueratosis ha sido principalmente estudiada en relación a sus dos formas más frecuentes, destacando un componente genético, un rol de la luz ultravioleta (si bien existe un caso reportado en que un paciente con DSAP presentó mejorías al tratarse con PUVA [11]) y un rol también de la inmunosupresión [2,12]. Asimismo, infección por virus papiloma humano y traumatismos han sido considerados como elementos con un posible rol patogénico [2]. No existen estudios aleatorizados sobre el tratamiento de la poroqueratosis y las respuestas suelen ser impredecibles: ningún tratamiento ha demostrado una eficacia consistente y de largo plazo [13,14]. Sin tratamiento, las lesiones persisten indefinidamente [10,13]. La regresión espontánea, si bien descrita, es rara [15]. Finalmente, dado el carácter premaligno de este desorden, es el seguimiento de los pacientes lo que resulta esencial. La única variante que constituye la excepción es la PP, sin casos de malignidades asociadas reportadas [10]. Muchos años después, en 1971, Guss describe un “tercer tipo de poroqueratosis” [21], diferente a las descripciones hechas por Mibelli y por Chernosky (quien describe en 1967 la variante DSAP [22]). DISCUSSION El La PP es una variante infrecuente de poroqueratosis que, a diferencia de las variantes más usuales, tiene una presentación clínica que se caracteriza por lesiones 183 © Our Dermatol Online 2.2018 www.odermatol.com cuadro de Guss terminaría correspondiendo a la variante palmaris et plantaris disseminata. La define como un cuadro autosómico dominante, de inicio alrededor de los 20 años y de compromiso tronco-palmo-plantar, morfológicamente similar a la DSAP. También en 1971, Brown publica su artículo titulado “Punctate keratoderma” (“Queratodermia punctata”) [23]. Este último ha sido considerado por diversos autores como el inicio de la confusión terminológica [3,16,17]. Brown comienza su artículo estableciendo que “la queratodermia punctata es un diagnóstico descriptivo que indica la presencia de pequeñas excreciencias puntiformes, cornudas, dispuestas irregularmente sobre palmas, plantas y la superficie flexora de los dedos”. A la histología destaca una hiperqueratosis con acantosis; de haber paraqueratosis, dice, es mínima e incidental. Brown define su cuadro como una patología hereditaria autosómica dominante y reporta un caso del San Diego Naval Hospital: hombre de 20 años, con lesiones palmo-plantares, con claro componente familiar. Brown comenta que fue sugerido el diagnóstico tentativo de “poroqueratosis punctata”, pero considerando la historia familiar, la edad de aparición y la falta de extensión periférica, decide finalmente considerar el caso como alguna forma de queratodermia punctata. Comenta no haber encontrado en la literatura ningún caso similar al que él se encontraba reportando. El mismo Brown relata que histológicamente le parece ver estructuras similares a la lámina cornoide de Mibelli. similitud clínica con la forma clásica de Mibelli, tal como el mismo Brown permite ver en su publicación. Rahbari considera que el diagnóstico diferencial de mayor dificultad radica en diferenciar tres entidades, en sus palabras: la PP, la queratodermia punctata y la queratosis arsenical. Himmelstein publica el año 1984 lo que considera el quinto caso reportado de PP [26]. Paciente de origen hispano, de 26 años, con lesiones recurrentes puntiformes de 1-2 milímetros, de tipo seed-like, en palma y planta izquierda. A la histología se describe una columna paraqueratósica considerada como lámina cornoide. Himmelstein realiza una revisión de la literatura que evidencia el ya presente problema terminológico: considera que el primer caso reportado de PP correspondería al reporte de Herman de 1973 titulado “Queratodermia poroqueratósica punctata” [27], y se suma a Rahbari en considerar que el caso de Brown probablemente correspondía a una poroqueratosis. DISCUSSION Sakas publica en 1985 otro caso de PP, en un paciente de 60 años con ascendencia coreana, considerándolo el sexto caso reportado de contabilizarse al de Brown como el primero [28]. Sakas nota, no obstante, que de los 6 casos de PP, solo 3 fueron exclusivamente palmo-plantares. Por esta razón extiende el nombre de su diagnóstico a PP palmaris et plantaris. De gran relevancia nos resulta el hecho de que Sakas es uno de los primeros autores en destacar activamente la presencia de queratinocitos vacuolados en la histología de su paciente. En la revisión bibliográfica realizada por el autor, destaca el compromiso tanto de hombres como de mujeres, la tendencia a no ser hereditaria (con la excepción de 1 caso), y la ausencia de algún predominio racial. La edad tendería a la pubertad, pero no sería exclusiva de ese rango etario. En 1974, Herman expresa la necesidad de un mayor reporte de casos que permita esclarecer las diferencias observadas en estos cuadros descriptivamente denominados queratodermias punctatas [24]. Comenta casos con predominio de hiperqueratosis y otros de paraqueratosis, así como también casos tanto hereditarios como esporádicos. En 1977, Rahbari postula de manera decisiva una nueva variante de la poroqueratosis de Mibelli: la poroqueratosis punctata, nombre ya utilizado en reportes previos, pero sin concretarse en un diagnóstico oficial [25]. A pesar de incorporar esta variante, destacando las lesiones de tipo seed-like, sus dos casos reportados no eran de ubicación exclusiva palmo-plantar. El primer reporte correspondía a una mujer de 16 años que presentaba lesiones en dedos y palma de la mano derecha y en axila derecha; el otro caso, un paciente de 59 años, presentaba lesiones en codo, muñeca y dedos. El autor considera que el caso de Brown correspondía a una poroqueratosis, y que el nombre no fue empleado tan solo por la falta de Friedmann comenta en 1988 que aún era necesaria una clasificación adecuada para estos casos [29]. El autor diferencia clínicamente las lesiones acuminadas, de tipo “caja musical”, y las lesiones deprimidas, denominadas pits. En el caso de las lesiones solevantadas palmo-plantares, considera solo 3 o posiblemente 4 casos previos. A pesar de los reportes anteriores, Friedmann apoya la categoría de Wolff-Schreiner, de 1987, en la que no consideran a la PP como una entidad por sí sola, sino una variante de la poroqueratosis linear o de la forma clásica de Mibelli [30]. DISCUSSION A los dos casos de su publicación se les atribuye histológicamente la presencia de lámina cornoide, pero en ausencia 184 © Our Dermatol Online 2.2018 www.odermatol.com de queratinocitos vacuolados y/o disqueratósicos a la microscopía de luz y electrónica, como ya se había descrito para las láminas cornoides de la forma clásica de Mibelli y de la DSAP [31,32]. Es por esto que Friedmann considera que sus reportes no son verdaderas poroqueratosis, catalogándolas más bien como queratodermias poroqueratósicas punctata, parte del grupo de lo que se denominó como dermatosis en caja musical (en inglés, music box spine dermatoses) [33]. Estas entidades que no satisfacían los criterios histológicos para ser denominadas verdaderas poroqueratosis tuvieron también sus respectivos cambios en términos de nomenclatura. Zarour realiza una clasificación en 1992 agrupándolas en lo que denomina queratosis filiformes [34] y luego McGovern y Gentry, en 1994, cambian aquel término por el de queratodermias espinosas (SK, del inglés spiny keratoderma), diferenciando los cuadros paraqueratósicos de los ortoqueratósicos [35]. en ausencia del resto de los elementos encontrados en la poroqueratosis. Algunos, por el contrario, se limitan a describir una columna paraqueratósica sin una denominación específica [16]. En este aspecto consideramos relevante exponer la extensa revisión realizada por Biswas el año 2015. En esta, el autor establece que para denominarse lámina cornoide propiamente tal, 3 características se deben encontrar: 1) presencia de una columna vertical paraqueratósica, 2) pérdida o disminución del estrato granular en el punto donde la paraqueratina indenta la superficia epidermal, y 3) disqueratosis y/o vacuolización de células en el estrato espinoso que subyace a la columna paraqueratósica. Siguiendo este criterio, la presencia de lámina cornoide queda excluida de las SK y sigue constituyendo el elemento unificador, pero no patognomónico, de las poroqueratosis [1]. Ya hemos mencionado previamente la patogenia de la poroqueratosis; en el caso de las SK, Hashimoto realiza en 1999 un análisis de 6 casos utilizando anticuerpos antiqueratinas AE13 y AE14, además de apoyarse con microscopía electrónica. Las muestras obtenidas fueron positivas para AE13, marcador de queratina inmadura del pelo. Los resultados sugieren que las SK pueden constituir enfermedades de formación ectópica abortiva de pelos en palmas y plantas [37]. Basta con estos artículos señalados para comprender la dificultad histórica de la nomenclatura utilizada y la gran cantidad de términos propuestos. DISCUSSION Consideramos que la revisión realizada por Urbani en 1998 ofrece un análisis comprensivo e integrativo de estos cuadros, plasmando y resumiendo un esquema aún vigente [36]. En su publicación diferencia categóricamente las PP de las SK en relación a la presencia o ausencia de queratinocitos vacuolados y/o disqueratósicos, respectivamente, utilizando como referencia el trabajo de Wade y Ackermann de 1980. Las SK además, a diferencia de las PP, no presentarían compromiso inflamatorio de la dermis, pero sí compartirían la disminución del estrato granular comprometido. Urbani comenta que en la literatura científica probablemente hay casos de SK descritos como PP y viceversa. Plantea además la hipótesis de 2 categorías de SK: una forma hereditaria que sería siempre benigna y una forma adquirida o idiopática que puede ser paraneoplásica o estar asociada a trastornos metabólicos. La asociación con distintas malignidades internas ha sido reportada particularmente en los casos idiopáticos de queratodermia poroqueratósica punctata (que pertenece a la categoría de SK paraqueratósica). Existen casos de asociación con carcinoma bronquial, cáncer de ovario, leucemia mieloide crónica, entre otros [13]. A partir del año 2000 se ha vislumbrado una clara tendencia a ocupar la nomenclatura actualizada de PP y SK, en especial destacándose la preferencia por el término SK por sobre otros términos históricamente utilizados [17]. Sin embargo, aún no existe una evidente homogeneización de la terminología [33]. Es importante recordar que ambas patologías son muy poco frecuentes y escasamente reportadas [1,17]. © Our Dermatol Online 2.2018 REFERENCES 28. Sakas EL, Gentry RH. Porokeratosis punctata palmaris et plantaris (punctate porokeratosis). Case report and literature review. J Am Acad Dermatol. 1985;13:908-12. 1. Biswas A. Cornoid lamellation revisited: apropos of porokeratosis with emphasis on unusual clinicopathological variants. Am J Dermatopathol. 2015;37:145-55. 29. Friedman SJ, Herman PS, Pittelkow MR, Su WP. Punctuate porokeratotic keratoderma. Arch Dermatol. 1988;124:1678-82. 2. Sertznig P, von Felbert V, Megahed M. Porokeratosis: present concepts. J Eur Acad Dermatol Venereol. 2012;26:404. 30. Wolff-Schreiner EC: Porokeratosis, in Fitzpatrick TB, Eisen AZ, Wolff K, et al (eds): Dermatology in General Medicine, ed 3. New York, McGraw-Hill International Book Co, 1987, pp 534-40. 3. Teixeira VB, Reis JP, Vieira R, Tellechea Ó, Figueiredo A. Unilateral punctate porokeratosis - Case report. An Bras Dermatol. 2013;88:441-6. 31. Mann PR, Cort DF, Fairburn EA, Abdel-Aziz A. Ultrastructural studies on two cases of porokeratosis of Mibelli. Br J Dermatol. 1974;90:607-17. 4. Mibelli V. Contributo allo studio della ipercheratosi dei canali sudoriferi (porokeratosis). G Ital Mal Vener Pelle. 1893;28:313–55. 32. Sato A, Anton-Lamprecht I, Schnyder UW. Ultrastructure of inborn errors of keratinization: VII. Porokeratosis Mibelli and disseminated superﬁ cial actinic porokeratosis. Arch Dermatol Res. 1976;255:271-84. 5. Lanka P, Lanka LR, Manivachagam D. Punctate Porokeratosis Palmaris et Plantaris. Indian J Dermatol. 2015;60:284-86. 6. Cockerel CJ, Larsen F. Benign epidermal tumours and proliferations. In: Bolognia JL, Jorizzo JL, Rapini PR, editors. Dermatology. Haryana: Mosby Elsevier; 2009. pp. 1661–80. 33. Saha A, Naskar B, Singha J, Chaterjee G. Music box spine keratoderma without any systemic manifestation. Indian Dermatol Online J. 2014;5:342-4. 7. Wade TR, Ackerman AB. Cornoid lamellation: a histologic reaction pattern. Am J Dermatopathol. 1980;2:5–15. 34. Zarour H, Grob J, Andrac L, Bonerandi J. Palmoplantar orthokeratotic ﬁ liform hyperkeratosis in a patient with associated Darier disease. Dermatology. 1992;185:205–9. 8. Magee JW, McCalmont TH, LeBoit PE. Over expression of P53 tumour suppressor protein in porokeratosis. Arch Dermatol. 1994;130:87. 35. McGovern TW, Gentry RH. Spiny Keratoderma: case report, classification, and treatment of music box spine dermatoses. Cutis. 1994;54:389–94. 9. Maubec E, Duvillard P, Margulis A, Bachollet B, Degois G, Avril MF. Common skin cancers in porokeratosis. Br J Dermatol. 2005;152:1389. 36. Urbani CE, Moneghini L. Palmar spiny keratoderma associated with type IV hyperlipoproteinemia. J Eur Acad Dermatol Venereol. 1998;10:262–6. 10. Sasson M, Krain AD. Porokeratosis and cutaneous malignancy. A review. Dermatol Surg. 1996;22:339. 37. Hashimoto K, Toi Y, Horton S, Sun TT. CONCLUSION En conclusión, y utilizando la nomenclatura que consideramos apropiada, el diagnóstico diferencial de lesiones puntiformes hiperqueratósicas palmo- plantares, ya sea acuminadas o deprimidas, en forma de caja musical o en forma de semillas, lo constituyen los 4 grandes grupos que hemos mencionado con anterioridad: 1) la PP, 2) las SK, 3) otras queratodermias punctatas [36], y 4) otras patologías de características distintivas [1,16]. Ya hemos argumentado la nomenclatura de las PP y las SK. En el caso del grupo 3, recalcamos la importancia de utilizar el concepto de queratodermia punctata solamente a modo descriptivo, como fue Tal como ya se ha expuesto, son varios los trabajos que describen casos de hiperqueratosis palmo-plantar puntiformes en los que histológicamente se ha descrito la presencia de una lámina cornoide, pero © Our Dermatol Online 2.2018 185 www.odermatol.com 16. Torres G, Behshad R, Han A, Castrovinci AJ, Gilliam AC. “I forgot to shave my hands”: a case of spiny keratoderma. J Am Acad Dermatol. 2008;58:344-8. concebido originalmente, y no como una patología o una entidad en sí misma. En este grupo, otras queratodermias punctatas incluyen patologías como la queratosis arsenical, la enfermedad de Buschke-Fisher- Brauer y la acroqueratoelastoidosis liquenoide [16]. En cuanto a otras patologías de características distintivas, se puede señalar la enfermedad de Darier, la enfermedad de Cowden, verrugas, queratolisis punctata (pitted keratolysis) y el síndrome del carcinoma basocelular nevoideo [1,16]. 17. Nagler A, Boyd KP, Patel RR, Lee HS. Spiny keratoderma. Dermatol Online J. 2013;19:20706. 18. Sweitzer S. Keratoder ma punctatum. Arch Der m Syphilol. 1923;8:687-94. 19. Davies-Colley N. Disseminated clavus of palms of hands and soles of feet. Trans Path Soc London. 1879;30:451. 20. Besnier. Kératodermie érythémateuse symétrique des extrémités; forme ponctuée; Kératose localisée a l’ostium sudorifere. Paume de la main. Museum of the St. Louis Hospital is a Baretta model. 1892;560. 21. Guss SB, Osbourn RA, Lutzner MA. Porokeratosis plantaris et Palmaris disseminate: A third type of porokeratosis. Arch Dermatol. 1971;104:366–73. La PP y las SK son patologías poco frecuentes con múltiples modalidades de tratamientos descritos para ambas, sin alguno particularmente establecido [13,14]. Más allá de las posibles molestias cosméticas o sintomáticas, resulta esencial el estudio histológico que permita un diagnóstico específico para el subsecuente seguimiento del paciente: en el caso de las PP por el riesgo teórico de CEC, y en el caso de las SK por las malignidades internas que se han visto asociadas. 22. CONCLUSION Chernosky ME, Freeman RG. Disseminated superﬁ cial actinic porokeratosis (DSAP). Arch Dermatol. 1967;96:611–24. 23. Brown FC. Punctate keratoderma. Arch Dermatol. 1971;104:682–3. 24. Herman PS. Letter: Punctate keratoderma. Arch Dermatol. 1974;109:10. 25. Rahbari H, Cordero AA, Mehregan AH. Punctate porokeratosis. A clinical variant of porokeratosis of Mibelli. J Cutan Pathol. 1977;4:338–41. 26. Himmelstein R, Lynfield YL. Punctate porokeratosis. Arch Dermatol. 1984;120:263–4. 27. Herman PS. Punctate porokeratotic keratoderma. Dermatologica. 1973;147:206-13. Copyright by Letty Pincay-Cedeño, 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. Source of Support: Nil, Conﬂ ict of Interest: None declared. REFERENCES Spiny keratoderma--a demonstration of hair keratin and hair type keratinization. J Cutan Pathol. 1999;26:25–30. 11. Schwarz T, Seiser A, Gschnait F. Disseminated superﬁ cial “actinic” porokeratosis. J Am Acad Dermatol. 1984;11:724. 12. Raychaudhuri SP, Smoller BR. Porokeratosis in immunosuppressed and nonimmunosuppressed patients. Int J Dermatol. 1992;31:781-2. 13. Alikhan A, Burns T, Zargari O. Punctate porokeratotic keratoderma. Dermatol Online J. 2010;16:13. Copyright by Letty Pincay-Cedeño, 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. Source of Support: Nil, Conﬂ ict of Interest: None declared. 14. Kanitakis J. Porokeratoses: an update of clinical, aetiopathogenic and therapeutic features. Eur J Dermatol. 2014;24:533-44. 15. Kanitakis J, Euvrard S, Faure M, Claudy A. Porokeratosis and immunosuppression. Eur J Dermatol. 1998;8:459. © Our Dermatol Online 2.2018 186
https://openalex.org/W4385287867	https://journals.plos.org/plospathogens/article/file?id=10.1371/journal.ppat.1011528&type=printable	English	null	Suppression of viral RNA polymerase activity is necessary for persistent infection during the transformation of measles virus into SSPE virus	PLOS pathogens	2,023	cc-by	17,822	OPEN ACCESS Subacute sclerosing panencephalitis (SSPE) is a fatal neurodegenerative disease caused by measles virus (MV), which typically develops 7 to 10 years after acute measles. During the incubation period, MV establishes a persistent infection in the brain and accumulates mutations that generate neuropathogenic SSPE virus. The neuropathogenicity is closely associated with enhanced propagation mediated by cell-to-cell fusion in the brain, which is principally regulated by hyperfusogenic mutations of the viral F protein. The molecular mechanisms underlying establishment and maintenance of persistent infection are unclear because it is impractical to isolate viruses before the appearance of clinical signs. In this study, we found that the L and P proteins, components of viral RNA-dependent RNA poly- merase (RdRp), of an SSPE virus Kobe-1 strain did not promote but rather attenuated viral neuropathogenicity. Viral RdRp activity corresponded to F protein expression; the suppres- sion of RdRp activity in the Kobe-1 strain because of mutations in the L and P proteins led to restriction of the F protein level, thereby reducing cell-to-cell fusion mediated propagation in neuronal cells and decreasing neuropathogenicity. Therefore, the L and P proteins of Kobe- 1 did not contribute to progression of SSPE. Three mutations in the L protein strongly sup- pressed RdRp activity. Recombinant MV harboring the three mutations limited viral spread in neuronal cells while preventing the release of infectious progeny particles; these changes could support persistent infection by enabling host immune escape and preventing host cell lysis. Therefore, the suppression of RdRp activity is necessary for the persistent infection of the parental MV on the way to transform into Kobe-1 SSPE virus. Because mutations in the genome of an SSPE virus reflect the process of SSPE development, mutation analysis will provide insight into the mechanisms underlying persistent infection. Citation: Sakamoto K, Konami M, Kameda S, Satoh Y, Wakimoto H, Kitagawa Y, et al. (2023) Suppression of viral RNA polymerase activity is necessary for persistent infection during the transformation of measles virus into SSPE virus. PLoS Pathog 19(7): e1011528. https://doi.org/ 10.1371/journal.ppat.1011528 Editor: Alexander Bukreyev, University of Texas Medical Branch / Galveston National Laboratory, UNITED STATES Received: December 19, 2022 Accepted: July 3, 2023 Published: July 26, 2023 Copyright: © 2023 Sakamoto 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. PLOS PATHOGENS PLOS PATHOGENS PLOS PATHOGENS Suppression of viral RNA polymerase activity is necessary for persistent infection during the transformation of measles virus into SSPE virus Kento Sakamoto1, Miho Konami1, Shinra Kameda1, Yuto Satoh1, Hiroshi Wakimoto1, Yoshinori Kitagawa2, Bin Gotoh2, Da-Peng Jiang3, Hak Hotta3, Masae Itoh1* Kento Sakamoto1, Miho Konami1, Shinra Kameda1, Yuto Satoh1, Hiroshi Wakimoto1, Yoshinori Kitagawa2, Bin Gotoh2, Da-Peng Jiang3, Hak Hotta3, Masae Itoh1* 1 Department of Microbiology, Faculty of Bio-Science, Nagahama Institute of Bio-Science and Technology, Nagahama, Shiga, Japan, 2 Division of Microbiology and Infectious Diseases, Department of Pathology, Shiga University of Medical Science, Otsu, Shiga, Japan, 3 Graduate School of Medicine, Kobe University, Kobe, Hyogo, Japan 1 Department of Microbiology, Faculty of Bio-Science, Nagahama Institute of Bio-Science and Technology, Nagahama, Shiga, Japan, 2 Division of Microbiology and Infectious Diseases, Department of Pathology, Shiga University of Medical Science, Otsu, Shiga, Japan, 3 Graduate School of Medicine, Kobe University, Kobe, Hyogo, Japan a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 * m_itoh@nagahama-i-bio.ac.jp * m_itoh@nagahama-i-bio.ac.jp * m_itoh@nagahama-i-bio.ac.jp Introduction Measles is a highly contagious acute infectious disease caused by measles virus (MV). Despite the availability of a safe and effective attenuated live virus vaccine, a worldwide resurgence of measles occurred between 2017 and 2019; the global incidence of measles reached 120 per mil- lion in 2019, and there were approximately 207,500 deaths [1]. Although low case numbers were reported in 2020 and 2021 because of the COVID-19 pandemic, delays and reductions in both vaccination and surveillance programs have increased the risk of additional measles resurgence in the near future [2,3]. MV also causes chronic persistent central nervous system (CNS) infection, subacute sclerosing panencephalitis (SSPE) in fully immunocompetent per- sons, which occurs 7 to 10 years after the initial MV infection [4–8]. SSPE is rare, but recent estimated incidences range from 22 to 30–59 per 100,000 in children who acquire measles before the age of 5 years [9–11]. Because the SSPE burden reflects the epidemiology of natural MV infection, an increased incidence of measles may be indicative of a future increase in the number of SSPE cases. MV, a member of the genus Morbillivirus in the Paramyxoviridae family of the Mononega- virales order, contains a single-stranded, non-segmented, negative-sense RNA genome com- posed of six genes encoding eight proteins. The phospho (P) and large (L) proteins form the RNA-dependent RNA polymerase (RdRp) complex, which acts as a viral transcriptase or repli- case by binding to the RNA genome encapsidated by the nucleocapsid (N) protein; the result is the RNase-resistant ribonucleoprotein (RNP) complex. The P gene encodes two non-struc- tural proteins: V and C [12–14]. The matrix (M) protein facilitates the assembly of the RNP and two envelope glycoproteins [hemagglutinin (H) and the fusion (F) protein], leading to viral particle budding [14,15]. The H and F proteins form an H/F protein complex, which functions in a coordinated manner as fusion machinery [16]. H protein binding to a receptor induces a conformational change in the F protein, triggering viral envelope fusion with the plasma membrane to introduce the RNP into the host cell and initiate infection [17–22]. The H and F proteins, expressed on the surface of infected cells, also support the fusion of the plasma membrane to the membranes of adjacent cells to form multinuclear giant cells (i.e., syncytia), thereby spreading the infection via cell-to-cell fusion [13,23]. Author summary Subacute sclerosing panencephalitis (SSPE) is a rare but fatal disease in apparently healthy children, which develops 7 to 10 years after acute measles. Measles virus (MV) persistently infects the brain, accumulates mutations, and transforms into neuropathogenic SSPE virus. The World Health Organization is promoting vaccination against MV to achieve global eradication of measles; this is also the most effective approach for prevention of SSPE. The live MV vaccine strains do not cause SSPE and all SSPE viruses isolated thus far have been derived from wild-type MV strains, but it remains unclear why live MV vaccine strains do not cause SSPE. Recently hyperfusogenic mutations of the MV F protein were proved to account for the enhanced propagation of the virus in the brain leading to the neurovirulence. In this study we showed that suppression of viral RNA polymerase is implicated in the establishment of persistent wild-type MV infection within neurons. Fur- ther investigations of the mechanisms by which wild-type MV transforms into SSPE virus will shed light on the reason why MV vaccine strains do not acquire neurovirulence. Competing interests: The authors have declared that no competing interests exist. PLOS PATHOGENS Role of viral RNA polymerase in SSPE development 19K08941) from the Japan Society for the Promotion of Science (JSPS) (https://www.jsps. go.jp). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. OPEN ACCESS Data Availability Statement: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by Grant-in-Aid for Young Scientists to YS (Grant number 19K16678) and Grants-in-Aid for Scientific Research to MI (Grant numbers 16K09112 and 1 / 28 PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1011528 July 26, 2023 PLOS PATHOGENS PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1011528 July 26, 2023 Introduction MVs infect immune and epithelial cells using signaling lymphocyte activation molecule (SLAM, also known as CD150) and nectin 4 as respective receptors [24–30]. Very rarely, MV PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1011528 July 26, 2023 2 / 28 PLOS PATHOGENS Role of viral RNA polymerase in SSPE development invades the CNS where it persists independently of SLAM and nectin 4; neither receptor is expressed in the human CNS [31,32]. This can cause the rare and fatal neurodegenerative dis- ease SSPE several years after recovery from primary acute measles [13], although it is unclear how MV establishes a chronic persistent CNS infection. MV variants obtained from the brains of SSPE patients (SSPE viruses) differ from wild-type MV and have properties such as lack of infectious viral particle production, enhanced cell-to-cell fusion ability, and neuropathogeni- city in rodents [33–37]. SSPE viruses harbor characteristic mutations throughout their genomes, particularly in the F and M genes, during infection of the brain [38–45]. Notably, mutations in the ectodomain of the F protein lead to F protein-specific hyperfusogenicity, which is associated with viral spread by cell-to-cell fusion in cells that do not express MV receptors (SLAM and nectin 4); such cells include human neurons [46–48]. This F protein- mediated viral hyperfusogenic phenotype causes the neuropathogenicity exhibited by SSPE viruses [49–52]. Contrarily, recombinant MVs bearing the hypermutant M protein derived from SSPE viruses are not neuropathogenic in rodents [49,53]. The SSPE virus Kobe-1 strain carries viral elements responsible for reducing pathogenicity. The neurovirulence of the Kobe-1 strain was reportedly lower than the neurovirulence of a chimeric recombinant MV possessing the M, F and H genes of the Kobe-1 strain, along with the N, P, and L genes of the wild-type MV [54]. This finding implicated N, P, and L protein- mediated viral RdRp activity in the attenuation of neuropathogenicity. To our knowledge, there is no available information concerning the roles of mutations that restrict the neuro- pathogenicity of SSPE viruses during the course of SSPE development. Here, we generated chi- meric viruses by exchanging the N, P, and L genes of the MV ICB strain and the SSPE virus Kobe-1 strain. Replacement of the L and P genes of MV with the corresponding genes of Kobe-1 restricted viral propagation in neuronal cells by reducing RdRp activity. Introduction By evaluating the effect of each amino acid mutation in the L and P proteins on RdRp activity, we identified a potential role for the suppression of RdRp activity in the establishment and/or maintenance of persistent infection in the brain during the transformation of MV into SSPE virus before appearance of clinical signs. PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1011528 July 26, 2023 The L gene and N and/or P genes of the SSPE virus Kobe-1 strain (SSPEV-L gene and SSPEV-N and/or SSPEV-P genes) did not promote but rather attenuated neuropathogenicity Six suckling mice were infected with 7 × 102 PFU of virus and monitored for 21 days. For the p-value calculations, log-rank tests were performed using the survival package in R. If a p-value was less than 0.05, the difference between the survival curves was considered statistically significant. Log-rank tests. , P < 0.05; n.s., not significant. https://doi.org/10.1371/journal.ppat.1011528.g001 Fig 1. Recombinant MV (rMV) harboring the L gene of the SSPE virus Kobe-1 strain (SSPEV-L gene) attenuated neurovirulence in suckling mice. (A) Schematic of the genomes of recombinant chimeric viruses (rMV). Protein- coding regions derived from the MV ICB strain [MV(ICB)] are shown as gray boxes, whereas protein-coding regions derived from the SSPE virus Kobe-1 strain [SSPEV(Kobe-1)] are shown as red boxes. (B) Mortality of suckling mice intracerebrally inoculated with the viruses in (A). Six suckling mice were infected with 7 × 102 PFU of virus and monitored for 21 days. For the p-value calculations, log-rank tests were performed using the survival package in R. If a p-value was less than 0.05, the difference between the survival curves was considered statistically significant. Log-rank tests. , P < 0.05; n.s., not significant. Fig 1. Recombinant MV (rMV) harboring the L gene of the SSPE virus Kobe-1 strain (SSPEV-L gene) attenuated neurovirulence in suckling mice. (A) Schematic of the genomes of recombinant chimeric viruses (rMV). Protein- coding regions derived from the MV ICB strain [MV(ICB)] are shown as gray boxes, whereas protein-coding regions derived from the SSPE virus Kobe-1 strain [SSPEV(Kobe-1)] are shown as red boxes. (B) Mortality of suckling mice intracerebrally inoculated with the viruses in (A). Six suckling mice were infected with 7 × 102 PFU of virus and monitored for 21 days. For the p-value calculations, log-rank tests were performed using the survival package in R. If a p-value was less than 0.05, the difference between the survival curves was considered statistically significant. Log-rank tests. , P < 0.05; n.s., not significant. https://doi.org/10.1371/journal.ppat.1011528.g001 https://doi.org/10.1371/journal.ppat.1011528.g001 PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1011528 July 26, 2023 The L gene and N and/or P genes of the SSPE virus Kobe-1 strain (SSPEV-L gene and SSPEV-N and/or SSPEV-P genes) did not promote but rather attenuated neuropathogenicity The L gene and N and/or P genes of the SSPE virus Kobe-1 strain (SSPEV-L gene and SSPEV-N and/or SSPEV-P genes) did not promote but rather attenuated neuropathogenicity By generating enhanced green fluorescent protein (EGFP)-expressing chimeric recombinant MVs of the ICB strain (rMVs), we demonstrated that the rMV harboring the M, F, and H genes of the SSPE virus Kobe-1 strain was more virulent than the Kobe-1 strain in mice when inoculated in the brain [54]. This suggested that the N, P, and/or L genes of the Kobe-1 attenu- ate neuropathogenicity. To confirm the observation and identify the responsible gene(s), we introduced the L gene of Kobe-1 (SSPEV-L gene) into rMV bearing the M, F, and H genes of Kobe-1 (rMV/sMFH) and generated rMV/sMFHL (Fig 1A). As shown in Fig 1B, although rMV/sMFH was highly lethal in suckling mice that were inoculated intracerebrally, mice inoculated with rMV/sMFHL died significantly more slowly than mice inoculated with rMV/sMFH; mice inoculated with SSPE virus Kobe-1 strain [SSPEV(Kobe-1)] died even more slowly than those inoculated with rMV/sMFHL although statistically not significant. Therefore, we concluded that the SSPEV-L gene attenuates the neurovirulence of chimeric MV in mice, and the N and/or P genes of the SSPE virus Kobe-1 strain (SSPEV-N and/or SSPEV-P genes) may decrease neuropathogenicity. PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1011528 July 26, 2023 3 / 28 PLOS PATHOGENS Role of viral RNA polymerase in SSPE development The SSPEV-L gene and the SSPEV-N and/or SSPEV-P genes suppressed cell-to-cell fusion and restricted viral propagation in human neuronal cells The neuropathogenicity of SSPE viruses is associated with viral hyperfusogenicity, which enables cell-to-cell spread of infection among neuronal cells lacking authentic viral receptors [49–52,54]. Although viral hyperfusogenicity is regulated by the fusion activity of the F protein [5,48,55], we examined the role of the L gene by introducing the SSPEV-L gene into EGFP- expressing rMV (rMV) and into rMV bearing the M F and H genes of the Kobe 1 (rMV/ Fig 1. Recombinant MV (rMV) harboring the L gene of the SSPE virus Kobe-1 strain (SSPEV-L gene) attenuated neurovirulence in suckling mice. (A) Schematic of the genomes of recombinant chimeric viruses (rMV). Protein- coding regions derived from the MV ICB strain [MV(ICB)] are shown as gray boxes, whereas protein-coding regions derived from the SSPE virus Kobe-1 strain [SSPEV(Kobe-1)] are shown as red boxes. (B) Mortality of suckling mice intracerebrally inoculated with the viruses in (A). The SSPEV-L gene and the SSPEV-N and/or SSPEV-P genes suppressed cell-to-cell fusion and restricted viral propagation in human neuronal cells The neuropathogenicity of SSPE viruses is associated with viral hyperfusogenicity, which enables cell-to-cell spread of infection among neuronal cells lacking authentic viral receptors [49–52,54]. Although viral hyperfusogenicity is regulated by the fusion activity of the F protein [5,48,55], we examined the role of the L gene by introducing the SSPEV-L gene into EGFP- expressing rMV (rMV) and into rMV bearing the M, F and H genes of the Kobe-1 (rMV/ sMFH); alternatively, we replaced the L gene of the EGFP-expressing recombinant SSPE virus Kobe-1 strain (rSSPEV) with the L gene of the MV ICB strain (rMV/sNPMFH) (Fig 2A). When inoculated into Vero/hSLAM cells, rMV/sL, rMV/sMFHL, and rSSPEV harboring the SSPEV-L gene demonstrated weaker cell-to-cell fusion ability compared with rMV, rMV/ sMFH, and rMV/sNPMFH, respectively (Fig 2B). Next, we used these viruses to inoculate SH-SY5Y human neuronal cells. As shown in Fig 2C, infections of rMV/sMFHL and rSSPEV PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1011528 July 26, 2023 4 / 28 PLOS PATHOGENS Role of viral RNA polymerase in SSPE development Fig 2. The SSPEV-L gene and the SSPEV-N and/or SSPEV-P genes suppressed cell-to-cell fusion and propagation of rMVs in neuronal cells. (A) Schematic of the genomes of EGFP-expressing rMVs. Colors of protein-coding regions correspond to Fig 1A. (B) Cell-to-cell fusion of rMVs in Vero/hSLAM cells. Vero/hSLAM cells were infected with EGFP-expressing cell-free rMVs, and syncytia derived from individual infected cells were observed for 60 h under a fluorescence microscope. Representative photographs of syncytia at 48 h post-infection are shown (upper panel). Magnification, ×200. Time course of syncytia enlargement shown as the percentage area of EGFP-positive syncytia, calculated using ImageJ (lower panel). Representative photographs at each time point are presented in S1A Fig. Data from five images are shown as means ± standard deviations. sL, rMV/sL; sMFH, rMV/sMFH; sMFHL, rMV/sMFHL; sNPMFH, rMV/sNPMFH. Unpaired Student’s t-test. , P < 0.05; n.s., not significant. (C) Propagation of rMVs in human neuronal cells. SH-SY5Y cells were infected with EGFP-expressing cell-free rMVs, and the spread of viral infection derived from a single infected cell was observed for 72 h under a fluorescence microscope. Representative photographs of infected cells at 72 h post-infection are shown (upper panel). Magnification, ×200. Time course of infection expansion shown as the percentage area of EGFP-positive cells, calculated using ImageJ (lower panel). Photographs at each time point are presented in S1B Fig. Data from five images are shown as means ± standard deviations. PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1011528 July 26, 2023 https://doi.org/10.1371/journal.ppat.1011528.g002 The SSPEV-L gene and the SSPEV-N and/or SSPEV-P genes suppressed cell-to-cell fusion and restricted viral propagation in human neuronal cells sMFH, rMV/sMFH; sMFHL, rMV/sMFHL; sNPMFH, rMV/sNPMFH. Unpaired Student’s t-test. , P < 0 05; n s not significant Role of viral RNA polymerase in SSPE development Fig 2. The SSPEV-L gene and the SSPEV-N and/or SSPEV-P genes suppressed cell-to-cell fusion and propagation of rMVs in neuronal cells. (A) Schematic of the genomes of EGFP-expressing rMVs. Colors of protein-coding regions correspond to Fig 1A. (B) Cell-to-cell fusion of rMVs in Vero/hSLAM cells. Vero/hSLAM cells were infected with EGFP-expressing cell-free rMVs, and syncytia derived from individual infected cells were observed for 60 h under a fluorescence microscope. Representative photographs of syncytia at 48 h post-infection are shown (upper panel). Magnification, ×200. Time course of syncytia enlargement shown as the percentage area of EGFP-positive syncytia, calculated using ImageJ (lower panel). Representative photographs at each time point are presented in S1A Fig. Data from five images are shown as means ± standard deviations. sL, rMV/sL; sMFH, rMV/sMFH; sMFHL, rMV/sMFHL; sNPMFH, rMV/sNPMFH. Unpaired Student’s t-test. , P < 0.05; n.s., not significant. (C) Propagation of rMVs in human neuronal cells. SH-SY5Y cells were infected with EGFP-expressing cell-free rMVs, and the spread of viral infection derived from a single infected cell was observed for 72 h under a fluorescence microscope. Representative photographs of infected cells at 72 h post-infection are shown (upper panel). Magnification, ×200. Time course of infection expansion shown as the percentage area of EGFP-positive cells, calculated using ImageJ (lower panel). Photographs at each time point are presented in S1B Fig. Data from five images are shown as means ± standard deviations. sMFH, rMV/sMFH; sMFHL, rMV/sMFHL; sNPMFH, rMV/sNPMFH. Unpaired Student’s t-test. , P < 0.05; n.s., not significant. Fig 2. The SSPEV-L gene and the SSPEV-N and/or SSPEV-P genes suppressed cell-to-cell fusion and propagation https://doi.org/10.1371/journal.ppat.1011528.g002 https://doi.org/10.1371/journal.ppat.1011528.g002 5 / 28 PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1011528 July 26, 2023 PLOS PATHOGENS Role of viral RNA polymerase in SSPE development spread more slowly than infections of rMV/sMFH and rMV/sNPMFH, respectively. Therefore, the SSPEV-L gene restricted viral growth by suppressing cell-to-cell spread among neuronal cells, reducing neuropathogenicity in mice (Fig 1B). Comparisons of rMV/sMFH and rMV/sNPMFH, or rMV/sMFHL and rSSPEV, revealed that rMV/sNPMFH and rSSPEV carrying the SSPEV-N and SSPEV-P genes showed less efficient cell-to-cell fusion and viral growth in neuronal cells, indicating that the SSPEV-N and/or SSPEV-P genes have suppressive effects. The SSPEV-L gene and the SSPEV-N and/or SSPEV-P genes restricted cell- surface expression of the F protein The N, P, and L proteins (encoded by the N, P, and L genes, respectively) form the RNP com- plex for transcription of viral mRNA and replication of the viral genome. The restricted growth of rMVs harboring the SSPEV-L gene and the SSPEV-N and/or SSPEV-P genes in neu- ronal cells may be caused by altered RdRp activity, thereby affecting F-protein expression at the cell surface. As shown in Fig 3A, the F mRNA levels were lower in cells infected with rMV/ sL, rMV/sMFHL, or rSSPEV than in cells infected with rMV, rMV/sMFH, or rMV/sNPMFH, respectively [i.e., the corresponding rMVs bearing the L gene of the MV ICB strain (MV-L gene)]. Thus, the surface F protein levels were significantly reduced in cells infected with rMV/ sL, rMV/sMFHL, or rSSPEV (Fig 3B). When compared between rMV/sMFH and rMV/ sNPMFH, or rMV/sMFHL and rSSPEV, the F mRNA and protein levels were lower in cells infected with rMV/sNPMFH or rSSPEV carrying the SSPEV-N and SSPEV-P genes. The reductions of N mRNA, viral genome (+ and–senses), and F mRNA suggested that the SSPEV-L protein and the SSPEV-N and/or SSPEV-P proteins suppressed the RdRp activity of the RNP complex, thereby decreasing cell-surface F-protein expression. We next examined the effects of the SSPEV-L protein and the SSPEV-N and/or SSPEV-P proteins on viral cell-to-cell fusion. Relative fusion activity was calculated through division of the cell-to-cell fusion activity (Fig 2B) by the F mRNA or protein level (Fig 3A or 3B). As shown in Fig 3C, whereas the relative fusion activities of rMV and rMV/sL bearing the MV-M, MV-F, and MV-H genes were very low, the relative fusion activities of rMV/sMFH, rMV/ sMFHL, rMV/sNPMFH, and rSSPEV carrying the SSPEV-M, SSPEV-F, and SSPEV-H genes were high. These differences were presumably related to the enhanced fusogenicity of the SSPEV-F protein, under the cooperative assistance by the SSPEV-M protein [54]. The relative cell-to-cell fusion activities of rMV/sMFH, rMV/sMFHL, rMV/sNPMFH, and rSSPEV were not significantly different, suggesting that the SSPEV-L protein and the SSPEV-N and/or SSPEV-P proteins did not directly influence cell-to-cell fusion. The suppressed cell-to-cell fusion activities of rMV/sL, rMV/sMFHL, and rSSPEV compared with rMV, rMV/sMFH, and rMV/sNPMFH, respectively (Fig 2B), were related to the decreased cell-surface F protein level that was caused by the suppression of RdRp activity. The SSPEV-L gene and the SSPEV-N and/or SSPEV-P genes suppressed cell-to-cell fusion and restricted viral propagation in human neuronal cells rMV and rMV/sL bearing the M, F, and H genes of the MV ICB strain did not spread among neuronal cells. These findings confirmed that the principal growth determinants of rMVs in neuronal cells lacking MV receptors are the F and M genes of Kobe-1 (i.e., SSPEV-F and SSPEV-M genes) as previously proved [54], which may be modified by the L gene and the N and/or P genes. The SSPEV-L gene and the SSPEV-N and/or SSPEV-P genes restricted cell- surface expression of the F protein These results indicated that cell-to-cell fusion of rMVs is principally regulated by the fuso- genicity of the F protein, which may be modulated by the F protein level that is determined by the RdRp activity of the viral RNP complex. SSPEV-L and SSPEV-P proteins suppressed RdRp activity We analyzed the RdRp activity of the RNP complex composed of the N, P, and L proteins from the MV ICB strain or the SSPE virus Kobe-1 strain in various combinations (Fig 4A). PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1011528 July 26, 2023 6 / 28 PLOS PATHOGENS Role of viral RNA polymerase in SSPE development Fig 3. rMVs carrying the SSPEV-L gene and the SSPEV-N and/or SSPEV-P genes reduced viral RNA production and restricted cell-surface expression of the F protein. (A) Viral RNA levels in cells infected with rMVs. Vero/ hSLAM cells were infected with the rMVs depicted in Fig 2A and incubated at 37˚C for 48 h in the presence of fusion- inhibiting peptide. Total RNA extraction, reverse transcription, and quantitative PCR were performed. Data from three independent experiments are shown as means ± standard deviations. (-), negative-sense genome; (+), positive- sense genome. Unpaired Student’s t-test. , P < 0.05; n.s., not significant. (B) F protein expression at the surface of cells infected with rMVs. Vero/hSLAM cells were infected with the rMVs in Fig 2A. Cell-surface proteins were biotinylated to detect F protein before preparation of whole-cell lysates. Samples were subjected to immunoblot analysis using anti- MV F, anti-MV N, anti-MV M, and anti-β-actin primary antibodies. A representative image of several experiments is shown (left panel). Inactive F0 and active F1 forms of the F protein are shown; molecular markers are indicated on right. CS, cell-surface fraction; WC, whole-cell fraction. Intensities of F1, N, and M protein bands were quantified using ImageJ (right panel). Fs, F1 protein in CS; F, F1 protein in WC. (C) Specific viral cell-to-cell fusion activity Fig 3. rMVs carrying the SSPEV-L gene and the SSPEV-N and/or SSPEV-P genes reduced viral RNA production and restricted cell-surface expression of the F protein. (A) Viral RNA levels in cells infected with rMVs. Vero/ hSLAM cells were infected with the rMVs depicted in Fig 2A and incubated at 37˚C for 48 h in the presence of fusion- inhibiting peptide. Total RNA extraction, reverse transcription, and quantitative PCR were performed. Data from three independent experiments are shown as means ± standard deviations. (-), negative-sense genome; (+), positive- sense genome. Unpaired Student’s t-test. , P < 0.05; n.s., not significant. (B) F protein expression at the surface of cells infected with rMVs. Vero/hSLAM cells were infected with the rMVs in Fig 2A. PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1011528 July 26, 2023 SSPEV-L and SSPEV-P proteins suppressed RdRp activity Cell-surface proteins were biotinylated to detect F protein before preparation of whole-cell lysates. Samples were subjected to immunoblot analysis using anti- MV F, anti-MV N, anti-MV M, and anti-β-actin primary antibodies. A representative image of several experiments is shown (left panel). Inactive F0 and active F1 forms of the F protein are shown; molecular markers are indicated on right. CS, cell-surface fraction; WC, whole-cell fraction. Intensities of F1, N, and M protein bands were quantified using ImageJ (right panel). Fs, F1 protein in CS; F, F1 protein in WC. (C) Specific viral cell-to-cell fusion activity relative to F mRNA and F1 protein levels. Specific cell-to-cell fusion activity was calculated through division of cell-to- cell fusion at 48 h post-infection in Fig 2B by the F mRNA or F1 protein level in Fig 3A and 3B. The value of rMV or rMV/sL (sL) was set at 100%. Unpaired Student’s t-test. , P < 0.05; n.s., not significant. https://doi.org/10.1371/journal.ppat.1011528.g003 7 / 28 PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1011528 July 26, 2023 PLOS PATHOGENS Role of viral RNA polymerase in SSPE development Fig 4. SSPEV-L and SSPEV-P proteins decrease RdRp activity. (A) RdRp activities of the N, P, and L proteins of the MV ICB strain (MV) or SSPE virus Kobe-1 strain (SV) in various combinations. Vero/hSLAM cells were transfected with plasmids expressing the N, P, and L proteins of MV or SV in combination, together with the MV mini-genome plasmid encoding the firefly luciferase gene and the Renilla luciferase-expressing plasmid. After incubation at 37˚C for 24 h, RdRp activity was determined. Data from three independent experiments are shown as means ± standard Fig 4. SSPEV-L and SSPEV-P proteins decrease RdRp activity. (A) RdRp activities of the N, P, and L proteins of the MV ICB strain (MV) or SSPE virus Kobe-1 strain (SV) in various combinations. Vero/hSLAM cells were transfected with plasmids expressing the N, P, and L proteins of MV or SV in combination, together with the MV mini-genome plasmid encoding the firefly luciferase gene and the Renilla luciferase-expressing plasmid. After incubation at 37˚C for 24 h, RdRp activity was determined. Data from three independent experiments are shown as means ± standard Fig 4. SSPEV-L and SSPEV-P proteins decrease RdRp activity. (A) RdRp activities of the N, P, and L proteins of the MV ICB strain (MV) or SSPE virus Kobe-1 strain (SV) in various combinations. PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1011528 July 26, 2023 SSPEV-L and SSPEV-P proteins suppressed RdRp activity Vero/hSLAM cells were transfected with plasmids expressing the N, P, and L proteins of MV or SV in combination, together with the MV mini-genome plasmid encoding the firefly luciferase gene and the Renilla luciferase-expressing plasmid. After incubation at 37˚C for 24 h, RdRp activity was determined. Data from three independent experiments are shown as means ± standard PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1011528 July 26, 2023 8 / 28 PLOS PATHOGENS Role of viral RNA polymerase in SSPE development deviations. (B) Relative RdRp activities for evaluation of the effect of the L protein. RdRp activities in (A) were compared between the MV-L protein (100%) and the SV-L protein with the same combinations of N and P proteins. Unpaired Student’s t-test. , P < 0.05. (C) Relative RdRp activities for evaluation of the effect of the P protein. RdRp activities in (A) were compared between the MV-P protein (100%) and the SV-P protein with the same combinations of N and L proteins. Unpaired Student’s t-test. , P < 0.05. (D) Relative RdRp activities for evaluation of the effect of the N protein. RdRp activities in (A) were compared between the MV-N protein (100%) and the SV-N protein with the same combinations of P and L proteins. Unpaired Student’s t-test. , P < 0.05; n.s., not significant. https://doi.org/10.1371/journal.ppat.1011528.g004 deviations. (B) Relative RdRp activities for evaluation of the effect of the L protein. RdRp activities in (A) were compared between the MV-L protein (100%) and the SV-L protein with the same combinations of N and P proteins. Unpaired Student’s t-test. , P < 0.05. (C) Relative RdRp activities for evaluation of the effect of the P protein. RdRp activities in (A) were compared between the MV-P protein (100%) and the SV-P protein with the same combinations of N and L proteins. Unpaired Student’s t-test. , P < 0.05. (D) Relative RdRp activities for evaluation of the effect of the N protein. RdRp activities in (A) were compared between the MV-N protein (100%) and the SV-N protein with the same combinations of P and L proteins. Unpaired Student’s t-test. , P < 0.05; n.s., not significant. https://doi.org/10.1371/journal.ppat.1011528.g004 Mini-genome assays showed that the SSPEV-L and SSPEV-P proteins strongly suppressed RdRp activity (Fig 4B and 4C), but the SSPEV-N protein moderately reduced RdRp activity in the presence of the SSPEV-P protein (Fig 4D). Three amino acid mutations in the SSPEV-L protein sharply suppressed RdRp activity Indeed, the triple-mutant MV-L protein possessing S519N, I539V, and K601R [L(triple) protein] exhibited extremely low RdRp activity. Amino acid mutations in the SSPEV-L protein were located in the RdRp, methyltransferase (MTase), and C-terminal (CT) domains (Fig 5C). S519N, I539V, and K601R substantially decreased the RdRp activity of the MV-L protein to a level below the activity of the SSPEV-L protein (Fig 5D). These mutations were around the site of RdRp activity in the RdRp domain, suggesting a direct relationship with RNA synthesis. Indeed, the triple-mutant MV-L protein possessing S519N, I539V, and K601R [L(triple) protein] exhibited extremely low RdRp activity. SSPEV-L and SSPEV-P proteins suppressed RdRp activity There may be differences between the MV-P protein and the SSPEV-P protein in terms of their interactions with the N protein. When the MV-P gene of rMV/sMFH or rMV/sMFHL was replaced with the SSPEV-P gene (S2A Fig), the resulting rMV/sPMFH or rMV/sPMFHL exhibited substantial suppression of viral cell-to- cell fusion ability and growth in neuronal cells. In contrast, replacement of the MV-N gene with the SSPEV-N gene did not lead to a change in viral characteristics (S2B and S2C Fig). The results indicated that the suppressed propagation in neuronal cells and reduced cell-to- cell fusion of rMV/sNPMFH and rSSPEV bearing the SSPEV-N and SSPEV-P genes, com- pared with rMV/sMFH and rMV/sMFHL bearing the MV-P and MV-N genes (Fig 2), is caused by the reduction of RdRp activity (Fig 3A) in a manner mediated by the SSPEV-P pro- tein, rather than the SSPEV-N protein. PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1011528 July 26, 2023 Three amino acid mutations in the SSPEV-L protein sharply suppressed RdRp activity To identify mutations involved in the reduction of RdRp activity, mutations in the SSPEV-P and SSPEV-L proteins were sequentially introduced into the MV-P and MV-L proteins, respectively; the effects were evaluated by mini-genome assays. Mutations were located throughout the SSPEV-P protein. Among them, T140A and S313P significantly reduced RdRp activity; MV-P proteins with single point mutations other than T140A and S313P exhibited non-significant reductions of RdRp activity (Fig 5B). The RdRp activity of the SSPE virus Kobe-1 strain may have decreased through the accumulation of mutations in the P protein. Amino acid 313 is located in the multimerization domain (MD) (Fig 5A), and S313P loosens the α-helix of the MD, thereby decreasing RdRp activity [56]. Amino acid mutations in the SSPEV-L protein were located in the RdRp, methyltransferase (MTase), and C-terminal (CT) domains (Fig 5C). S519N, I539V, and K601R substantially decreased the RdRp activity of the MV-L protein to a level below the activity of the SSPEV-L protein (Fig 5D). These mutations were around the site of RdRp activity in the RdRp domain, Mutations were located throughout the SSPEV-P protein. Among them, T140A and S313P significantly reduced RdRp activity; MV-P proteins with single point mutations other than T140A and S313P exhibited non-significant reductions of RdRp activity (Fig 5B). The RdRp activity of the SSPE virus Kobe-1 strain may have decreased through the accumulation of mutations in the P protein. Amino acid 313 is located in the multimerization domain (MD) (Fig 5A), and S313P loosens the α-helix of the MD, thereby decreasing RdRp activity [56]. significantly reduced RdRp activity; MV-P proteins with single point mutations other than T140A and S313P exhibited non-significant reductions of RdRp activity (Fig 5B). The RdRp activity of the SSPE virus Kobe-1 strain may have decreased through the accumulation of mutations in the P protein. Amino acid 313 is located in the multimerization domain (MD) (Fig 5A), and S313P loosens the α-helix of the MD, thereby decreasing RdRp activity [56]. Amino acid mutations in the SSPEV-L protein were located in the RdRp, methyltransferase (MTase), and C-terminal (CT) domains (Fig 5C). S519N, I539V, and K601R substantially decreased the RdRp activity of the MV-L protein to a level below the activity of the SSPEV-L protein (Fig 5D). These mutations were around the site of RdRp activity in the RdRp domain, suggesting a direct relationship with RNA synthesis. Suppression of RdRp activity restricted progeny virus production and may promote persistent infection (C) Schematic of the locations of amino acid mutations in the SSPEV-L protein. RdRp, RdRp domain; Capping, capping domain; CD, connecting domain; MTase, methyltransferase domain; CT, C-terminal domain. (D) Effects of mutations of the SSPEV-L protein on RdRp activity. Vero/hSLAM cells were transfected with a plasmid Fig 5. Multiple amino acid mutations in the P and L proteins reduced RdRp activity. (A) Schematic of the locations of amino acid mutations in the SSPEV-P protein. NTD, N-terminal domain; MD, multimerization domain; XD, X domain. (B) Effects of the mutations of SSPEV-P protein on RdRp activity. Vero/hSLAM cells were transfected with a plasmid expressing the P protein with each mutation, together with plasmids expressing the N and L proteins of MV, an MV mini-genome plasmid encoding the firefly luciferase gene, and the Renilla luciferase-expressing plasmid. RdRp activity was determined as in Fig 4A. Data from three independent experiments are shown as means ± standard deviations. Unpaired Student’s t-test. , P < 0.05 compared with MV-P protein; n.s., not significant compared with MV-P protein. (C) Schematic of the locations of amino acid mutations in the SSPEV-L protein. RdRp, RdRp domain; Capping, capping domain; CD, connecting domain; MTase, methyltransferase domain; CT, C-terminal domain. (D) Effects of mutations of the SSPEV-L protein on RdRp activity. Vero/hSLAM cells were transfected with a plasmid Fig 5. Multiple amino acid mutations in the P and L proteins reduced RdRp activity. (A) Schematic of the locations of amino acid mutations in the SSPEV-P protein. NTD, N-terminal domain; MD, multimerization domain; XD, X domain. (B) Effects of the mutations of SSPEV-P protein on RdRp activity. Vero/hSLAM cells were transfected with a plasmid expressing the P protein with each mutation, together with plasmids expressing the N and L proteins of MV, an MV mini-genome plasmid encoding the firefly luciferase gene, and the Renilla luciferase-expressing plasmid. RdRp activity was determined as in Fig 4A. Data from three independent experiments are shown as means ± standard deviations. Unpaired Student’s t-test. , P < 0.05 compared with MV-P protein; n.s., not significant compared with MV-P protein. (C) Schematic of the locations of amino acid mutations in the SSPEV-L protein. RdRp, RdRp domain; Capping, capping domain; CD, connecting domain; MTase, methyltransferase domain; CT, C-terminal domain. (D) Effects of mutations of the SSPEV-L protein on RdRp activity. Vero/hSLAM cells were transfected with a plasmid Fig 5. PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1011528 July 26, 2023 Suppression of RdRp activity restricted progeny virus production and may promote persistent infection The suppression of RdRp activity by the SSPEV-L and SSPEV-P proteins suppressed viral cell- to-cell spread among neuronal cells. Because the onset and/or aggravation of SSPE are caused by and correlated with the propagation of SSPE viruses in the brain, reduced RdRp activity presumably did not contribute to these events. Although it is impossible to isolate and examine virus persisting in the brain of an SSPE patient before the appearance of clinical signs, we PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1011528 July 26, 2023 9 / 28 PLOS PATHOGENS Role of viral RNA polymerase in SSPE development Fig 5. Multiple amino acid mutations in the P and L proteins reduced RdRp activity. (A) Schematic of the location of amino acid mutations in the SSPEV-P protein. NTD, N-terminal domain; MD, multimerization domain; XD, X domain. (B) Effects of the mutations of SSPEV-P protein on RdRp activity. Vero/hSLAM cells were transfected with a plasmid expressing the P protein with each mutation, together with plasmids expressing the N and L proteins of MV, an MV mini-genome plasmid encoding the firefly luciferase gene, and the Renilla luciferase-expressing plasmid. RdRp activity was determined as in Fig 4A. Data from three independent experiments are shown as means ± standard deviations. Unpaired Student’s t-test. , P < 0.05 compared with MV-P protein; n.s., not significant compared with MV-P protein. (C) Schematic of the locations of amino acid mutations in the SSPEV-L protein. RdRp, RdRp domain; Capping, capping domain; CD, connecting domain; MTase, methyltransferase domain; CT, C-terminal domain. (D) Effects of mutations of the SSPEV-L protein on RdRp activity. Vero/hSLAM cells were transfected with a plasmid Role of viral RNA polymerase in SSPE developmen Fig 5. Multiple amino acid mutations in the P and L proteins reduced RdRp activity. (A) Schematic of the locations of amino acid mutations in the SSPEV-P protein. NTD, N-terminal domain; MD, multimerization domain; XD, X domain. (B) Effects of the mutations of SSPEV-P protein on RdRp activity. Vero/hSLAM cells were transfected with a plasmid expressing the P protein with each mutation, together with plasmids expressing the N and L proteins of MV, an MV mini-genome plasmid encoding the firefly luciferase gene, and the Renilla luciferase-expressing plasmid. RdRp activity was determined as in Fig 4A. Data from three independent experiments are shown as means ± standard deviations. Unpaired Student’s t-test. , P < 0.05 compared with MV-P protein; n.s., not significant compared with MV-P protein. Suppression of RdRp activity restricted progeny virus production and may promote persistent infection Multiple amino acid mutations in the P and L proteins reduced RdRp activity. (A) Schematic of the locations of amino acid mutations in the SSPEV-P protein. NTD, N-terminal domain; MD, multimerization domain; XD, X domain. (B) Effects of the mutations of SSPEV-P protein on RdRp activity. Vero/hSLAM cells were transfected with a plasmid expressing the P protein with each mutation, together with plasmids expressing the N and L proteins of MV, an MV mini-genome plasmid encoding the firefly luciferase gene, and the Renilla luciferase-expressing plasmid. RdRp activity was determined as in Fig 4A. Data from three independent experiments are shown as means ± standard deviations. Unpaired Student’s t-test. , P < 0.05 compared with MV-P protein; n.s., not significant compared with MV-P protein. (C) Schematic of the locations of amino acid mutations in the SSPEV-L protein. RdRp, RdRp domain; Capping, capping domain; CD, connecting domain; MTase, methyltransferase domain; CT, C-terminal domain. (D) Effects of mutations of the SSPEV-L protein on RdRp activity. Vero/hSLAM cells were transfected with a plasmid PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1011528 July 26, 2023 10 / 28 PLOS PATHOGENS Role of viral RNA polymerase in SSPE development expressing the L protein with each mutation, together with plasmids expressing the N and P proteins of MV, an MV mini-genome plasmid encoding the firefly luciferase gene, and the Renilla luciferase-expressing plasmid. RdRp activity was determined as in Fig 4A. Triple, L protein carrying S519N, I539V, and K601R mutations. Data from three independent experiments are shown as means ± standard deviations. Unpaired Student’s t-test. , P < 0.05 compared with MV-L protein; n.s., not significant compared with MV-L protein. https://doi.org/10.1371/journal.ppat.1011528.g005 expressing the L protein with each mutation, together with plasmids expressing the N and P proteins of MV, an MV mini-genome plasmid encoding the firefly luciferase gene, and the Renilla luciferase-expressing plasmid. RdRp activity was determined as in Fig 4A. Triple, L protein carrying S519N, I539V, and K601R mutations. Data from three independent experiments are shown as means ± standard deviations. Unpaired Student’s t-test. , P < 0.05 compared with MV-L protein; n.s., not significant compared with MV-L protein. https://doi.org/10.1371/journal.ppat.1011528.g005 suspect that MV suppresses the expression of viral proteins and the release of viral particles, thus establishing a persistent infection. This suppression may involve reduced RdRp activity. Suppression of RdRp activity restricted progeny virus production and may promote persistent infection Next, we evaluated the effect of L protein with S519N, I539V, and K601R mutations [L(tri- ple) protein] on progeny virus production and growth in neuronal cells. As shown in Fig 6A, rMV carrying the SSPEV-L protein (rMV/sL) and the L(triple) protein [rMV/L(triple)] produced < 10% and < 1% infectious rMV, respectively. Lack of progeny virus release, typical of SSPE viruses, is caused by mutations in the M protein [49,54], but replacement of the MV-M gene with the SSPEV-M gene did not abolish virus particle production (see rMV/sM in Fig 6A) [34]. The reduction of RdRp activity restricted virus release, and rMV/sML(triple) car- rying the SSPEV-M protein and the L(triple) protein lost the ability to release infectious virus. Because the SSPEV-F and SSPEV-M proteins are indispensable for propagation in neuronal cells [54], we next evaluated the effect of the triple mutant on viral cell-to-cell fusion and growth in neuronal cells using rMVs harboring the SSPEV-M, SSPEV-F, and SSPEV-H genes. The triple mutants strongly suppressed cell-to-cell fusion of rMV/sMFHL(triple) and limited viral spread in SH-SY5Y neuronal cells, compared with rMV/sMFH and rMV/sMFHL (Fig 6B). Therefore, the suppression of RdRp activity restricted viral particle release and cell-to-cell spread among neuronal cells, suggesting that the L(triple) protein (which had substantially reduced RdRp activity) promoted the establishment of persistent infection in the brain by lim- iting viral propagation. Suppression of RdRp activity by the L(triple) protein was restored by induction of other mutations in the SSPEV-L protein The L(triple) protein promoted persistent infection at the stage before onset of SSPE. To exam- ine the effects of other mutations in the SSPEV-L protein on the RdRp activity of the L(triple) protein, we introduced mutations into the L(triple) protein (Fig 7A). Mutations in groups 1, 2, and 3 (in the RdRp domain) significantly increased the RdRp activity of the L(triple) protein, which was enhanced by combinations of these mutations (Fig 7B). The substantial enhance- ments in groups 1+2 and 1+3 demonstrated that the suppressed RdRp activity of the L(triple) protein was restored by mutations in group 1, through synergistic interactions with mutations in group 2 or 3. The addition of all mutations led to an additional increase in RdRp activity, which was further enhanced by the addition of mutations in group 4 (SSPEV-L protein in Fig 7B); however, mutations in group 4 (i.e., MTase and CT domains) alone did not enhance activ- ity. Therefore, the RdRp activity of the L(triple) protein was substantially reduced by S519N, I539V, and K601R mutations; it was restored by the cumulative addition of other mutations. Restoration of RdRp activity corresponded to viral propagation in neuronal cells To determine whether restoration of the RdRp activity of the L(triple) protein affects viral behavior, we generated rMVs harboring the L(triple) protein with the other mutations (Fig 7) based on rMV/sMFHL(triple) (Fig 8A). The RdRp activity of the L(triple) protein was increased by the addition of the four groups of mutations (Fig 7B); the F mRNA level was PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1011528 July 26, 2023 11 / 28 PLOS PATHOGENS Role of viral RNA polymerase in SSPE development Fig 6. The triple-mutant L protein [L(triple) protein] limited progeny virus production and restricted viral propagation in neuronal cells. (A) Cell-free infectious viruses released from cells infected with rMVs. Schematic of the genomes of the rMVs is shown (upper panel). Colors of the protein-coding regions derived from MV or SSPEV correspond to Fig 1A. Triple (blue box) represents the MV-L gene harboring S519N, I539V, and K601R mutations. Vero/hSLAM cells were infected with rMVs and incubated at 37˚C for 4 days; the medium was changed every 24 h. After centrifugation, cell-free viruses in culture medium were titrated in Vero/hSLAM cells. Infectious virus titers at day 4 post-infection are shown. Titers over 4 days are presented in S3 Fig. Data from three independent experiments are shown as means ± standard deviations. Unpaired Student’s t-test. , P < 0.05; N.D., not detected. (B) Effects of the L(triple) protein on viral cell-to-cell fusion and propagation in neuronal cells. Schematic of the genomes of rMVs bearing the SSPEV-M, SSPEV-F, and SSPEV-H genes is shown (upper panel). Colors of the protein-coding regions are as in (A). Vero/hSLAM (left panel) and SH-SY5H cells (right panel) were infected with rMVs, incubated at 37˚C, and monitored under a fluorescence microscope. Representative photographs derived from a single infected cell were acquired at 48 and 72 h. Magnification, ×200. https://doi.org/10.1371/journal.ppat.1011528.g006 g 6. The triple-mutant L protein [L(triple) protein] limited progeny virus production and restricted viral Fig 6. The triple-mutant L protein [L(triple) protein] limited progeny virus production and restricted viral propagation in neuronal cells. (A) Cell-free infectious viruses released from cells infected with rMVs. Schematic of the genomes of the rMVs is shown (upper panel). Colors of the protein-coding regions derived from MV or SSPEV correspond to Fig 1A. Triple (blue box) represents the MV-L gene harboring S519N, I539V, and K601R mutations. Vero/hSLAM cells were infected with rMVs and incubated at 37˚C for 4 days; the medium was changed every 24 h. PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1011528 July 26, 2023 Restoration of RdRp activity corresponded to viral propagation in neuronal cells After centrifugation, cell-free viruses in culture medium were titrated in Vero/hSLAM cells. Infectious virus titers at day 4 post-infection are shown. Titers over 4 days are presented in S3 Fig. Data from three independent experiments are shown as means ± standard deviations. Unpaired Student’s t-test. , P < 0.05; N.D., not detected. (B) Effects of the L(triple) protein on viral cell-to-cell fusion and propagation in neuronal cells. Schematic of the genomes of rMVs bearing the SSPEV-M, SSPEV-F, and SSPEV-H genes is shown (upper panel). Colors of the protein-coding regions are as in (A). Vero/hSLAM (left panel) and SH-SY5H cells (right panel) were infected with rMVs, incubated at 37˚C, and monitored under a fluorescence microscope. Representative photographs derived from a single infected cell were acquired at 48 and 72 h. Magnification, ×200. https://doi.org/10.1371/journal.ppat.1011528.g006 https://doi.org/10.1371/journal.ppat.1011528.g006 12 / 28 PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1011528 July 26, 2023 PLOS PATHOGENS Role of viral RNA polymerase in SSPE development Fig 7. Reduced RdRp activity of the L(triple) protein was restored by introduction of other mutations into the SSPEV-L protein. (A) Schematic of the locations of amino acid mutations in the SSPEV-L protein. S519N, I539V, and K601R mutations are shown in blue. Other mutations (red): group 1 (H313Y, E368A, and K441R), group 2 (N504S and A521S), group 3 (S621I, R623K, and V720I), and group 4 (I1891V, I1995V, and V2130I). (B) RdRp activities of the L(triple) protein in combination with groups 1, 2, 3, and 4 mutations. Amino acid substitutions are listed in S1 Table. RdRp activities were measured as in Fig 5. Data from three independent experiments are shown as means ± standard deviations. Unpaired Student’s t-test. , P < 0.05 compared with triple-mutant L protein; n.s., not significant compared with triple-mutant L protein; †, P < 0.05. https://doi.org/10.1371/journal.ppat.1011528.g007 Fig 7. Reduced RdRp activity of the L(triple) protein was restored by introduction of other mutations into the SSPEV-L protein. (A) Schematic of the locations of amino acid mutations in the SSPEV-L protein. S519N, I539V, and K601R mutations are sho n in blue Other mutations (red) group 1 (H313Y E368A and K441R) group 2 (N504S Fig 7. Reduced RdRp activity of the L(triple) protein was restored by introduction of other mutations into the SSPEV-L protein. (A) Schematic of the locations of amino acid mutations in the SSPEV-L protein. S519N, I539V, and K601R mutations are shown in blue. Restoration of RdRp activity corresponded to viral propagation in neuronal cells Other mutations (red): group 1 (H313Y, E368A, and K441R), group 2 (N504S and A521S), group 3 (S621I, R623K, and V720I), and group 4 (I1891V, I1995V, and V2130I). (B) RdRp activities of the L(triple) protein in combination with groups 1, 2, 3, and 4 mutations. Amino acid substitutions are listed in S1 Table. RdRp activities were measured as in Fig 5. Data from three independent experiments are shown as means ± standard deviations. Unpaired Student’s t-test. , P < 0.05 compared with triple-mutant L protein; n.s., not significant compared with triple-mutant L protein; †, P < 0.05. https://doi.org/10.1371/journal.ppat.1011528.g007 increased in cells infected with each virus (S4A Fig), and the level of cell-surface F-protein expression was also increased (S4B Fig). Accordingly, rMVs increased cell-to-cell fusion (Fig 8B) and the spread of infection among neuronal cells (Fig 8C) in a manner that corresponded to RdRp activity. Control virus (rMV/sMFH) bearing the MV-L protein with the highest RdRp activity produced the greatest cell-surface F-protein expression and caused the greatest propa- gation in neuronal cells. Therefore, RdRp activity corresponds to viral cell-to-cell fusion and regulates viral propagation in neuronal cells if the virus can successfully infect neuronal cells (i.e., it contains SSPEV-F and SSPEV-M proteins). Because growth in neuronal cells is associ- ated with viral neuropathogenicity (Figs 1 and 2) [54], the neurovirulence of rMV/sMFHL(tri- ple) should have been extremely low and increased with the enhancement of RdRp activity in a manner mediated by the accumulation of other mutations in the SSPEV-L protein. increased in cells infected with each virus (S4A Fig), and the level of cell-surface F-protein expression was also increased (S4B Fig). Accordingly, rMVs increased cell-to-cell fusion (Fig 8B) and the spread of infection among neuronal cells (Fig 8C) in a manner that corresponded to RdRp activity. Control virus (rMV/sMFH) bearing the MV-L protein with the highest RdRp activity produced the greatest cell-surface F-protein expression and caused the greatest propa- gation in neuronal cells. Therefore, RdRp activity corresponds to viral cell-to-cell fusion and regulates viral propagation in neuronal cells if the virus can successfully infect neuronal cells (i.e., it contains SSPEV-F and SSPEV-M proteins). Because growth in neuronal cells is associ- ated with viral neuropathogenicity (Figs 1 and 2) [54], the neurovirulence of rMV/sMFHL(tri- ple) should have been extremely low and increased with the enhancement of RdRp activity in a manner mediated by the accumulation of other mutations in the SSPEV-L protein. PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1011528 July 26, 2023 Restoration of RdRp activity corresponded to viral propagation in neuronal cells (A) Schematic of EGFP-expressing rMV/sMFH genomes harboring amino acid substitutions in the L protein in Fig 7. (B) Cell-to-cell fusion. Vero/hSLAM cells were infected with the cell-free rMVs in (A) and enlargement of fused cells was monitored under a fluorescence microscope; photographs were obtained at 12-h intervals. Representative photographs are shown in S5A Fig. Cell-to-cell fusion activity was quantified as in Fig 2B. Data from five images are shown as means ± standard deviations. (C) Viral propagation in neuronal cells. SH-SY5Y cells were infected with the cell-free rMVs in (A) and the spread of infection from a single infected cell was observed under a fluorescence microscope; photographs were obtained at 12-h intervals. Representative photographs are shown in S5B Fig. Spread of viral infection was determined as in Fig 2C. Data from five images are shown as means ± standard deviations. Fig 8. Restoration of RdRp activity was associated with enhancement of cell-to-cell fusion and propagation of rMVs in neuronal cells. (A) Schematic of EGFP-expressing rMV/sMFH genomes harboring amino acid substitutions in the L protein in Fig 7. (B) Cell-to-cell fusion. Vero/hSLAM cells were infected with the cell-free rMVs in (A) and enlargement of fused cells was monitored under a fluorescence microscope; photographs were obtained at 12-h intervals. Representative photographs are shown in S5A Fig. Cell-to-cell fusion activity was quantified as in Fig 2B. Data from five images are shown as means ± standard deviations. (C) Viral propagation in neuronal cells. SH-SY5Y cells were infected with the cell-free rMVs in (A) and the spread of infection from a single infected cell was observed under a fluorescence microscope; photographs were obtained at 12-h intervals. Representative photographs are shown in S5B Fig. Spread of viral infection was determined as in Fig 2C. Data from five images are shown as means ± standard deviations. https://doi.org/10.1371/journal.ppat.1011528.g008 Restoration of RdRp activity corresponded to viral propagation in neuronal cells PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1011528 July 26, 2023 13 / 28 PLOS PATHOGENS Role of viral RNA polymerase in SSPE development Fig 8. Restoration of RdRp activity was associated with enhancement of cell-to-cell fusion and propagation of rMVs in neuronal cells. (A) Schematic of EGFP-expressing rMV/sMFH genomes harboring amino acid substitutions in the L protein in Fig 7. (B) Cell-to-cell fusion. Vero/hSLAM cells were infected with the cell-free rMVs in (A) and enlargement of fused cells was monitored under a fluorescence microscope; photographs were obtained at 12-h intervals. Representative photographs are shown in S5A Fig. Cell-to-cell fusion activity was quantified as in Fig 2B. Data from five images are shown as means ± standard deviations. (C) Viral propagation in neuronal cells. SH-SY5Y cells were infected with the cell-free rMVs in (A) and the spread of infection from a single infected cell was observed under a fluorescence microscope; photographs were obtained at 12-h intervals. Representative photographs are shown in S5B Fig. Spread of viral infection was determined as in Fig 2C. Data from five images are shown as means ± standard deviations. htt //d i /10 1371/j l t 1011528 008 Fig 8. Restoration of RdRp activity was associated with enhancement of cell-to-cell fusion and propagation of rMVs in neuronal cells. (A) Schematic of EGFP-expressing rMV/sMFH genomes harboring amino acid substitutions in the L protein in Fig 7. (B) Cell-to-cell fusion. Vero/hSLAM cells were infected with the cell-free rMVs in (A) and enlargement of fused cells was monitored under a fluorescence microscope; photographs were obtained at 12-h intervals. Representative photographs are shown in S5A Fig. Cell-to-cell fusion activity was quantified as in Fig 2B. Data from five images are shown as means ± standard deviations. (C) Viral propagation in neuronal cells. SH-SY5Y cells were infected with the cell-free rMVs in (A) and the spread of infection from a single infected cell was observed under a fluorescence microscope; photographs were obtained at 12-h intervals. Representative photographs are shown in S5B Fig. Spread of viral infection was determined as in Fig 2C. Data from five images are shown as means ± standard deviations. Fig 8. Restoration of RdRp activity was associated with enhancement of cell-to-cell fusion and propagation of l ll ( ) h f h b d b Fig 8. Restoration of RdRp activity was associated with enhancement of cell-to-cell fusion and propagation of rMVs in neuronal cells. PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1011528 July 26, 2023 Effects of mutations in the SSPE virus Kobe-1 strain on SSPE progression Based on our findings, we constructed a model of the contributions of mutations in the SSPE virus Kobe-1 strain to the progression of SSPE (Fig 9). The finding that the SSPEV-L protein attenuated (rather than promoted) viral neuropathogenicity prompted us to consider the role of the L protein in persistent infection before the onset of SSPE. Under the assumption that the tracks of mutations must be present in the genome of an SSPE virus, we analyzed the PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1011528 July 26, 2023 14 / 28 PLOS PATHOGENS Role of viral RNA polymerase in SSPE development Fig 9. Possible roles of mutations accumulated in the SSPE virus Kobe-1 strain in SSPE. Mutations in blue suppress the functions of viral proteins and may contribute to the establishment and maintenance of persistent infection. Mutations in red enhance viral protein functions to promote viral propagation in the brain, resulting in the onset of SSPE. Mutations in the M protein were described previously [34,54]. Fig 9. Possible roles of mutations accumulated in the SSPE virus Kobe-1 strain in SSPE. Mutations in blue suppress the functions of viral proteins and may contribute to the establishment and maintenance of persistent infection. Mutations in red enhance viral protein functions to promote viral propagation in the brain, resulting in the onset of SSPE. Mutations in the M protein were described previously [34,54]. https://doi.org/10.1371/journal.ppat.1011528.g009 Fig 9. Possible roles of mutations accumulated in the SSPE virus Kobe-1 strain in SSPE. Mutations in blue suppress the functions of viral proteins and may contribute to the establishment and maintenance of persistent infection. Mutations in red enhance viral protein functions to promote viral propagation in the brain, resulting in the onset of SSPE. Mutations in the M protein were described previously [34,54]. https://doi.org/10.1371/journal.ppat.1011528.g009 https://doi.org/10.1371/journal.ppat.1011528.g009 effects of mutations of the SSPEV-L protein on RdRp activity. RdRp activity was substantially reduced by the presence of S519N, I539V, and K601R mutations, but it was restored by the addition of other mutations (Fig 7). The reduction of RdRp activity could promote persistence by hindering progeny virus production and viral protein expression, thereby mediating immune escape. Two mutations in the SSPEV-P protein that suppressed viral RdRp activity also promoted the establishment and maintenance of persistent infection. However, the resto- ration of RdRp activity may explain efficient Kobe-1 virus spread in the brain after the acquisi- tion of fusogenic mutations in the F protein. PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1011528 July 26, 2023 Effects of mutations in the SSPE virus Kobe-1 strain on SSPE progression Mutations in the SSPEV-M protein hindered viral particle production (Fig 6A) under the suppressed viral RdRp activity, thereby avoiding host immunity, but enhanced cell-to-cell fusion, thereby promoting viral spread among neuro- nal cells, and increasing neuropathogenicity under synergistic cooperation with the fusogenic SSPEV-F protein [54]. The accelerated fusion activity of the SSPEV-F protein was indispens- able for cell-to-cell spread in the brain, which was enhanced by mutations in the SSPEV-L and SSPEV-M proteins. Neuropathogenicity would presumably increase as mutations effects of mutations of the SSPEV-L protein on RdRp activity. RdRp activity was substantially reduced by the presence of S519N, I539V, and K601R mutations, but it was restored by the addition of other mutations (Fig 7). The reduction of RdRp activity could promote persistence by hindering progeny virus production and viral protein expression, thereby mediating immune escape. Two mutations in the SSPEV-P protein that suppressed viral RdRp activity also promoted the establishment and maintenance of persistent infection. However, the resto- ration of RdRp activity may explain efficient Kobe-1 virus spread in the brain after the acquisi- tion of fusogenic mutations in the F protein. Mutations in the SSPEV-M protein hindered viral particle production (Fig 6A) under the suppressed viral RdRp activity, thereby avoiding host immunity, but enhanced cell-to-cell fusion, thereby promoting viral spread among neuro- nal cells, and increasing neuropathogenicity under synergistic cooperation with the fusogenic SSPEV-F protein [54]. The accelerated fusion activity of the SSPEV-F protein was indispens- able for cell-to-cell spread in the brain, which was enhanced by mutations in the SSPEV-L and SSPEV-M proteins. Neuropathogenicity would presumably increase as mutations PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1011528 July 26, 2023 15 / 28 PLOS PATHOGENS Role of viral RNA polymerase in SSPE development Fig 10. Clusters of amino acid mutations in the P protein of SSPE viruses. Sequence alignment of the region in the P protein surrounding the T140A and S313P mutations (marked in light blue) of the SSPEV Kobe-1 strain. Several strains of MV and SSPE virus of the same genotype (D3, D6, C2, C1, B3 and A) are compared. The red letters indicate mutations specific to SSPE viruses. DM domain of the P protein is marked in yellow. -, equivalent amino acid to that of ICB. Accession numbers in DDBJ/EMBL/GenBank: ICB, AB016162; MVi/Tokyo.JPN/37.99(Y), GQ376026; Kobe-1, AB254456; WA.USA/17.98, DQ227321; 97–45881, DQ227319; MVs/Alberta.CAN/22.14, MF775733; MVs/Zagreb. Effects of mutations in the SSPE virus Kobe-1 strain on SSPE progression CRO/08.03, DQ227320; MVs/Zagreb.CRO/47.02, DQ227318; JM, M90469; ZH, AB453187; Mvi/Lyon.FRA/77, HM562908; Mvs/Toulon.FRA/08.07, HM562909; Masusako, LC655230; Nagahata, D63927; Osaka-1/Fr/V, LC655226; Osaka-2/Fr/V, LC655228; Osaka-3/Bs/V, LC655229; Yamagata-1, D10635; Kitaken-1, AB453045; KS, HM439386; MVs/CapeTown.ZAF/16.10/2, KT851526; MVs/CapeTown.ZAF/52.14, KT851534; Edmonston, K01711; AIK-C, AB046218. Fig 10. Clusters of amino acid mutations in the P protein of SSPE viruses. Sequence alignment of t Fig 10. Clusters of amino acid mutations in the P protein of SSPE viruses. Sequence alignment of the region in the P protein surrounding the T140A and S313P mutations (marked in light blue) of the SSPEV Kobe-1 strain. Several strains of MV and SSPE virus of the same genotype (D3, D6, C2, C1, B3 and A) are compared. The red letters indicate mutations specific to SSPE viruses. DM domain of the P protein is marked in yellow. -, equivalent amino acid to that of ICB. Accession numbers in DDBJ/EMBL/GenBank: ICB, AB016162; MVi/Tokyo.JPN/37.99(Y), GQ376026; Kobe-1, AB254456; WA.USA/17.98, DQ227321; 97–45881, DQ227319; MVs/Alberta.CAN/22.14, MF775733; MVs/Zagreb. CRO/08.03, DQ227320; MVs/Zagreb.CRO/47.02, DQ227318; JM, M90469; ZH, AB453187; Mvi/Lyon.FRA/77, HM562908; Mvs/Toulon.FRA/08.07, HM562909; Masusako, LC655230; Nagahata, D63927; Osaka-1/Fr/V, LC655226; Osaka-2/Fr/V, LC655228; Osaka-3/Bs/V, LC655229; Yamagata-1, D10635; Kitaken-1, AB453045; KS, HM439386; MVs/CapeTown.ZAF/16.10/2, KT851526; MVs/CapeTown.ZAF/52.14, KT851534; Edmonston, K01711; AIK-C, AB046218. https://doi.org/10.1371/journal.ppat.1011528.g010 https://doi.org/10.1371/journal.ppat.1011528.g010 accumulated, leading to the onset of SSPE. We are conducting further analyses of mutations in the SSPEV-F protein that are responsible for its fusogenicity. accumulated, leading to the onset of SSPE. We are conducting further analyses of mutations in the SSPEV-F protein that are responsible for its fusogenicity. Discussion SSPE is a very rare late complication of MV infection that occurs in apparently healthy chil- dren, 7 to 10 years after acute measles [4–8]. Although virus cannot be isolated during the PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1011528 July 26, 2023 16 / 28 PLOS PATHOGENS Role of viral RNA polymerase in SSPE development transformation of MV into SSPE virus before the appearance of clinical signs, the development of SSPE may occur as follows. First, MV enters the brain, presumably during the acute exanthematous phase [57]. Next, MV infects neurons that lack receptors for MV. Nectin-elic- ited cytoplasm transfer, which transports transmembrane and cytoplasmic proteins via cell-to- cell contacts established by the nectin adhesive interface, can spread MV infection from epithe- lial cells to primary neurons [58]. Within a neuron, MV undergoes mutations to avoid immune recognition [5,59]. The M gene is highly mutated in nearly all SSPE cases, impairing the formation of viral particles and promoting viral escape from neutralizing antibodies [8]. MV reproduces intracellularly in a non-cytopathic manner to avoid destroying host neurons, thereby establishing persistent infection [7]. The mutations and mechanisms involved in this step are unclear. Then the F protein acquires hyperfusogenicity, which facilitates cell-to-cell fusion and transneuronal viral spread in the absence of MV receptors [47]. Cell adhesion mol- ecules 1 and 2 [60,61], or neurokinin-1 [62], may enable MV to induce neuronal fusion. Muta- tion of the F protein is essential for the advancement from persistent to reproductive infection. When clinical signs of neurological disease occur (e.g., behavioral changes, decreased school performance, and/or seizures), SSPE virus is widely distributed in neurons of the CNS [8,11]. The inflammatory response in the brain to persistent SSPE virus leads to extensive tissue dam- age and cerebral atrophy. Clinically, SSPE is characterized by florid panencephalitis [7,8]. The role of the RdRp activity of the L and P proteins in SSPE development is unclear. Catta- neo et al. [63] reported that transcription decreases at each gene junction in the MV genome in the brains of SSPE patients, presumably reducing the expression of viral envelope proteins on the surface of brain cells. This phenomenon may explain the lack of viral budding and the ability to escape from immune surveillance, thereby enabling MV persistent infection. Nota- bly, they presumed that the phenomena were brain-specific based on several host factors and did not evaluate mutations in the L and/or P proteins [64]. PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1011528 July 26, 2023 Discussion In the present study, we showed that suppression of the RdRp activity by the SSPEV-L and SSPEV-P proteins decreased the expression levels of viral proteins, enabling the establishment of persistent infection in the brain during the incubation period. The virus could then evade host immunity and suppress neuronal destruction by avoiding excessive propagation. The reduction of RdRp activity may be necessary to establish and/or maintain persistent MV infection in the brain. The RdRp activity of the SSPEV-P protein progressively decreased as mutations (e.g., T140A and S313P) accumulated. The T140A and S313P mutations are unique to the SSPEV Kobe-1 strain and are not found in the P protein of other sequenced SSPE viruses. Alignment analysis, however, identified clusters of mutations at around amino acid 110–150 and 270– 320, in which T140A and S313P are included, respectively (Fig 10). Amino acid 313 is located at the N-terminal end of DM domain of the P protein and mutations accumulated especially around this region. It is of interest whether these mutations in the other SSPE viruses than the Kobe-1 strain alter RdRp activity and are involved in the establishment of the persistent MV infection. We did not find any clusters of mutations in the L protein sequence of SSPE viruses. Mutations in the L protein, S519N, I539V and K601R, were specific to the SSPEV Kobe-1 strain. Comparison of the P and L proteins between the MV vaccine strains (genotype A) and the wild-type viruses revealed no marked characteristics in regards to amino acids with which mutation occurred in SSPE viruses. It is currently impossible to determine the order in which mutations accumulated in pro- teins of an SSPE virus. In the L protein of the SSPEV Kobe-1 strain, the K601R, S519N, and I538V mutations were introduced during the very early stage of persistent infection because each mutation decreased RdRp activity to a level below the activity of the SSPEV-L protein. The L(triple) protein had extremely low RdRp activity and enabled the establishment and maintenance of persistent infection; virus possessing the L(triple) protein released few PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1011528 July 26, 2023 17 / 28 PLOS PATHOGENS Role of viral RNA polymerase in SSPE development infectious viral particles and exhibited restricted growth in neuronal cells (Fig 6). Ethics statement Animal experiments were reviewed and approved by the Committee of the Institute for Exper- imental Animals, Kobe University Graduate School of Medicine (permit number 23–67), and all procedures were performed in accordance with relevant guidelines. PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1011528 July 26, 2023 Discussion These changes are followed by reinforcement at the late stage (after the F protein has become hyper- fusogenic), which facilitates the spread of infection by cell-to-cell fusion (Fig 9). The reinforce- ment of RdRp activity was experimentally confirmed by addition of other mutations in the SSPEV-L protein (Fig 7), which corresponded to the enhancement of viral propagation in neu- ronal cells (Fig 8). In a previous study, we isolated a variant of the SSPE virus Kobe-1 strain that replaced the Kobe-1 strain after long-term passage in human neuronal cells because of accelerated cell-to-cell fusion [56]. The enhanced RdRp activity of the variant increased the expression of viral proteins and conferred robust cell-to-cell fusion ability. Variants of SSPE virus with improved growth in the brain may be selected during disease progression, and the isolation of the Kobe-1 variant suggested that RdRp activation could provide selective pressure. Therefore, MV establishes persistent infection by suppressing its RdRp activity, spreads via cell-to-cell fusion after hyperfusogenic mutation of the F protein, and promotes further viral propagation by enhancing RdRp activity. Fig 9 shows a hypothetical model of the transforma- tion of MV into the SSPE Kobe-1 strain. The isolation of virus during the transformation of MV into SSPE virus is impractical before the appearance of clinical signs. It is also difficult to repeatedly isolate a series of viruses from a single patient because virus can be isolated only via biopsy or during autopsy [33,44,65–68]. Therefore, we cannot currently trace the accumulation of mutations in the MV genome during disease progression. Although mutations in the SSPE virus genome contain information regarding its transformation from the parental MV, the large number of such mutations may preclude analysis of their effects. Because the Kobe-1 strain was isolated 6 weeks after disease onset from a patient who had contracted acute measles 5 years prior, and thus the virus bears only 49 amino acid substitutions [65], we could chase the effect of each mutation. The mutation process of the L protein we have proposed here is hypothetical, and analyses of other SSPE viruses will provide additional insights. Plasmid construction The plasmid p(+)MV323, which contained all MV ICB strain genes, was a gift from K. Takeu- chi [71]. The cDNA of the genome of the SSPE virus Kobe-1 strain (GenBank: AB254456) was synthesized by reverse transcription polymerase chain reaction (PCR). The SalI–SpeI fragment [nucleotides (nt) 3365–9175 according to the ICB strain genome sequence (GenBank: AB016162)] [76] of p(+)MV323 was replaced with the corresponding region of the Kobe-1 strain, yielding a plasmid with the full-length genome of the ICB strain carrying the M, F, and H genes of the SSPE virus Kobe-1 strain [p(+)MV323/sMFH]. The SpeI–Eco47III fragment (nt 9176–15767) of p(+)MV323/sMFH was replaced with the corresponding regions of the Kobe- 1 strain, yielding a plasmid with the genome of the ICB strain carrying the M, F, H, and L genes of the SSPE virus Kobe-1 strain [p(+)MV323/sMFHL]. The plasmid p(+)MV323/SSPEV containing all genes of the Kobe-1 strain was constructed by replacing the SmaI–SalI fragment (nt 839–3364) of p(+)MV323/sMFHL with the corresponding region of the Kobe-1 strain. AB016162)] [76] of p(+)MV323 was replaced with the corresponding region of the Kobe-1 strain, yielding a plasmid with the full-length genome of the ICB strain carrying the M, F, and H genes of the SSPE virus Kobe-1 strain [p(+)MV323/sMFH]. The SpeI–Eco47III fragment (nt 9176–15767) of p(+)MV323/sMFH was replaced with the corresponding regions of the Kobe- 1 strain, yielding a plasmid with the genome of the ICB strain carrying the M, F, H, and L genes of the SSPE virus Kobe-1 strain [p(+)MV323/sMFHL]. The plasmid p(+)MV323/SSPEV containing all genes of the Kobe-1 strain was constructed by replacing the SmaI–SalI fragment (nt 839–3364) of p(+)MV323/sMFHL with the corresponding region of the Kobe-1 strain. The plasmid p(+)MV323-EGFP, which contained all MV ICB strain genes and an addi- tional EGFP transcriptional unit, was a gift from Y. Yanagi [77]. The plasmids p(+) MV323-EGFP/SSPEV, p(+)MV323-EGFP/sM, and p(+)MV323-EGFP/sMFH, which con- tained all genes, only the M gene, and only the M, F, and H genes of the SSPE virus Kobe-1 strain, respectively, were described previously [54]. To prepare chimeric N–P genes units, the MV-N (nt 98–1826) and MV-P (nt 1807–3369) gene fragments, or the SSPEV-N (nt 98–1826) and SSPEV-P (nt 1807–3369) gene fragments, were amplified by PCR using p(+) MV323-EGFP or p(+)MV323-EGFP/SSPEV as templates. Cells and viruses Vero cells constitutively expressing human SLAM (Vero/hSLAM; a gift from Y. Yanagi) [25] were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 8% fetal bovine serum. SH-SY5Y human neuroblastoma cells were maintained in Dulbec- co’s modified Eagle’s medium/Ham’s F-12 medium (Wako Pure Chemical, Osaka, Japan) sup- plemented with 8% fetal bovine serum. Baby hamster kidney fibroblast (BHK) cells constitutively expressing T7 RNA polymerase (BHK/T7-9; a gift from N. Ito and M. Sugiyama) [69] were maintained in high-glucose Dulbecco’s modified Eagle’s medium supplemented with 5% fetal bovine serum, 10% tryptose phosphate broth solution (Sigma-Aldrich, St. Louis, MO, USA), and 0.6 mg/mL hygromycin B. B95a cells (marmoset B cells transformed with Epstein–Barr virus) [70] were maintained in high-glucose Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. The isolation of the SSPE virus Kobe-1 strain was described previously (DDBJ/EMBL/Gen- Bank: AB254456.1) [65]. The Kobe-1 strain was derived from the MV of the genotype D3, and the ICB strain [71] (referred to as the 84–01 strain before [70]) is one of the representative PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1011528 July 26, 2023 18 / 28 PLOS PATHOGENS Role of viral RNA polymerase in SSPE development strains of the D3 MV [72]. Recombinant MVs (rMVs) were generated in accordance with the method of Seki et al. [73] by transfecting BHK/T7-9 cells with plasmids containing the full- length MV genome described below, as well as three support plasmids: pCITE-IC-N, pCI- TE-IC-PΔC, and pCITEko-9301B-L (gifts from M. Takeda) [74]. The generated rMVs were propagated in Vero/hSLAM cells, and cell-free infectious rMV particles were collected as described previously for viruses carrying the M gene of SSPE virus. Briefly, the culture medium was replaced with medium containing 1 μM cytochalasin D (Sigma-Aldrich); the cells were incubated overnight at 37˚C, and infected cells were scraped and pipetted vigorously. The resulting suspension was stored at –80˚C until downstream processing. T A l ( T ) f f [ ] T7 RNA polymerase-expressing vaccinia virus (vTF7-3) was a gift from B. Moss [75]. PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1011528 July 26, 2023 Cell-to-cell fusion assay Vero/hSLAM cells cultured in T25 flasks were infected with cell-free rMVs and incubated at 37˚C. At the indicated time points, photographs of representative syncytia were obtained using a fluorescence microscope. The percentage area occupied by syncytia in the images was quantified using ImageJ software (http://imagej.nih.gov/ij/) and expressed as cell-to-cell fusion activity. Plasmid construction Next, the MV-N and SSPEV-P gene fragments, or the SSPEV-N and MV-P gene fragments, were connected and amplified by over- lap extension PCR to obtain chimeric N–P genes units (nt 98–3369). The full-length genome plasmids for generation of various chimeric viruses were constructed by using the BspT104I– SalI fragment (nt 100–3364) or the SpeI–Eco47III fragment (nt 9176–15767) to replace the N– P genes unit or L gene unit of the MV ICB strain and the SSPE virus Kobe-1 strain, respec- tively, on the p(+)MV323-EGFP, p(+)MV323-EGFP/SSPEV, p(+)MV323-EGFP/sM, and p(+) MV323-EGFP/sMFH plasmids. To introduce the triple mutations (S519N, I539V, and K601R) or triple plus additional mutations into the L protein of the rMVs, the SpeI–Eco47III fragment (nt 9176–15767) of the plasmids p(+)MV323-EGFP, p(+)MV323-EGFP/sM, or p(+) MV323-EGFP/sMFH was replaced with the corresponding fragment from pGEM/MV-L(tri- ple) or pGEM/MV-L(triple) with the additional mutations described below. To construct pCA7/MV-N for expression of the N protein of MV, the N gene fragment (nt 108–1685) was amplified by PCR using p(+)MV323-EGFP and cloned into the pCA7 vector. pCA7/MV-P-ΔCV was constructed by cloning the P gene fragment (nt 1807–3330) of the MV ICB strain with nucleotide substitutions causing C and V protein deletion into the pCA7 vec- tor. The SpeI–Eco47III fragment of p(+)MV323-EGFP was cloned into the pBluescript II SK PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1011528 July 26, 2023 19 / 28 PLOS PATHOGENS Role of viral RNA polymerase in SSPE development (-) vector to obtain pBS/MV-L. Next, the EcoRI–EagI fragment of pBS/MV-L was cloned into the pGEM vector to produce pGEM/MV-L for MV-L protein. pCA7/SSPEV-N, pCA7/ SSPEV-P-ΔCV, and pGEM/SSPEV-L expressing the N, P, and L proteins of the SSPE virus Kobe-1 strain, respectively, were described previously [56]. To introduce mutations into the P and L proteins of MV, fragments with nucleotide substitutions causing the corresponding amino acid mutations were amplified by PCR using pCA7/MV-P-ΔCV and pGEM/MV-L as templates. Plasmids harboring the mutations were generated using In-Fusion Snap Assembly Master Mix (Takara Bio, Shiga, Japan) based on pCA7/MV-P-ΔCV and pGEM/MV-L, in accordance with the manufacturer’s protocol. pGEM/MV-L(triple), prepared for expression of the L protein with S519N, I539V, and K601R mutations, was used as the template to introduce further mutations in the same manner. pcDNA3/R-Luc was constructed by cloning the Renilla luciferase gene fragment into the pcDNA3 vector. Primer sequences used to construct these plasmids are available upon request. Virus titration Monolayers of Vero/hSLAM cells in 24-well plates were infected with serially diluted virus samples. After incubation for 1 h at 37˚C, the virus samples were removed, and medium con- taining 100 μg/mL fusion-inhibiting peptide (4092; Peptide Institute, Osaka, Japan) was added to block secondary infections. After 60 h, spots expressing EGFP were counted using a fluores- cence microscope (Axioskop2; Zeiss, Oberkochen, Germany); the number of the fluorescent spots was regarded as plaque-forming unit (PFU) [78]. Virus challenge BALB/c suckling mice, purchased from CLEA Japan, Inc. (Tokyo, Japan), were observed for health condition for a week and used prior to the age of 3 weeks. Mice were anesthetized, then intracerebrally inoculated with 7 × 102 PFU of each recombinant chimeric virus in a 20 μL sus- pension of B95a cells. After inoculation, clinical signs were observed daily, and moribund mice were euthanized. PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1011528 July 26, 2023 Quantitative PCR (qPCR) Monolayers of Vero/hSLAM cells in 24-well plates were infected with rMVs at a multiplicity of infection (MOI) of 0.04. After 2 h of incubation at 37˚C, the virus samples were removed, and medium containing 100 μg/mL of fusion-inhibiting peptide was added. At 48 h post-infection, total RNA was extracted from virus-infected Vero/hSLAM cells using a NucleoSpin RNA kit (Macherey-Nagel); cDNA was prepared by reverse transcription using ReverTra Ace (Toyobo) in accordance with the manufacturer’s protocol, using specific primers for mRNA, (-) genomic RNA, and (+) genomic RNA. qPCR was performed using FastStart Essential DNA Green Mas- ter Mix and LightCycler 96 (Roche Diagnostics, Basel, Switzerland) at 95˚C for 600 s, followed by 45 cycles of 95˚C for 10 s, 55˚C for 20 s, and 72˚C for 20 s. Melting curve analysis was per- formed after amplification. Relative expression levels of viral RNAs were calculated by the PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1011528 July 26, 2023 20 / 28 PLOS PATHOGENS Role of viral RNA polymerase in SSPE development ΔΔCt method, using β-actin mRNA as the internal control. Primer sets for the target genes were described previously [56]. Mini-genome assay Subconfluent monolayers of Vero/hSLAM cells in 24-well plates were transfected with 80 ng of the N protein-expressing pCA7/MV-N or pCA7/SSPEV-N; 120 ng of the P protein-express- ing pCA7/MV-P-ΔCV, pCA7/SSPEV-P-ΔCV, or pCA7/MV-P-ΔCV with a mutation; 300 ng of the L protein-expressing pGEM/MV-L, pGEM/SSPEV-L, or pGEM/MV-L with mutations; and 350 ng of the MV mini-genome plasmid p18MGFLuc01 encoding the firefly luciferase gene (a gift from K. Komase) [81], along with 10 pg of the Renilla luciferase-expressing pcDNA3/R-Luc, then incubated at 37˚C. At 24 h post-transfection, luciferase activity was mea- sured using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) and Centro XS3 LB960 (Berthold Technologies, Bad Wildbad, Germany) in accordance with the manufacturers’ protocols. Relative RdRp activity was calculated through division of firefly luciferase activity by Renilla luciferase activity. In Fig 4A, the value in the absence of N, P, and L proteins was shown as the control. In other Figs, the results were demonstrated with the value after subtracting the value of control (in the absence of N, P, and L proteins). Cell-surface biotinylation and Western blot analysis Vero/hSLAM cells were infected with rMVs as described above for qPCR. At 48 h post-infec- tion, cells were incubated with 0.5 mg EZ-Link Sulfo-NHS-SS-Biotin (Thermo Fisher Scien- tific, Waltham, MA, USA) at room temperature for 30 min, then lysed in 1 mL of lysis buffer (5 mM sodium chloride, 0.5% Triton X-100, 0.5% sodium deoxycholate, and 10 mM Tris- hydrochloric acid, pH 7.5) at 4˚C for 1 h. Next, lysates were centrifuged at 13,000 × g for 10 min at 4˚C, and supernatants were collected. A small amount (24 μL) of each cell extract was mixed with sodium dodecyl sulfate (SDS) loading buffer. To detect the M protein, cell extracts were concentrated by chloroform/methanol precipitation using the method of Saito et al. [79]. Briefly, 200 μL of cell extract were mixed with 200 μL of methanol and 50 μL of chloroform, then centrifuged at 13,000 × g for 5 min at 4˚C. The protein pellet was washed with methanol, then mixed with SDS loading buffer. For collection of cell-surface samples, 800 μL of each cell extract were mixed with Streptavidin Sepharose High Performance (GE Healthcare, Chicago, IL, USA) and incubated at 4˚C for 2 h. The adsorbed biotinylated protein was used as the cell- surface sample. Samples were separated by SDS-polyacrylamide gel electrophoresis in 12% polyacrylamide gels, then electroblotted onto polyvinylidene difluoride membranes. Proteins were detected by incubating the membranes with a rabbit polyclonal antibody against MV-F protein [80], mouse monoclonal antibody against MV-M protein (MAB8910; Merck Millipore, Burlington, MA, USA), or mouse monoclonal antibody against β-actin (#3700; Cell Signaling Technology, Danvers, MA, USA). Membranes were then incubated with a horseradish peroxidase-conju- gated goat anti-rabbit IgG (#7074; Cell Signaling Technology) or goat anti-mouse IgG (sc- 2005; Santa Cruz Biotechnology, Dallas, TX, USA) secondary antibody. Proteins were visual- ized using ImmunoStar Zeta (Fujifilm Wako Pure Chemical Corp., Tokyo, Japan) and C-DiGit Chemiluminescent Western Blot Scanner (LI-COR, Lincoln, NE, USA). PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1011528 July 26, 2023 Statistical analysis Comparisons between two groups were conducted using unpaired two-tailed Student’s t-tests. P-values < 0.05 were considered indicative of statistical significance. We performed the log-rank tests using the survival package in the R Software to analyze dif- ferences between survival curves. If a p-value was less than 0.05, the difference between the survival curves was considered statistically significant. Infectious virus particle assay Monolayers of Vero/hSLAM cells in 24-well plates were infected with cell-free rMVs at a MOI of 0.04. After 2 h of incubation at 37˚C, virus samples were removed; the cells were washed with phosphate-buffered saline and incubated at 37˚C. The culture medium was collected and PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1011528 July 26, 2023 21 / 28 PLOS PATHOGENS Role of viral RNA polymerase in SSPE development replaced with fresh medium every 24 h. After the medium had been centrifuged at 2,000 × g for 5 min at 4˚C, the numbers of infectious cell-free viruses in supernatant were measured as described above for virus titration. PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1011528 July 26, 2023 Supporting information S1 Fig. The SSPEV-L gene and the SSPEV-N and/or SSPEV-P genes suppressed cell-to-cell fusion and propagation of rMVs in neuronal cells. (A) Enlargement of syncytia formed by rMV-infected Vero/hSLAM cells. Vero/hSLAM cells were infected with the EGFP-expressing cell-free rMVs in Fig 2A and a syncytium derived from a single infected cell was observed at 12-h intervals under a fluorescence microscope. A representative photograph at each time point is presented. Magnification, ×200. mock, uninfected cells. n.a., not applicable. (B) Spread of rMV infection in human neuronal cells. SH-SY5Y cells were infected with the EGFP- expressing cell-free rMVs in Fig 2A and the spread of infection from a single infected cell was observed at 12-h intervals under a fluorescence microscope. A representative photograph at each time point is presented. Magnification, ×200. mock, uninfected cells. n.a., not applicable. (TIFF) S2 Fig. Suppression of RdRp activity is associated with the reduction of viral cell-to-cell S2 Fig. Suppression of RdRp activity is associated with the reduction of viral cell-to-cell fusion and growth in neuronal cells. (A) Schematic of the genomes of the EGFP-expressing rMV/sMFH variants after exchanges of N, P, and L genes between the MV ICB strain and the SSPE virus Kobe-1 strain. (B) Viral cell-to-cell fusion. Vero/hSLAM cells were infected with the rMVs in (A). After incubation at 37˚C for 36 h, a syncytium derived from a single infected cell was observed and photographed under a fluorescence microscope. Magnification, ×200. Cell-to-cell fusion was quantified as in Fig 2B. Data from five images are shown as means ± standard deviations. (C) Viral propagation in human neuronal cells. SH-SY5Y cells were infected with the rMVs in (A). After incubation at 37˚C for 72 h, the spread of infection from a single infected cell was observed and photographed under a fluorescence microscope. Magnification, ×200. Spread of viral infection was determined as in Fig 2C. Data from five images are shown as means ± standard deviations. (TIFF) S3 Fig. The L(triple) protein restricted the release of infectious virus particles. Vero/ hSLAM cells were infected with rMVs bearing the MV-M protein (A) or the SSPEV-M protein (B) in Fig 6A, then incubated at 37˚C. Culture medium was collected every 24 h until 4 days post-infection. Cell-free viruses in the supernatant after centrifugation were titrated in Vero/ hSLAM cells. Data from three independent experiments are shown as means ± standard devia- tions. S4 Fig. Restoration of RdRp activity improved viral RNA production and expression of F protein on the cell surface. (A) Viral RNA levels in cells infected with rMVs harboring the L (triple) gene and its variants. Vero/hSLAM cells were infected with the rMVs in Fig 8A, and viral mRNA and genomic RNA were quantified as in Fig 3A. Data from three independent 22 / 28 PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1011528 July 26, 2023 PLOS PATHOGENS Role of viral RNA polymerase in SSPE development experiments are shown as means ± standard deviations. (-), negative-sense genome; (+), posi- tive-sense genome. (B) Surface F protein expression of cells infected with rMVs. Vero/hSLAM cells were infected with the rMVs in Fig 8A, cell-surface proteins were biotinylated to detect F protein, and viral proteins were quantified as in Fig 3B. A representative image of several experiments is shown (left panel). Acknowledgments We would like to thank Kaoru Takeuchi (Tsukuba University, Japan) for providing p(+) MV323, Yusuke Yanagi (Kyushu University, Japan) for providing Vero/hSLAM cells, p(+) MV323-EGFP and pCA7, Makoto Takeda (National Institute of Infectious Diseases, Japan) for providing pCITE-IC-N, pCITE-IC-PΔC and pCITEko9301B-L, Naoto Ito and Makoto Sugiyama (Gifu University, Japan) for providing BHK/T7-9 cells, Katsuhiro Komase (National Institute of Infectious Diseases, Japan) for providing p18MGFLuc01, and Bernard Moss (National Institute of Allergy and Infectious Diseases, National Institutes of Health, USA) for providing vTF7-3. We thank Jun-ichi Miyazaki (Osaka University, Japan) for the permission to use the CAG promoter of pCA7. We also thank Masafumi Shionyu (Nagahama Institute of Bio-Science and Technology, Japan) for the assist in performing statistical analyses. Author Contributions Conceptualization: Hak Hotta, Masae Itoh. Data curation: Yoshinori Kitagawa, Bin Gotoh. Formal analysis: Kento Sakamoto, Masae Itoh. Funding acquisition: Yuto Satoh, Masae Itoh. Investigation: Kento Sakamoto, Miho Konami, Shinra Kameda, Hiroshi Wakimoto, Da-Peng Jiang. Methodology: Kento Sakamoto, Yuto Satoh, Hiroshi Wakimoto. Project administration: Kento Sakamoto, Yuto Satoh. Resources: Masae Itoh. S2 Fig. Suppression of RdRp activity is associated with the reduction of viral cell-to-cell Inactive F0 and active F1 forms of the F protein are shown; molecular markers are indicated on right. CS, cell-surface fraction; WC, whole-cell fraction. Intensities of F1, N, and M protein bands were quantified using ImageJ (right panel). Fs, F1 protein in CS; F, F1 protein in WC. (TIFF) S5 Fig. Cell-to-cell fusion and viral propagation in neuronal cells were improved by resto- ration of RdRp activity. (A) Representative photographs of viral cell-to-cell fusion. Vero/ hSLAM cells were infected with the rMVs in Fig 8A and enlargement of fused cells was moni- tored under a fluorescence microscope; photographs were obtained at 12-h intervals. Magnifi- cation, ×200. n.a., not applicable. (B) Representative photographs of viral propagation in neuronal cells. SH-SY5Y cells were infected with the rMVs in Fig 8A and the spread of infec- tion from a single infected cell was observed under a fluorescence microscope; photographs were obtained at 12-h intervals. Magnification, ×200. n.a., not applicable. (TIFF) PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1011528 July 26, 2023 References 1. Patel MK, Goodson JL, Alexander JP, Kretsinger K, Samir Sodha V, et al. 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https://openalex.org/W2907524811	https://discovery.ucl.ac.uk/10071537/8/Rees_Influences%20on%20the%20Implementation%20of%20Mobile%20Learning%20for%20Medical%20and%20Nursing%20Education_VoR.pdf	English	null	Influences on the Implementation of Mobile Learning for Medical and Nursing Education: Qualitative Systematic Review by the Digital Health Education Collaboration	JMIR. Journal of medical internet research/Journal of medical internet research	2,019	cc-by	15,420	Corresponding Author: Corresponding Author: Rebecca Rees, PhD Evidence for Policy and Practice Information and Co-ordinating Centre, Social Science Research Unit, Department of Social Science University College London Institute of Education University College London 18 Woburn Square London, WCIH 0NR United Kingdom Phone: 44 07932 243030 Email: rebecca.rees@ucl.ac.uk Rebecca Rees, PhD Evidence for Policy and Practice Information and Co-ordinating Centre, Social Science Research Unit, Department of Social Science U i i C ll L d I i f Ed i University College London Institute of Education Abstract Background: In the past 5 decades, digital education has increasingly been used in health professional education. Mobile learning (mLearning), an emerging form of educational technology using mobile devices, has been used to supplement learning outcomes through enabling conversations, sharing information and knowledge with other learners, and aiding support from peers and instructors regardless of geographic distance. Objective: This review aimed to synthesize findings from qualitative or mixed-methods studies to provide insight into factors facilitating or hindering implementation of mLearning strategies for medical and nursing education. Methods: A systematic search was conducted across a range of databases. Studies with the following criteria were selected: examined mLearning in medical and nursing education, employed a mixed-methods or qualitative approach, and published in English after 1994. Findings were synthesized using a framework approach. Results: A total of 1946 citations were screened, resulting in 47 studies being selected for inclusion. Most studies evaluated pilot mLearning interventions. The synthesis identified views on valued aspects of mobile devices in terms of efficiency and personalization but concerns over vigilance and poor device functionality; emphasis on the social aspects of technology, especially in a clinical setting; the value of interaction learning for clinical practice; mLearning as a process, including learning how to use a device; and the importance of institutional infrastructure and policies. Conclusions: The portability of mobile devices can enable interactions between learners and educational material, fellow learners, and educators in the health professions. However, devices need to be incorporated institutionally, and learners and educators need additional support to fully comprehend device or app functions. The strategic support of mLearning is likely to require procedural guidance for practice settings and device training and maintenance services on campus. JOURNAL OF MEDICAL INTERNET RESEARCH JOURNAL OF MEDICAL INTERNET RESEARCH Lall et al Original Paper (J Med Internet Res 2019;21(2):e12895) doi:10.2196/12895 (J Med Internet Res 2019;21(2):e12895) doi:10.2196/12895 Influences on the Implementation of Mobile Learning for Medical and Nursing Education: Qualitative Systematic Review by the Digital Health Education Collaboration Priya Lall1, PhD; Rebecca Rees2, PhD; Gloria Chun Yi Law3, PhD; Gerard Dunleavy3, MSc; Živa Cotič4, MSc; Josip Car3,5, MSc, MD, PhD, DIC, FFPH, FRCP https://www.jmir.org/2019/2/e12895/ Priya Lall1, PhD; Rebecca Rees2, PhD; Gloria Chun Yi Law3, PhD; Gerard Dunleavy3, MSc; Živa Cotič4, MSc; Josip Car3,5, MSc, MD, PhD, DIC, FFPH, FRCP 1School of Geography, Queen Mary University of London, London, United Kingdom 2Evidence for Policy and Practice Information and Co-ordinating Centre, Social Science Research Unit, Department of Social Science, University College London Institute of Education, University College London, London, United Kingdom 3Centre of Population Health Sciences, Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Singapore 4Faculty of Social Sciences, University of Ljubljana, Ljubljana, Slovenia 5Faculty of Medicine, School of Public Health, Imperial College London, London, United Kingdom J Med Internet Res 2019 \| vol. 21 \| iss. 2 \| e12895 \| p.1 (page number not for citation purposes) Background In the past 5 decades, digital education has increasingly been used in health professional education, and technological advances have produced various forms of digital education modalities such as computer-based simulations, virtual patients, and internet-based courses and interactive contents [1,2]. Adoption of these digital education modalities in health professional education is rapidly expanding before the establishment of a robust evidence base for consideration of multiple dimensions and outcomes [3,4]. A noteworthy trend within digital education is mobile learning (mLearning), which can be defined as follows [5]: Our review considered the broad issue of implementation of mLearning. This is important because mLearning is a relatively new area of development compared with other forms of digital education. We are in the early stages of learning what happens and what might be helpful when mLearning is introduced into real-world settings. Having a systematic and in-depth exploration of the range of potential barriers to and facilitatorsof mLearning in health professional education should deepen the understanding of the topic and allow insights to be obtained for effective implementation and positive outcomes. It is also important to understand mLearning in terms of the underlying assumptions about teaching and learning (ie, pedagogy and andragogy) of different approaches, to maximize the potential richness of the learning process in mobile environments and enable teachers to plan for optimal learning [16]. ...consuming, interacting with or creating information, mediated through a compact digital portable device that the individual carries on a regular basis, has reliable connectivity, and fits in a pocket or purse. This is enabled by a growth of capabilities in mobile devices (eg, smartphones) and the convenience they offer, such as omnipresent usability and accessibility to the internet, while mobile. Approximately 1.1 billion people living in rural areas [6] and 73% of the total world population [7] are now covered by mobile broadband. Koole’s Framework for the Rational Analysis of Mobile Education (FRAME) model guides the qualitative synthesis for this study (see Figure 1) [17]. This model considers how features of mobile technology, along with learner capacities and social interaction, influence learning processes occurring in an information context. Within the FRAME model, mLearning is conceived as the convergence of the following aspects: (1) device, signifying functional characteristics of a mobile device, for example, processor speed; (2) learner, accounting for individuals’ cognitive abilities and learning styles; and (3) social, referring to elements of social interaction and culture-influencing learning processes. KEYWORDS medical education; nursing education; distance education; qualitative research; systematic review https://www.jmir.org/2019/2/e12895/ XSL•FO RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Lall et al Lall et al Lall et al characteristics of the educational intervention, problems addressed by the intervention, features of the health system, the adopting system, and other contextual factors [15]. However, no review has been identified which examines systematically the factors influencing the use of mLearning interventions for health professional education. J Med Internet Res 2019 \| vol. 21 \| iss. 2 \| e12895 \| p.2 (page number not for citation purposes) Protocol A protocol was developed so as to establish the review’s scope and methods before evaluating existing literature. This was registered with PROSPERO, the international prospective register of systematic reviews (record number CRD42016035411 Multimedia Appendix 1) [20]. Objectives This study aimed to synthesize insights from empirical research using qualitative and mixed methods on mLearning implementation in medical and nursing education. Our study employed systematic methods to identify, appraise, and synthesize qualitative findings from studies to explore mLearning strategies for medical and nursing education. These studies can allow us to better understand the nature of material and sociocultural influences (eg, cultural norms) and causal pathways [18] to delineate a more complete picture of the phenomenon under study [19]. Qualitative findings from existing studies are used to uncover the perspectives of learners and other key actors with experience of mLearning strategies. Particular attention is paid to perceptions of implementation processes. The broad research question for this review was as follows: What are the views of educators, learners, and other key actors with experience of mLearning in medical and nursing education about perceived factors which facilitate/enhance or hinder its implementation? Background Framework for the Rational Analysis of Mobile Education (FRAME) model. involve some form of qualitative data collection or analysis (eg, focus group interviews), to collect data from learners in medical or nursing education who were involved in mLearning as defined by Wexler et al [5], and to be published after 1994. Identifying Relevant Studies We conducted a comprehensive search that combined terms for the concepts of digital technology, education, and health professionals. This search was conducted in February 2015 and was repeated in March 2017 on 8 electronic databases (MEDLINE, EMBASE, Cochrane Library, PsycINFO, ERIC, CINAHL, Web of Science, and International Clinical Trials Platform). Databases were searched from and including the year 1995 to March 2017 (see Multimedia Appendix 2). All references identified were uploaded to the specialist systematic review software EPPI-Reviewer 4 (University College London) [21], and data deduplication was performed within this program. A second phase of searches was then run to identify qualitative studies of mLearning using the EPPI-Reviewer search function. These searches looked for items that had terms related to qualitative research and to mLearning (see Multimedia Appendix 2). The resulting set of references was assessed against our predefined inclusion and exclusion criteria. The criteria were developed by all authors and piloted by 4 authors (GD, GL, PL, and ZC) on a randomly selected sample of studies. The pilot was completed after there was a high level of agreement (over 90%) on the selection of studies between all 4 authors. Abstracts and full texts were each independently screened by 2 of these same 4 authors. In cases where there were difficulties reaching consensus on inclusion of a particular text, a third provided the deciding judgment. Figure 2 shows the Preferred Reporting Items for Systematic Reviews and Meta-Analyses flowchart for the study. https://www.jmir.org/2019/2/e12895/ J Med Internet Res 2019 \| vol. 21 \| iss. 2 \| e12895 \| p.3 (page number not for citation purposes) Background Mobile devices can offer a variety of functions and be used across contexts. For instance, mLearning can provide access to educational content and information in daily clinical practice [8-10]; enable conversations and the sharing of information and knowledge with other learners; and elicit support from peers and instructors regardless of geographic distance [8-10]. Handheld computers can be used to keep track of students’ skill development and progress in assignments [11]; promote self-directed and self-regulated learning [12,13]; display audio-visual information relating to a specific place, scene, or situation; and aid situated learning [10]. In terms of interactions between these aspects, first, Device Usability is thought of as containing aspects belonging to the device and learner and describes how an individual relates to the device. For example, learners can express satisfaction with a particular device because of its esthetic qualities. Second, Social Technology covers the intersection between device and social aspects and accounts for how mobile devices enable connection between multiple interfacing individuals and systems, such as the use of collaborative tools. Finally, Interaction Learning spans the intersection between learner and social aspects, describing how the learner interacts with other individuals. For instance, a mobile device could enable interaction between a learner and their instructor on long-distance educational courses. The culmination of all 3 aspects is envisioned as the eventual process of mLearning. Evaluations of the effects of digital education and specifically mLearning as a whole raise more questions than they answer. For example, a meta-analysis by Free et al [14] included 7 randomized controlled trials and investigated the educational outcomes associated with the use of personal digital assistants (PDAs) and portable media players in medical and nursing education. The studies incorporated into the systematic review examined the effectiveness of mLearning in improving knowledge and attitudes; however, the meta-analysis showed no clear evidence of benefit. There are many factors influencing the effectiveness of digital education and mLearning interventions that warrant closer investigation. The implementation of digital education can be influenced by J Med Internet Res 2019 \| vol. 21 \| iss. 2 \| e12895 \| p.2 (page number not for citation purposes) https://www.jmir.org/2019/2/e12895/ XSL•FO RenderX XSL•FO RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Lall et al Lall et al Figure 1. Framework for the Rational Analysis of Mobile Education (FRAME) model. Figure 1. Framework for the Rational Analysis of Mobile Education (FRAME) model. Figure 1. Describing Studies and Appraising Their Quality Describing Studies and Appraising Their Quality Features of the included studies were described according to the following characteristics: (1) aim; (2) sample characteristics (ie, size, country, and study population); (3) type of mLearning device and apps used; (4) type of study (ie, study of an intervention or inquiry into an existing phenomenon); (5) type of mLearning (eg, reference repository); and (6) study design, such as the type of data collection and sampling approach. Inclusion Criteria Studies were included if they examined medical and/or nursing students’ (or their educators’) perspectives on or experiences of mLearning. They also needed to be written in English, to J Med Internet Res 2019 \| vol. 21 \| iss. 2 \| e12895 \| p.3 (page number not for citation purposes) https://www.jmir.org/2019/2/e12895/ XSL•FO RenderX XSL•FO RenderX Lall et al Lall et al JOURNAL OF MEDICAL INTERNET RESEARCH Figure 2. Preferred Reporting Items for Systematic Reviews and Meta-Analyses chart. igure 2. Preferred Reporting Items for Systematic Reviews and Meta-Analyses chart. study (high, medium, or low for each dimension) and then compared judgments before coming to a consensus. Studies that met the inclusion criteria were included in the review regardless of the study quality, with ratings described alongside other characteristics of the papers. https://www.jmir.org/2019/2/e12895/ J Med Internet Res 2019 \| vol. 21 \| iss. 2 \| e12895 \| p.4 (page number not for citation purposes) Analysis and Synthesis Themes were identified using a framework analysis approach in which data are reduced through the development of a matrix, comparing categories of data or cases, and a synthesis is developed using an initial theoretical framework. Data were analyzed according to phases of analysis identified by Pope et al [24], starting with 3 authors (PL, GL, and GD) familiarizing themselves with the selected studies. These authors coded selected studies according to the FRAME model. The information gathered from coded text was distilled into a chart containing summaries of the themes. After the search update, 3 authors (PL, GL, and GD) undertook the same process, further Quality of the final set of studies was assessed using a tool used in previous studies [22,23], where the quality of each study is rated using a total of 7 questions set within 2 dimensions—trustworthiness and usefulness of the findings. The first dimension captures the degree to which the methods were used to ensure rigor; the second, for the purposes of this review, focused on the complexity of analysis of the mLearning strategy. Furthermore, 2 reviewers independently rated each J Med Internet Res 2019 \| vol. 21 \| iss. 2 \| e12895 \| p.4 (page number not for citation purposes) XSL•FO RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Lall et al [27,39,40]. The remaining evaluations examined a variety of specific mLearning strategies, including the use of multimodal techniques (eg, those using videos of clinical skills, whiteboards, and presentation software) for group or individual activities (n=5) [29,42,55,59,64], augmented reality (n=2) [38,43], messaging services (n=4) [26,44,46,66], a social media–enabled discussion group (n=1) [63], and a mobile app to prompt specific clinical behaviors [58]. modifying the themes and subthemes. When findings were found in texts which addressed an area not covered in the initial framework, the framework themes were added to or modified accordingly. Two authors (PL and GL) wrote a narrative to describe and illustrate the themes and their relationships. In addition, 1 author (RR) then familiarized herself with all the studies and worked further with the lead author on the synthesis narrative and themes, using study texts to check and further incorporate references to individual studies. Of the remaining studies, a further 4 explored the co-design of, or needs for, specific future mLearning interventions [45,50,51,67]. A final set of 11 studies were not conducted with the purpose of designing or evaluating a specific intervention. Analysis and Synthesis Instead, these studies explored students’ experiences of using mobile devices to enable their own learning in the absence of an institutionally planned mLearning initiative [2,30,34-36,48,52-54,56,57]. Research Questions Being Addressed and Quality of Studies The 47 studies in this synthesis [2,10,12,25-68] varied according to study context and participant types, the research questions being addressed, types of mLearning strategies used, and aspects of study design (see Multimedia Appendix 3). A total of 37 studies were conducted in high-income countries [2,10,12,25,27-42,45-50,52,56-58,60-62,64-66], for example, the United Kingdom (n=9) [10,12,29,31,34,40,41,61,66], with the remainder set in lower-income settings [26,43,44,51, 53-55,59,63,67,68], including India [44,55,59], South Africa [53,63], Botswana [26,68], and Rwanda [67]. Studies predominantly employed a mixed-methods research design (n=33). These studies used one or a mix of qualitative data collection methods, such as focus group discussions (n=13) [12,26,27,33,37-40,55,59,60,65,68], group or individual interviews (n=15) [25,34-36,41-44,49,51,52,58,64,66,67], and analysis of textual survey responses (n=9) [12,27,29,32, 34,36,40,46,67]. A smaller number of studies used only qualitative methods (n=14) [28,30,31,45,47,48,50,53,54, 56,57,61-63], which included focus group discussions (n=6) [28,47,48,50,54,63], group or individual interviews (n=7) [30,45,53, 56,57,61,63], textual reflection or journals (n=4) [31,47,61,63], and participant observation [57]. Studies mainly sought views of learners, but some also included educators (n=11) [26,30,39,45-48,53,61,64,65] or focused solely on educators (n=4) [30,47,48,61]. Most studies focused solely on the experience of medical staff or students (n=24) [10,12,25,27,28,30,31,33,36,40,42-45,49,51,54-56,58,60,62,67,68] whereas a smaller number of studies sampled either solely from nursing staff and students (n=19) [2,29,32,35,37,38,46-48,50, 52,53,57,59,61,63-66] or from a mixture of both doctors and nurses (n=4) [26,34,39,41]. Students were at different stages of education and so were learning in different settings. A small number of studies looked at device use aimed at supporting learning in undergraduate lecture, seminar, or laboratory environments (n=5) [29,38,43,61,63]. In all, 7 studies sought views on mLearning for the further professional and/or academic development of fully qualified doctors or nurses [31,35,52,55,57,67]. Most studies, however, sought the views of nursing and medical students, or educators, about mLearning during various clinical placements before health professional registration. Multimedia Appendix 4 displays the quality appraisal of studies included in this synthesis in terms of quality. Ten studies were judged to have highly reliable findings [12,28,47,51-54, 58,59,62] whereas 12 were deemed to be highly useful for this review [12,29,31,39,49,53,54,56,57,62,63,68]. Only 4 studies were considered both highly reliable and useful [12,53,54,62]. The ratings for each study are listed in Multimedia Appendix 3 and Multimedia Appendix 4. https://www.jmir.org/2019/2/e12895/ J Med Internet Res 2019 \| vol. 21 \| iss. 2 \| e12895 \| p.5 (page number not for citation purposes) Social technology 6] Negotiating the social aspect of mLearninga “As the patient was an elderly gentleman I was slightly apprehensive that he wouldn’t appreciate me using a phone during the consultation however with explanation of my actions he was perfectly content with my use of [the device].” [31, p. 6] “When you are dealing with a patient it is easy to access that list and decide on the right medication to- gether. It is also handy when you have a laboratory result and you want to find out what you can do in terms of additional laboratory research.” [25, p. 332] Quotes (from learners unless otherwise specified) “Much, much quicker than flicking through the paper version. . . Looking things up in the paper BNF [British National Formulary] for the n-th time on ward rounds puts time pressure on the junior doctor causing stress and increasing risk of errors.” [10, p. 8] “You could do that [feedback] in a few minutes on your phone, rather than doing it or on a piece of paper that you lose.” [40, p. 928] “Carrying books is a drag, now I’m a ‘lightweight’.” [28, p. 614] “Carrying books is a drag, now I’m a ‘lightweight’.” [28, p. 614] “The places I feel uncomfortable using [the mobile device] are outside, like in the mall or in a kombi [public transportation], because it’s sort of a big thing, and I think it could attract thieves.” [68, p. 75] “I preferred working on the e-portfolio and entering data via computer as the screen was too small on the PDA to be practical and efﬁcient.” [39, p. 652] “The use of the device got me thinking what I actually needed and the sheer fact that a laptop is too large and cumbersome to carry around with you. I wanted something that I could boot up quite instantly and get on the Wi-Fi; go transfer ﬁles and this is ideal.” [62, p. 574] “I think [a tablet] would be better than a [smartphone] because if it was an [tablet] you could actually have lectures on there and it would be big enough to read and work on.” [40, p. 928] Ownership, personalization, and sense of self “I can access it [the mobile device] anytime ... and it is mine to use ...” [28, p. 613] “I’ve sometimes forgotten my handheld and had the feeling of being naked in a way.” [28, p. 616] “It is part of my life now […] a means of contact, a means of learning. You know, people who have phones just learn a lot.” [53, p. 1401] “I find I am having more and more problems with exams because I cannot look up easily what I normally look up... everyday on my [smartphone].” [33, p. 134] Substantive Findings Looking things up in the paper BNF [British National Formulary] for the n-th time on ward rounds puts time pressure on the junior doctor causing stress and increasing risk of errors.” [10, p. 8] Portability means efficiency but also vigilance “You could do that [feedback] in a few minutes on your phone, rather than doing it or on a piece of paper that you lose.” [40, p. 928] “Carrying books is a drag, now I’m a ‘lightweight’.” [28, p. 614] “The places I feel uncomfortable using [the mobile device] are outside, like in the mall or in a kombi [public transportation], because it’s sort of a big thing, and I think it could attract thieves.” [68, p. 75] “I preferred working on the e-portfolio and entering data via computer as the screen was too small on the PDA to be practical and efﬁcient.” [39, p. 652] Fit for purpose hardware, software, and data “The use of the device got me thinking what I actually needed and the sheer fact that a laptop is too large and cumbersome to carry around with you. I wanted something that I could boot up quite instantly and get on the Wi-Fi; go transfer ﬁles and this is ideal.” [62, p. 574] “I think [a tablet] would be better than a [smartphone] because if it was an [tablet] you could actually have lectures on there and it would be big enough to read and work on.” [40, p. 928] “I can access it [the mobile device] anytime ... and it is mine to use ...” [28, p. 613] Ownership, personalization, and sense of self “I’ve sometimes forgotten my handheld and had the feeling of being naked in a way.” [28, p. 616] “It is part of my life now […] a means of contact, a means of learning. You know, people who have phones just learn a lot.” [53, p. 1401] “I find I am having more and more problems with exams because I cannot look up easily what I normally look up... everyday on my [smartphone].” [33, p. 134] Table 1. Illustrative quotes according to theme. Quotes (from learners unless otherwise specified) Fit for purpose hardware, software, and data Devices can impact care and learning relationships Social technology “Well, it’s not that I don’t use a [PDA], I use it for looking up drugs and things, but I think in a conver- sation it is kind of awkward to kind of pull it out and break eye contact.” [58, p. 5] “Well, it’s not that I don’t use a [PDA], I use it for looking up drugs and things, but I think in a conver- sation it is kind of awkward to kind of pull it out and break eye contact.” [58, p. 5] Devices can impact care and learning relationships “Because [the doctors] think that I’m not concentrating with them while using technology, whether it’s [a smartphone or tablet]… I’m writing notes or something, but … in the beginning they didn’t like the fact that I’m using this.” [57, p. 5] “Because [the doctors] think that I’m not concentrating with them while using technology, whether it’s [a smartphone or tablet]… I’m writing notes or something, but … in the beginning they didn’t like the fact that I’m using this.” [57, p. 5] Devices raise issues of professionalism and practice boundaries “These days with the younger generation, if you pull out your [tablet or PDA] and you come up with the information, you are seen as competent. You are seen as having the advanced knowledge. If you say 'well just a minute, I have to go find my book' and you are flipping through the book then you are seen as old fashioned and that you aren't as current as you should be.” [35, p. 12] “You know someone will say ‘Hey put your phone down’ or ‘Check your message later’ or something and you can’t say ‘Oh I’m actually looking…’ it just looks unprofessional so to be honest I don’t use it when I’m in front of a patient or with the doctors…When we…on an actual round I am very careful not to pull my phone out because it’s still a phone you know so I think the stigma is that you’re then distracted because it’s a phone and it could be…you know if the doctor is talking.” [56, p. 5] “I think some doctors have made comments about ‘What are you doing on that, are you texting someone, or playing games’.” [12, p. 6] think some doctors have made comments about ‘What are you doing on that, are you texting someone, playing games’.” [12, p. https://www.jmir.org/2019/2/e12895/ Devices raise issues of professionalism and practice boundaries Substantive Findings The narrative below presents an overview of study participants’ views of mLearning organized under the spaces in which the device, learner, and their social setting interact (device usability, social technology, interaction learning, mLearning processes and implementation in clinical contexts). Table 1 provides illustrative quotations. The full synthesis narrative, which includes citations to the studies that support each theme, is available as Multimedia Appendix 5. The purpose of the majority of studies was to evaluate pilot mLearning approaches (n=32) [10,12,25-29,31-33,37-44, 46,47,49,55,58-66,68] that were implemented in medical and nursing contexts. Furthermore, 2 of these evaluations examined the provision of mobile hardware without describing specific software arrangements [33,60]. In a further 9 evaluations, mobile devices had been designed primarily to be reference repositories [10,12,28,31, 32,37,41,49,68], for example, students were loaned a PDA with preloaded medical texts by their institutions [28]. In a further 8 evaluations, devices were aimed at supporting learning through use of a suite of recommended apps or software [25,27,39,40,47,61,62,65]. In 3 of these 8, the studies focused in particular on students’ use of electronic logs or ePortfolios to reflect on and/or evaluate their experiences or learning Analysis revealed that the progress of mLearning strategies in medical and nursing education often appeared to be shaped by processes that were out of the hands of learners and their teaching staff. Instead, issues raised sometimes related to other actors in the institutional contexts in which learning was taking place and the implementation of policies within these learning settings. An additional factor shaping the operation of mLearning strategies was social norms governing the use of mobile devices in clinical and classroom settings. J Med Internet Res 2019 \| vol. 21 \| iss. 2 \| e12895 \| p.5 (page number not for citation purposes) J Med Internet Res 2019 \| vol. 21 \| iss. 2 \| e12895 \| p.5 (page number not for citation purposes) XSL•FO RenderX XSL•FO RenderX Lall et al Lall et al JOURNAL OF MEDICAL INTERNET RESEARCH Table 1. Illustrative quotes according to theme. Quotes (from learners unless otherwise specified) FRAME model themes Device usability “Much, much quicker than flicking through the paper version. . . Looking things up in the paper BNF [British National Formulary] for the n-th time on ward rounds puts time pressure on the junior doctor causing stress and increasing risk of errors.” [10, p. Substantive Findings 8] Portability means efficiency but also vigilance “You could do that [feedback] in a few minutes on your phone, rather than doing it or on a piece of paper that you lose.” [40, p. 928] “Carrying books is a drag, now I’m a ‘lightweight’.” [28, p. 614] “The places I feel uncomfortable using [the mobile device] are outside, like in the mall or in a kombi [public transportation], because it’s sort of a big thing, and I think it could attract thieves.” [68, p. 75] “I preferred working on the e-portfolio and entering data via computer as the screen was too small on the PDA to be practical and efﬁcient.” [39, p. 652] Fit for purpose hardware, software, and data “The use of the device got me thinking what I actually needed and the sheer fact that a laptop is too large and cumbersome to carry around with you. I wanted something that I could boot up quite instantly and get on the Wi-Fi; go transfer ﬁles and this is ideal.” [62, p. 574] “I think [a tablet] would be better than a [smartphone] because if it was an [tablet] you could actually have lectures on there and it would be big enough to read and work on.” [40, p. 928] “I can access it [the mobile device] anytime ... and it is mine to use ...” [28, p. 613] Ownership, personalization, and sense of self “I’ve sometimes forgotten my handheld and had the feeling of being naked in a way.” [28, p. 616] “It is part of my life now […] a means of contact, a means of learning. You know, people who have phones just learn a lot.” [53, p. 1401] “I find I am having more and more problems with exams because I cannot look up easily what I normally look up... everyday on my [smartphone].” [33, p. 134] Social technology “Well it’s not that I don’t use a [PDA] I use it for looking up drugs and things but I think in a conver- Devices can impact care and learning Table 1. Illustrative quotes according to theme. Quotes (from learners unless otherwise specified) FRAME model themes Device usability “Much, much quicker than flicking through the paper version. . . Ownership, personalization, and sense of self Negotiating the social aspect of mLearninga mLearning processes Changes in pedagogy and learning Changes in pedagogy and learning Changes in pedagogy and learning “In contrast to the previously mentioned statements made by teachers about students’ uncritical and non- reflective use of ICT, the teachers also acknowledged positive changes with respect to the division of labour, as indicated in the following statement by a teacher: ‘There has been a dramatic change. We don’t have to teach everything now. It’s not teacher based learning. It is student based learning. We just tell them and guide them. We give them topics. We tell them to look up and search those topics on the internet and we ask them to verify them from the textbooks. If they find something new and interesting they can ask us. The students are helping us. They are stimulating us to study more. It’s a two way con- versation. And the students are also contributing’.” [54, p. 1161] “The use of the [tablet] allowed for the shared construction of knowledge between the teachers and the students. One comment was ‘I found the immediacy of this learning immensely powerful for my own learning and the student's … able to look together. In fact, one student pulled their [smartphone] and said, ‘I'll race you!’ While another commented, ‘off into the internet to ﬁnd out together!’ to ﬁnd the answer to a clinical question that neither knew the answer to’.” [47, p. 4] “I was quite averse to it at first –I was one of the haters... [interviewer: What changed your mind?]… I think it’s actually finding I did use the PDA and it did come in handy several times. It just makes life a bit easier.” [12, p. 7] “Actually, I was shown by my daughter at home. […] So I showed my colleagues, yeah.” [53, p. 1401] “[talking about not being able to view past assessments on a smartphone] If I actually saved it on the phone it would be useful to actually learn from, because before I went to do my next [clinical evaluation exercise], I could look at my last [one] and go okay, several times doctors have said that I should say this.” [40, p. 928] Interaction learning 116] Use of the mobile device during downtime, such as skim reading meeting agendas while on the train …was mentioned as 1 of the main benefits of having the portable device (eg, “…instead of having a paper base you can just scroll through the minutes just to remind yourself”). [61, p. 573] Use of the mobile device during downtime, such as skim reading meeting agendas while on the train …was mentioned as 1 of the main benefits of having the portable device (eg, “…instead of having a paper base you can just scroll through the minutes just to remind yourself”). [61, p. 573] “I don’t use my phone immediately. I will write down the things we didn’t know, we nod our heads and then when we leave we’ll sit on our tea break and look them up quickly to make sure we understand or we know what we are talking about.” [56, p. 4] “When we are together [in school settings], we share and discuss the photos. Some [conditions] we learn in school take a long time to see [in practice settings]. So, when you witness this condition and you are not together with your colleagues, you take this picture. […] Then you look at the picture and [later] discuss it, if it corresponds with what we have learned.” [53, p. 1400] “[written scenario] When teaching is impromptu, conventional multimedia equipment may be either unavailable or inappropriate. … The portability of the Smartphone facilitated teaching anatomy in the context of its clinical application within general surgery. It provided visual stimuli to enrich several ad hoc teaching experiences in a single day.” [Educator] [10, p. 7] “A lot of people also discovered that you could use Facebook on it, and also games and stuff … I feel that when you are in the hospital, or actually when you are in the OR, and you are doing something on your iPod, whatever it is, you will be distracted from the process, and it takes longer to react on the things that are happening.” [57, p. 1106] Interaction learning 1161] Changes in pedagogy and learning “The use of the [tablet] allowed for the shared construction of knowledge between the teachers and the students. One comment was ‘I found the immediacy of this learning immensely powerful for my own learning and the student's … able to look together. In fact, one student pulled their [smartphone] and said, ‘I'll race you!’ While another commented, ‘off into the internet to ﬁnd out together!’ to ﬁnd the answer to a clinical question that neither knew the answer to’.” [47, p. 4] “I was quite averse to it at first –I was one of the haters... [interviewer: What changed your mind?]… I think it’s actually finding I did use the PDA and it did come in handy several times. It just makes life a bit easier.” [12, p. 7] Learning to mLearn “Actually, I was shown by my daughter at home. […] So I showed my colleagues, yeah.” [53, p. 1401] Quotes (from learners unless otherwise specified) Quotes (from learners unless otherwise specified) FRAME model themes “[Describing a social media facilitated student group]… Sometimes you use the group afterwards, after you have managed the patient, to see how you went, where you went wrong, how you did, or sometimes they say I messed up. Then, they give you the reasons, or sometimes they will tell you, oh, well done, but you missed that and that.” [53, p. 1400] “[describing peer evaluation of clinical skills via Skype] I have learnt a lot and by students asking me questions. I feel my own knowledge has improved.” [Educator] [51, p. 467] “[describing peer evaluation of clinical skills via Skype] I have learnt a lot and by students asking me questions. I feel my own knowledge has improved.” [Educator] [51, p. 467] “…sharing information and allocating tasks to different members …it can allow that interaction to happen across distance. … PDAs would help keeping the interaction that coordinate the [problem based learning] process, in tagging people (peers, clinicians and the …faculty)” [Educator]. [45, p. 116] “…sharing information and allocating tasks to different members …it can allow that interaction to happen across distance. … PDAs would help keeping the interaction that coordinate the [problem based learning] process, in tagging people (peers, clinicians and the …faculty)” [Educator]. [45, p. Interaction learning Facilitated interaction and learning Facilitated interaction and learning “The students explained… ‘[We show the picture] to flat mates. This is the case I have seen. [...] The whole batch gets it. [.. .] We proudly show it to the others’.” [54, p. 1160] “I liked the fact that it was anonymous, so it gave me the freedom to ask anything without the fear of being criticised without it feeling as if I’m asking a stupid question.” [63, p. 5] “I liked the fact that it was anonymous, so it gave me the freedom to ask anything without the fear of being criticised without it feeling as if I’m asking a stupid question.” [63, p. 5] “[names a social media discussion group], I love it. …I’m part of the group… He [the group convenor] asks questions to medical students and gives the correct answers… there are more than 15000 people.” [54, p. 1160] https://www.jmir.org/2019/2/e12895/ J Med Internet Res 2019 \| vol. 21 \| iss. 2 \| e12895 \| p.6 (page number not for citation purposes) XSL•FO RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Lall et al Lall et al Quotes (from learners unless otherwise specified) FRAME model themes “[Describing a social media facilitated student group]… Sometimes you use the group afterwards, after you have managed the patient, to see how you went, where you went wrong, how you did, or sometimes they say I messed up. Then, they give you the reasons, or sometimes they will tell you, oh, well done, but you missed that and that.” [53, p. 1400] “[describing peer evaluation of clinical skills via Skype] I have learnt a lot and by students asking me questions. I feel my own knowledge has improved.” [Educator] [51, p. 467] “…sharing information and allocating tasks to different members …it can allow that interaction to happen across distance. … PDAs would help keeping the interaction that coordinate the [problem based learning] process, in tagging people (peers, clinicians and the …faculty)” [Educator]. [45, p. 116] Organizing learning using mobile de- vices Use of the mobile device during downtime, such as skim reading meeting agendas while on the train …was mentioned as 1 of the main benefits of having the portable device (eg, “…instead of having a paper base you can just scroll through the minutes just to remind yourself”). [61, p. 573] “I don’t use my phone immediately. Interaction learning I will write down the things we didn’t know, we nod our heads and then when we leave we’ll sit on our tea break and look them up quickly to make sure we understand or we know what we are talking about.” [56, p. 4] Reflective learning for clinical practice “When we are together [in school settings], we share and discuss the photos. Some [conditions] we learn in school take a long time to see [in practice settings]. So, when you witness this condition and you are not together with your colleagues, you take this picture. […] Then you look at the picture and [later] discuss it, if it corresponds with what we have learned.” [53, p. 1400] “[written scenario] When teaching is impromptu, conventional multimedia equipment may be either unavailable or inappropriate. … The portability of the Smartphone facilitated teaching anatomy in the context of its clinical application within general surgery. It provided visual stimuli to enrich several ad hoc teaching experiences in a single day.” [Educator] [10, p. 7] “A lot of people also discovered that you could use Facebook on it, and also games and stuff … I feel that when you are in the hospital, or actually when you are in the OR, and you are doing something on your iPod, whatever it is, you will be distracted from the process, and it takes longer to react on the things that are happening.” [57, p. 1106] mLearning processes “In contrast to the previously mentioned statements made by teachers about students’ uncritical and non- reflective use of ICT, the teachers also acknowledged positive changes with respect to the division of labour, as indicated in the following statement by a teacher: ‘There has been a dramatic change. We don’t have to teach everything now. It’s not teacher based learning. It is student based learning. We just tell them and guide them. We give them topics. We tell them to look up and search those topics on the internet and we ask them to verify them from the textbooks. If they find something new and interesting they can ask us. The students are helping us. They are stimulating us to study more. It’s a two way con- versation. And the students are also contributing’.” [54, p. FRAME model themes “[Training could be improved] If the [training workshop] hour was tailored to the tool [mobile device]… interviewing each other did not work… we just talked.” [45, p. 116] “[Training could be improved] If the [training workshop] hour was tailored to the tool [mobile device]… interviewing each other did not work… we just talked.” [45, p. 116] “…have some base level training…for everybody…specifically on knowing how to turn it on and ma- nipulate it, how it should be used and how it benefits medical education, how the faculty or school expect it to be used. …you need drop-in sessions, extra assistance or individual assistance for people struggling with the technology…” [Educator] [45, p. 116] mLearning needs course planning and institutional leadership “Focus on the areas where you really feel like the [tablet] is an appropriate tool for the thing you want to do, but do not try to wedge [it] into areas where it may or may not be the best thing to use… there are things you can do and things you cannot do at each step along the way.” [Educator] [30, p. 1154] “the participants categorised the teachers as being either old school, a term they frequently assigned to the older generation, or new school, a term afforded to more youthful practitioners—the former being described as paper-dependent and being offended when interviewees used their devices in front of them. Many of the old school doctors did not appear to understand the reliance that the younger student gener- ation have on their mobile devices as learning tools.” [56, p. 5] “It’s things like that [teacher advocacy] which encourage you, maybe I will bring it with me tomorrow and take it on the ward round with me.” [12, p. 7] “…it is easy to see the value of some technologies where it works very well and it is very easy to get over-enthusiastic about it and then not realize that people might not be ready to actually use that technol- ogy for whatever reasons…” [Educators] [45, p. 116] amLearning: mobile learning. mLearning in clinical learning contexts in Figure 3). Otherwise, the synthesis produced themes that could be grouped under the aspects of learning represented in Koole’s original model, and we used subthemes under each aspect to help illustrate attributes that relate to the specifics of doctors’ and nurses’ learning. The FRAME model was adapted to account for these differing findings. The implementation of mLearning in clinical contexts 5] “It’s things like that [teacher advocacy] which encourage you, maybe I will bring it with me tomorrow and take it on the ward round with me.” [12, p. 7] “…it is easy to see the value of some technologies where it works very well and it is very easy to get over-enthusiastic about it and then not realize that people might not be ready to actually use that technol- ogy for whatever reasons…” [Educators] [45, p. 116] amLearning: mobile learning. The FRAME model was adapted to account for these differing mLearning in clinical learning contexts in Figure 3) Otherwise Quotes (from learners unless otherwise specified) Quotes (from learners unless otherwise specified) Quotes (from learners unless otherwise specified) The implementation of mLearning in clinical contexts “Loss of carrier signal or connection was a recurring event. … One lecturer described their experience, ‘this week I had a problem with 3G connection, so missed a day using [my tablet] while sorting that out’.” [Educators] [47, p. 4] Institutional infrastructure and re- sources Institutional infrastructure and re- sources “Several schools talked about the importance of all the sites having Wi-Fi. … [one reported that a] ‘commonly cited reason for our clerkship students to not use them was if they were at a site where the Wi-Fi was unreliable or unavailable’.” [Educators] (30, p. 1154] J Med Internet Res 2019 \| vol. 21 \| iss. 2 \| e12895 \| p.7 (page number not for citation purposes) https://www.jmir.org/2019/2/e12895/ https://www.jmir.org/2019/2/e12895/ XSL•FO RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Lall et al Lall et al Quotes (from learners unless otherwise specified) FRAME model themes “[Training could be improved] If the [training workshop] hour was tailored to the tool [mobile device]… interviewing each other did not work… we just talked.” [45, p. 116] mLearning training and technical sup- port “…have some base level training…for everybody…specifically on knowing how to turn it on and ma- nipulate it, how it should be used and how it benefits medical education, how the faculty or school expect it to be used. …you need drop-in sessions, extra assistance or individual assistance for people struggling with the technology…” [Educator] [45, p. 116] “Focus on the areas where you really feel like the [tablet] is an appropriate tool for the thing you want to do, but do not try to wedge [it] into areas where it may or may not be the best thing to use… there are things you can do and things you cannot do at each step along the way.” [Educator] [30, p. 1154] mLearning needs course planning and institutional leadership “the participants categorised the teachers as being either old school, a term they frequently assigned to the older generation, or new school, a term afforded to more youthful practitioners—the former being described as paper-dependent and being offended when interviewees used their devices in front of them. Many of the old school doctors did not appear to understand the reliance that the younger student gener- ation have on their mobile devices as learning tools.” [56, p. J Med Internet Res 2019 \| vol. 21 \| iss. 2 \| e12895 \| p.8 (page number not for citation purposes) JOURNAL OF MEDICAL INTERNET RESEARCH Lall et al Social Technology This theme encompassed participants’ perspectives on social responses to mobile devices and many studies were conducted in clinical contexts. Within these settings, students were expected to combine their learning with practice, which resulted in the device influencing social interactions with a number of actors, including their supervisors, patients, and peers. Mobile devices seemed to hold the symbolic value of being a form of technology for recreational use rather than for learning, owing to multiple functions enabling information retrieval alongside highly social activities, such as sending and viewing messages. Discussion Regarding device-enabled group work, online study groups were described as enabling case discussions, and participants commented upon the pros and cons of structured, cooperative peer assessment approaches. It also enabled students to remotely The Implementation of Mobile Learning in Clinical Contexts Study participants reported challenges with mLearning that had little to do with interactions between students, devices and their contents, patients, and tutors. Here, what was implicated were insufficient institutional structures and resources, a lack of device-focused training and support, and limited planning and leadership of mLearning programs. For example, the importance—and yet variability—of network connectivity was emphasized by both tutors and students, and concerns were raised over program provision of ill-suited devices. Mobile devices, thus, affected students’ relationships with patients and their professional identity. For example, although mobile devices were seen as potentially strengthening communication between clinicians and patients, concerns were raised about possible interference with activities at the bedside. There were reports of feeling rude [37,56,58] or awkward [31,58]. Although some feared being viewed as unprofessional by either patients or colleagues, others linked device use with perceptions of increased competence. Although these social norms did result in some students being reluctant to openly use the device, others developed strategies for negotiating device use including asking for permission, explaining device use, and jointly using devices with patients. The use of mLearning strategies did not always appear to have been planned with course content or pedagogy in mind, or with consideration of the attributes required by teaching staff. Students reported they were offered little guidance on how to integrate mobile devices into their learning activities as well as a lack of device knowledge among clinical instructors. Experiential or ongoing training and local technical support were particularly valued; participants reported forgetting functions covered during orientation, and support had been experienced by some as fragmented. Reports were made across numerous studies of disapproval for device use among supervising staff in clinical settings and of students, as a result, being hesitant to use a device openly. A range of proposals were made across the studies, including initiatives to improve staff awareness about the value of portable devices and the development of codes of conduct. Mobile Learning Processes Some participants reflected upon mLearning as a whole process. Subthemes here represent views on how educational roles could be changing and the process of adapting one’s learning to the mobile device. In terms of the former theme, both students and tutors described how they were participating on more equal terms. Enthusiasm, however, was far from universal, and positive comparisons were made with more traditional forms of learning. Frustration and impatience were expressed about the process of learning how to use a device. Participants described a reliance on others, in particular peers and friends, and although familiarity reportedly improved over time, the need for support and repeat training was emphasized. Uncertainty was voiced over the trustworthiness or reliability of information being distributed through mLearning apps or websites. Interaction Learning Studies highlighted how mobile use enabled learning processes contingent on students’ interaction with their academic institution, peers, and practice. Students used these multiple forms of interaction to learn cooperatively with their peers, organize competing demands of clinical practice and study, and situate their learning within clinical contexts. These forms of mLearning encompassed individual device use for the purposes of information retrieval and organization to device-enabled group work. Device Usability contact supervisors while working in clinical settings. Meanwhile, students and staff described using mobile devices to help them organize their learning, for example, to access information on learning activities when in a clinical setting. As such, students emphasized the value of access to immediately relevant or difficult-to-access clinical cases or using devices to prepare immediately before encounters. Participants referred to the physical, technical, and functional characteristics of mobile devices in relation to an individual’s learning, which involved access, manipulation, and storage of information. Subthemes explored possible positive and unintended consequences of devices being mobile, views on the sufficiency of device functionality, and ideas about the individualistic nature of device use. Enthusiasm for a mobile device focused on efficiency yet was accompanied by an awareness of the need for caution, in terms of a risk of loss of device or contamination in certain settings. Reports of problems attributed to hardware and software were seen in a range of studies, with some participants noting that screens were too small for reading documents [37,40,61] or that they lost information owing to system crashes [12,27,38]. Learners reported wanting devices that suited their own specific needs, describing device use as either a way of life (p. 111 [65]) or as a part of my life now (p. 1401 [53]). https://www.jmir.org/2019/2/e12895/ FRAME model themes Social technology was altered to account for the impact of mobile devices on social interaction, rather than to describe how it enabled connection between multiple interfacing entities. We also added an additional circle that contained the model’s overlapping 3 circles (see the theme Implementation of Figure 3. Framework for the Rational Analysis of Mobile Education (FRAME) model adapted for this study. J Med Internet Res 2019 \| vol. 21 \| iss. 2 \| e12895 \| p.8 https://www.jmir.org/2019/2/e12895/ (page number not for citation purposes) L•FO https://www.jmir.org/2019/2/e12895/ J Med Internet Res 2019 \| vol. 21 \| iss. 2 \| e12895 \| p.8 (page number not for citation purposes) XSL•FO RenderX JOURNAL OF MEDICAL INTERNET RESEARCH J Med Internet Res 2019 \| vol. 21 \| iss. 2 \| e12895 \| p.9 (page number not for citation purposes) Principal Findings To our knowledge, this is the first systematic review synthesizing qualitative research findings about health professionals’ experiences of mLearning. The review identified J Med Internet Res 2019 \| vol. 21 \| iss. 2 \| e12895 \| p.9 (page number not for citation purposes) XSL•FO RenderX Lall et al JOURNAL OF MEDICAL INTERNET RESEARCH Lall et al a total of 47 studies that varied in the types of health professionals involved, their stage of learning, and the mLearning strategies considered. Qualitative data in the majority of studies had been sought so as to pilot mLearning approaches or examine nonspecific use of mobile devices for learning. In many studies, qualitative findings were slim and provided little explanatory detail but across this body of work, it is possible to identify recurring themes about experiences and some explanatory narratives from both learners and educators. enable communication and collaboration among multiple individuals and systems (p. 34), whereas findings from studies within this synthesis instead identify impacts of mLearning on interactions with patients and the management of professional identity. The FRAME model [17] represents the mLearning processes as an integration of the device, learner, and social aspects that provides for enhanced collaboration between learners, access to information, and deeper contextualization of learning (p. 38). Although there were some positive accounts of device use for situated learning and of cooperative learning activities, accounts from studies in our synthesis placed more emphasis on the process of learning how to apply devices for the purposes of learning. Qualitative research into mLearning for health professional education appears still to be in its infancy, with few studies referring to the supported integration of mLearning within a pedagogically informed program of study. Our synthesis of findings from these studies illustrates some of the potentials of mLearning but also some of the challenging realities for students, doctors, and nurses who are learning in contexts where mobile devices have either formally been introduced or tend to be common. Early commentators on mLearning envisaged methods of delivery that would be highly suited to the just enough, just in time, and just for me demands of twenty-first century learners [69]. Students in the studies we reviewed did indeed value devices for the possibility of lessening cognitive loads and helping to make good use of time. They also described device use in terms of individualized needs and preferences. Limitations This study provided a comprehensive overview of current qualitative research on mLearning strategies in medical and nursing education. Its strengths include a sensitive search strategy encompassing several bibliographic databases and independent screening by pairs of reviewers, both lowering the likelihood that relevant literature would be overlooked as well as coding and synthesis work done independently and in pairs, aimed at bringing a variety of perspectives to the act of making sense of a heterogeneous set of study findings. The review is, nevertheless, limited by the qualities of the reviewed studies, especially those employing mixed-methods designs, wherein the quantitative component was given far heavier weighting than qualitative findings. Few studies described in sufficient detail the steps taken by researchers to ensure confidence in the quality of their findings. The majority of studies offered little explanation of methods used to sample participants and collect or analyze data. Moreover, there were many studies in which authors provided little evidence as to how they arrived at their findings. These studies offered few quotes from participants, sometimes making it difficult to decipher whether results were guided by the perspectives of respondents. This synthesis identifies additional social and institutional factors that seem key for understanding how mLearning for health professionals might be implemented to the best effect. In particular, throughout much of their training, medical and nursing students need to combine learning with professional caring responsibilities. The social aspects of learning that are already complex within more formal education settings become considerably multilayered when students are, for example, at the bedside or in an operating theatre. On top of interactions with information, fellow learners, and formal educators come interactions with a variety of other health professionals and with patients. Learning can happen through peripheral participation in clinical activities, observation, role modeling, and reflective activities, as well as through work with lecturers, supervisors, and other students, and with text books and other information sources. mLearning needs to fit into this mix of interactions but instead our synthesis contains accounts of reluctance, told by both students and educators, toward the use of mobile devices in the clinical workplace because of existing, often implicit, rules for practice. Although negotiation was said sometimes to enable device use for learning, participants in more than one study identified a need for procedural guidance on device use, echoing calls from education more broadly [73,74]. Principal Findings However, although both learners and educators described the potential value of devices for accessing, organizing, and enhancing learning, limitations in hardware were reported across the full time period covered by our included studies. Researchers in other spheres of education have also emphasized the need for devices to be fit for purpose [70-72]. Future mLearning strategies for medical and nursing education should, therefore, be developed with an awareness of device affordances for the learning activities required. J Med Internet Res 2019 \| vol. 21 \| iss. 2 \| e12895 \| p.10 (page number not for citation purposes) https://www.jmir.org/2019/2/e12895/ Limitations Another methodological limitation was that nearly all of the studies explored learning within clinical settings. Owing to this, much of the mLearning described would be classified as informal learning, that is, learning which results from incidental day-to-day activities. Our synthesis, therefore, contains little detail sourced from experiences of programs set up to encourage mLearning in university settings. Finally, few studies explicitly referred to educators’ learning theories or described course structures in any detail, which meant study findings could not be explored in terms of different objectives for students’ learning. Efforts should be made in future qualitative studies to clearly define the educational purposes of the mLearning programs concerned to make findings more applicable to given learning circumstances. We found Koole’s conceptualization of mLearning, involving a combination of learner, device, and social aspects, to be helpful when organizing findings. However, the themes of social technology and mLearning processes in our synthesis diverged from that of Koole’s conceptualization. With regard to social technology, Koole’s [17] model emphasizes how mobile devices J Med Internet Res 2019 \| vol. 21 \| iss. 2 \| e12895 \| p.10 (page number not for citation purposes) J Med Internet Res 2019 \| vol. 21 \| iss. 2 \| e12895 \| p.10 (page number not for citation purposes) https://www.jmir.org/2019/2/e12895/ XSL•FO RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Lall et al Lall et al as private data can be potentially disseminated to unintended audiences [74,78,79]. In terms of medical and nursing education, there is the added concern that the welfare of patients might be compromised. The need identified above for guidance for health professionals’ device use, consequently, will require a strong ethical component. Conclusions The findings of our review have underlined that there is still much to be understood about what is involved in mLearning for medical and nursing education. Our review has indicated that mLearning can potentially play a substantial role as students are already likely to be using mobile devices for a number of differing purposes associated with their learning, ranging from communication with supervisors to organization of tasks. The multipurpose nature of mobile devices means that students can personalize these tools toward their learning needs, which entails a process of learning within itself. As with any complex tool used for educational purposes, mobile devices should be appropriately incorporated into the structures of academic and medical institutions and steps need to be taken to ensure that learners fully comprehend the functions of each mobile device or app used for learning. These 2 considerations can only be addressed by paying close attention to the process of implementing mLearning strategies in medical and nursing scholarship and the building of an educational infrastructure that enables use of mLearning techniques. This review also starts to identify gaps in the literature where additional studies might throw light on a more complete range of mLearning practices within medical and nursing education. For example, study authors made no mention of discussion by study participants of ethical concerns over patient privacy and data security. Educational experts, however, raise concerns vis-à-vis use of mLearning strategies in other settings, arguing that these interventions can compromise students’ confidentiality Comparison With Earlier Work Findings about a lack of device training, technical support, and other forms of institutional support led to one of the biggest modifications to the FRAME model seen in our synthesis, which was the development of an additional aspect—implementation in a clinical context. This theme highlighted that even when mobile devices had been introduced for the purpose of evaluation, this appeared to have been done with insufficient consideration of course content or needs at the institutional level, including both sufficient Wi-Fi coverage and the alignment and capacity of teaching staff to use mLearning. Insights might be gained through the study of device maintenance services on campus [45] and the implementation of mLearning strategies with learning outcomes as well as a wider curriculum in mind [30,45,47]. Studies of change management around learning technology in higher education outside the field of health might also be relevant here, as they have explored the potential for initiatives, such as staff as champions, and strategic contextual analyses [75-77]. Multimedia Appendix 1 Protocol for the review. [PDF File (Adobe PDF File), 450KB - jmir_v21i2e12895_app1.pdf ] Conflicts of Interest None declared. Acknowledgments We gratefully acknowledge support for this work by National Healthcare Group, Singapore, the Lee Kong Chian, School of Medicine, Nanyang Technological University, Singapore, and UCL Institute of Education, University College London. Conflicts of Interest None declared. Multimedia Appendix 1 Protocol for the review. [PDF File (Adobe PDF File), 450KB - jmir_v21i2e12895_app1.pdf ] Multimedia Appendix 2 Search strategy. [DOCX File, 59KB - jmir_v21i2e12895_app2.docx ] Multimedia Appendix 3 Details of included studies. [DOCX File, 37KB - jmir_v21i2e12895_app3.docx ] Multimedia Appendix 4 Quality appraisal of studies included in the synthesis. [DOCX File, 47KB - jmir_v21i2e12895_app4.docx ] J Med Internet Res 2019 \| vol. 21 \| iss. 2 \| e12895 \| p.11 https://www.jmir.org/2019/2/e12895/ (page number not for citation purposes) •FO derX We gratefully acknowledge support for this work by National Healthcare Group, Singapore, the Lee Kong Chian, School of Medicine, Nanyang Technological University, Singapore, and UCL Institute of Education, University College London. J Med Internet Res 2019 \| vol. 21 \| iss. 2 \| e12895 \| p.11 (page number not for citation purposes) Multimedia Appendix 2 Search strategy. [DOCX File, 59KB - jmir_v21i2e12895_app2.docx ] [DOCX File, 59KB - jmir_v21i2e12895_app2.docx ] Multimedia Appendix 3 Details of included studies. Details of included studies. [DOCX File, 37KB - jmir_v21i2e12895_app3.docx ] [DOCX File, 37KB - jmir_v21i2e12895_app3.docx ] Multimedia Appendix 4 Quality appraisal of studies included in the synthesis. Quality appraisal of studies included in the synthesis. [DOCX File, 47KB - jmir_v21i2e12895_app4.docx ] J Med Internet Res 2019 \| vol. 21 \| iss. 2 \| e12895 \| p.11 (page number not for citation purposes) https://www.jmir.org/2019/2/e12895/ XSL•FO RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Lall et al Lall et al [DOCX File, 23KB - jmir_v21i2e12895_app5.docx ] [DOCX File, 23KB - jmir_v21i2e12895_app5.docx ] References 1. Triola MM, Huwendiek S, Levinson AJ, Cook DA. New directions in e-learning research in health professions education: report of two symposia. Med Teach 2012;34(1):e15-e20. 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Abbreviations FRAME: Framework for the Rational Analysis of Mobile Education mLearning: mobile learning PDA: Personal Digital Assistant Please cite as: Lall P, Rees R, Law GCY, Dunleavy G, Cotič Ž, Car J Influences on the Implementation of Mobile Learning for Medical and Nursing Education: Qualitative Systematic Review by the Digital Health Education Collaboration J Med Internet Res 2019;21(2):e12895 URL: https://www.jmir.org/2019/2/e12895/ doi:10.2196/12895 PMID:30816847 ©Priya Lall, Rebecca Rees, Gloria Chun Yi Law, Gerard Dunleavy, Živa Cotič, Josip Car. Originally published in the Journal of Medical Internet Research (http://www.jmir.org), 28.02.2019. 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 the Journal of Medical Internet Research, is properly cited. The complete bibliographic information, a link to the original publication on http://www.jmir.org/, as well as this copyright and license information must be included. J Med Internet Res 2019 \| vol. 21 \| iss. 2 \| e12895 \| p.15 (page number not for citation purposes) https://www.jmir.org/2019/2/e12895/ XSL•FO RenderX
https://openalex.org/W2047321243	https://zenodo.org/records/1872645/files/article.pdf	German	null	Über das Auftreten yon Polyphemus pediculus in der “alten Donau” bei Wien	Internationale Revue der gesamten Hydrobiologie	1,913	public-domain	6,737	9.) Uber das Auftreten yon Polyphemus pedieulus in der ,,alten Donau" bei Wien. Von H. Joseph (Wien). In einer ungcmein ausfuhrlichen und grundlichen Arbeit, einem Resultate mehrjiihriger Untersuchungen , hat A. S te u er die Entomostrakenfauna der sogenannten ,, alten Donau' bei Wien (Zoo]. Jahrbucher Syst., Bd. 15, 190 1) zum Ausgangspunkte weit ausgreifender okologischer und tiergeographischer Betrachtungen gemacht und unter anderem auch auf die Fremdlinge, nament- lich in der lininetischen Tierwelt, hingewiesen, welche in Gewissern, wie es das erwiihnte ist, niimlich vom Hauptbette abgetrennten FluDarmen, zunachst sporadisch auftreten, die aber bei der immer weiter fortschreitenden Bnderung dcr Lebensbedingungen schlie5lich zu autochthonen Formen werden konnen ; diese Voraussage konnte er speziell fur die Daphniden in besonders zuver- sichtliclier Form aussprechen. Wir durfen nicht im geringsten daran zweifeln, da8 S teuer bei seinen durch lange Zeit kontinuierlich angestellten Fangen, kein wesentliches und markantes Glied der Entomostrakenfauna der alten Donau entgangen ist. Seine Untersuchungen durften im Jahre 1899 abgeschlossen worden sein wid spielen sich also in einem Zeitraume ab, der hochstens bis 23 Jahre iiacli erfolgter Abtrennung des Gebietes vom Hauptstrome reicht. 13s haben sich in dieser Periode die naturlichen Bedingungen in der ,,alten Donau" bereits in auffallender Art den Verhaltnissen gro8er stehender Ge- wasser genihert, und wie wir oben sclion betonten, Stener zur Annahme einer noch weiter fortschreitenden Entwicklung im gleichen Sinne veranlaDt. Im Jahre 1910 nun waren seit der im Jahre 1876 abgeschlossenen Los- losung der ,alten Doiiau' durch das Werk der Donauregulierung 34 Jahre verflossen , urn 11 Jahre mehr als beiiu Abschlusse der Untersuchungen Steuers, und es ware zu erwarten gewesen, da5 die vorausgesetzten Ver- Snderungen in der Zusammensetzung der Flora und Fauna hatten nachge- wiesen merden konnen. Dies ist nun leider nicht der Fall, da sich in den1 angegebenen Zeitraum niemand fand, der die Untersuchungen Steuers in gleicher Richtung fortgesetzt hatte und jene die gelegentlichen Fiinge vorlagen, welche von den Wiener biologischen Instituten lediglich zur Gewinnung von 1Jntersnchungsmaterial angestellt wurden. Es erscheint mir daher von einigem Stationsnachrichteii und Notizen. 482 Interesse, einen ini Jahre 1910 gemachten Fund, dem gelegdlich der freilicli nicht in syttematischer n'eise angestellten Fange der Zwischenzeit kein Bhn- licher vorausgegangen war, hier mitzuteilen, da er einen gewissen Beitrag zu den Voraussagungen S t eu e r s liefert. g g In der ersten Junihalfte des genannten Jahres fand ich am Nordostufcr des als ,,Bruckenwasser' bezeiclineten Absclinittes (vgl. 9.) Uber das Auftreten yon Polyphemus pedieulus in der ,,alten Donau" bei Wien. Tafel 2 in S t e u e r s Arbeit) in der Uferzone vor den Ruderklubhausern dicht am nordostlichen Ropfe der Kagraner Holzbriicke zwischen und uber cler reichlichen submersen Vegetation zieinlich groBe Mengen von P o l y p h e m u s pediculus, welchc Cladocere meines Wissens bisher uberhaupt nicht im genannten Gebiete bc- obachtet wurde und die auch demgema8 in Steuers Zusammenstellungen lehlt. Das Briickenwasser gehort zu dem von Steuer wegen des Dorninierens von Clathrocystis im Plankton als ,, Chroococcaceensee" bezeichneten weitaus groDeren Teil des Untersuchungsgebietf s und stellt zugleich die Lo- lralitat dar, auf welche der genannte Autor die intensivste Aufmerksamkeit 1-erwendete. Es erscheint ausgeschlossen, daD S t e u e r den P o l y p h e m u s iibersehen habe und wir niussen dieses Tier daher als ein Novum dieses Ge- wassers ansehen. Es wird nicht uninteress;tnt sein, auf die Details meinei- Ereilich nicht sehr ausgedehnten Befunde naher einzugehen. ZunLchst sei be- merkt, daD der genannte Fundplatz am Ufer bei clcn Ruderklubhausern drr einzige blieb und daB nanientlich weder drauBen im freien Wasser noch aiir Grunde die Form aufgefunden werden konnte. In Gesellsclinft des I'oly - pliemus fanden sich auBer ziemlich zahlreichen, von inir nicht nilier be- stimmten Cop ep ode n (namentlich C y clo p s und N a up1 i u s stadien) in reiclicr Menge folgende schon von S t e u e r bemerkte Formen: Chydorus und Bos- niina, daneben in geringerer Menge S i d a , Scapholeberis und Clerio- d a p h ni a , ganz vereinzelt €1 y a 1 o do 11 h n i ;t c ii c u 11 at a , A cr o 1) e r u s , A1 o n :L c o s t a t a , Leptodora, sowie Arguluslnrven. Zir dieser Liste sei folgeiides bernerkt: Scayholeberis gibt S t e u e r bIoD fur das von ihm als ,,Dine- bryonsee '' bezeichnete ,, K a r p f e n w n s s e r " und fur einen der beideii, in der Nihe meines Fundortes gelegenen , wither jedoch versch~vundenen ,,TroIvos - t u m p e l " in nicht bedeutender Zahl an. Meinen Erfahrungen nacli gehort sie jetzt zu den gewohnlichen, wenn auch noc~h imnier nicht massenhaften Be- wohnern des Clhrooconcaceengebietes. 9.) Uber das Auftreten yon Polyphemus pedieulus in der ,,alten Donau" bei Wien. Und es darf wohl nuch weiter geschlossen werden, dab die Verhalt- nisse fur Polyphemus derzeit noch nicht derartige sind, daB er als ein lieimatsberechtigtes Glied der lokalen Fauna angesehw werden kann , da er offenbar seinen Cyclus nicht volleriden konnte und wieder verschwand. Auch dic sehr geringe (maximal 7) Zahl der Subitaiikeime spricht fur eine derartige Deutung. Solches sporudisches Auftreten und Wiederverschwinden einer der- zeit noch fremden Form , walirscheinlich veranlal3t durch passive Einwande- rung, mu8 wohl in der Regel einer definitiven Einbiirgerung vorangegangeii sein. Es wird natiirlich der Frage nach dem Vorkommen des Polyphemus iu dem besprochenen Gebiete in Zukunft weitere A ufmerksamkeit zu schenken und namentlich auf die vollige Absolvierung des Entwicklungszyklus zu achten sein. Herbst nicht wieder auf und wurden in den folgenden Jahren auch nicht mehr beobachtet. Das Auftreten im Juni 1910 muD also nach meinen Erfahrungen als ein vereinzeltss bezeichnet werden. Hervorgehoben sei, daD mein Fundort eine dem direkten Sonnenlicht wahrend eines groDen Teils des Tages aus- gesetzte Lokalitit ist. Freilich konnten die Tiere zwischen dem dichten Pflanzenwuchs der Uferregion penugend Schatten finden ; ich habe leider auf die genauere Verteilung ibres Vorkommens nicht geachtet, da mir damals die zwischen S t r o h l (Zool. Anz., Bd. 22, 1907, Internat. Revue f. Hydrobiol. u. Hydrogr., Bd. 1, 1908/09) und l s s a k 6 w i t s c h (Biol. Centralbl., Bd. 28, 1908) gef uhrte und unter anderem auch auf diesen Punkt bezugliche Diskussion nicht gegenwartig war. Obwohl nun meine Beobachtungen uber GroDe und Menge der Subitaneier des Polyphemus im Weseutlicheu nichts News bieten, mag es immerhin gerechtfertigt erscheinen, diesen Nachtrag zu S t e u e r s Mono- graphie vorzubringen, donn es mag immerhin die Annahme zulassig sein, daiD in den letzten 11 Jahren weitere Veranderungen in dem physikalisch-bio- loqischen Verhelten der Donaualtwisser eingetreten sind, welche die Ansiede- lung neuer limnetischer Fornien gestatteten; hierfiir mag unter anderem aucli das Steuer noch nicht bekannte Vorkommen von Scapholeberis zu rechnen sein. Und es darf wohl nuch weiter geschlossen werden, dab die Verhalt- nisse fur Polyphemus derzeit noch nicht derartige sind, daB er als ein lieimatsberechtigtes Glied der lokalen Fauna angesehw werden kann , da er offenbar seinen Cyclus nicht volleriden konnte und wieder verschwand. Auch dic sehr geringe (maximal 7) Zahl der Subitaiikeime spricht fur eine derartige Deutung. 9.) Uber das Auftreten yon Polyphemus pedieulus in der ,,alten Donau" bei Wien. Lep tod o r a f m d ich in vielen Proben niir ganz vereinzelt und in winzigen Exemplaren. Die gafundenen P o l y p he mu s maren durchweg Weibcheri von iiiaIii,ger GrODe, ganz entsprechend den bisher von anderen ilutoren fiir sudlichc wid tiefgelegene Fundorte mgegebenen hlaRen. Die groDten Esemplare ninOen vom vorderen Kopfrande bis zum Hinterende des Brutraumes nicht inelir a1.s 930 p, erreichten :dso eben die GroDe der yon nordlichen oder hodigelegenen Orten stammenden Mannchen. Dabei befanden sich die Tiere in voller Fortpflanzungs- tatigkeit, so daD man alle GroIien vom eben freigewordenen Tiere his zum erwLhnten Maximum finden konnte. Konform init den Angabeii friihercr Aiitoren konnte ich auch die relntiv geringe Mengc von Eiern resii. Embry- onen im Brutranme feststellen, die Maximalzahl war 7. Es fnnden sich nur Subitaneier; trotz eifrigen Suchens gelang es nicht , Dauereier oder Mannclien zu finden. Die Funde von Polyphemus wiederholten sich im Juni 1910 rioch mehrere Male, dnrin verschwanden die Tiere, traten im Spatsommer imtl Stationsnachrichten und Notmizen 483 Herbst nicht wieder auf und wurden in den folgenden Jahren auch nicht mehr beobachtet. Das Auftreten im Juni 1910 muD also nach meinen Erfahrungen als ein vereinzeltss bezeichnet werden. Hervorgehoben sei, daD mein Fundort eine dem direkten Sonnenlicht wahrend eines groDen Teils des Tages aus- gesetzte Lokalitit ist. Freilich konnten die Tiere zwischen dem dichten Pflanzenwuchs der Uferregion penugend Schatten finden ; ich habe leider auf die genauere Verteilung ibres Vorkommens nicht geachtet, da mir damals die zwischen S t r o h l (Zool. Anz., Bd. 22, 1907, Internat. Revue f. Hydrobiol. u. Hydrogr., Bd. 1, 1908/09) und l s s a k 6 w i t s c h (Biol. Centralbl., Bd. 28, 1908) gef uhrte und unter anderem auch auf diesen Punkt bezugliche Diskussion nicht gegenwartig war. Obwohl nun meine Beobachtungen uber GroDe und Menge der Subitaneier des Polyphemus im Weseutlicheu nichts News bieten, mag es immerhin gerechtfertigt erscheinen, diesen Nachtrag zu S t e u e r s Mono- graphie vorzubringen, donn es mag immerhin die Annahme zulassig sein, daiD in den letzten 11 Jahren weitere Veranderungen in dem physikalisch-bio- loqischen Verhelten der Donaualtwisser eingetreten sind, welche die Ansiede- lung neuer limnetischer Fornien gestatteten; hierfiir mag unter anderem aucli das Steuer noch nicht bekannte Vorkommen von Scapholeberis zu rechnen sein. 9.) Uber das Auftreten yon Polyphemus pedieulus in der ,,alten Donau" bei Wien. Solches sporudisches Auftreten und Wiederverschwinden einer der- zeit noch fremden Form , walirscheinlich veranlal3t durch passive Einwande- rung, mu8 wohl in der Regel einer definitiven Einbiirgerung vorangegangeii sein. Es wird natiirlich der Frage nach dem Vorkommen des Polyphemus iu dem besprochenen Gebiete in Zukunft weitere A ufmerksamkeit zu schenken und namentlich auf die vollige Absolvierung des Entwicklungszyklus zu achten sein. zoologischen und botanischen Ferial- kurse zu und deshalb erscheint es zweckmaiDig, an dieser Stelle zur Infor- mation der interessierten Kreise eine brziigliche Mitteilung zu machen. zoologischen und botanischen Ferial- kurse zu und deshalb erscheint es zweckmaiDig, an dieser Stelle zur Infor- mation der interessierten Kreise eine brziigliche Mitteilung zu machen. Zunachst mochte hinsichtlich der Historik dieser Kurse erwahnt werden, daD sie durvh den Schreiber dieser Zeilen bei der ubernahme der Leitung der Triester zoologischen Station, d. i. seit Sommer 1898 eingefiihrt wurden, da sich ergab, daB die Studenten und jungen Naturforscher, welche das erstemal ans Meer studienhalber kommen, die meist knapp bemessene Zeit ohne eine entsprechende Anleitung und ohne ein vorlieaendes Arbeitsprogramm unmoglich in der fur sie wiinschenswerten Form ausnutzen konnen. Der Anfanger wird bei einem Erstlingsaufenthalt am Meere in den meisten Fallen durch den Umfang g g Zunachst mochte hinsichtlich der Historik dieser Kurse erwahnt werden, daD sie durvh den Schreiber dieser Zeilen bei der ubernahme der Leitung der Triester zoologischen Station, d. i. seit Sommer 1898 eingefiihrt wurden, da sich ergab, daB die Studenten und jungen Naturforscher, welche das erstemal ans Meer studienhalber kommen, die meist knapp bemessene Zeit ohne eine entsprechende Anleitung und ohne ein vorlieaendes Arbeitsprogramm unmoglich in der fur sie wiinschenswerten Form ausnutzen konnen. Der Anfanger wird bei einem Erstlingsaufenthalt am Meere in den meisten Fallen durch den Umfang Stationsnachrichten und Notizeii. 484 uud die Mannigfaltigkeit des Materials und durch die Menge der neuen Eindrucke iiberwaltigt, so daD er dann nicht weiD, wo er anfangen soll. Tatsachlich hat die nun vieljshrige Erfahrung gezeigt, daO unter den zahlreichen Kursteilnrhmern die Zahl derjenigen eine verschwindend kleine ist, die ein derartiges Wissen mitbrachten und eine derartige Selbstandigkeit besaBen, um ohne weitgehendt! Hilfe mit Erfolg einen solchen Studienaufenthalt am Meere zu absolvieren. Endlich sind diese Kurse auch in dem Sinne gedacht, daB sie eine Erganzunp zum Unterrichte an der Universitat bilden sollen, da die marine Fauna und Flora bei den Arbeiten im Laboratorium dock nur im beschrankten Umfange beruck- sichtigt werden kann. 9.) Uber das Auftreten yon Polyphemus pedieulus in der ,,alten Donau" bei Wien. Es handelt sich also im vorliegenden Fall nicht uni Spezialkurse, sondern die Triester Kurse verfolgen vielmehr den Zweck eine Orientierung uber die Meeresfauna und -Flora zu bieten. g Der Titel der Kurse ist: Ferialkurs iiber die Anatomie , Entwicklungs- geschichte und Biologie der niarinen Fauna, beziehungsweise Ferialkurs uber die Anatomie, Entwicklungsgeschichte, Systematik und Biologie der marinen Algen. Den zoologischen Kurs halt der Gefertigte unter Mithilfe seiner beiden lissistenten fur Zoologie ab, wahrend den botanischen Kurs der jeweilige A%ssistent fur Botanik leitet. Es sei darauf aufmerksam gemacht, daB einc. Teilnahme an beiden Kursen nicht moglich ist, da diese zur selben Zeit ab- gehalten werden. Die Kurse finden zweimal im Jahre statt und zwar in den Osterferien und in den Sommerferien. Die Osteikurse fallen rnit den oster- reichischen Hochschulosterferien zusanimen, die sich an das Osterfest halten. Daher beginnen die Osterkurse immer Montag 3 Wochen Tor Ostersonntag und enden rnit der ersten oder zweiten Woche nach Ostern. In den Sommerferien beginnen die Kurse regelmaBig am 1. September und werden gewohnlich mit tler ersten Woche im Oktober geschlossen. Die t8gliche Arbeitszeit ist an Wochentagen von 8l/%-12 Uhr vormittag und von 2l/? bis 5 Uhr nach- mittag. An Sonn- und Feiertagen wird nicht gearbeitet und werden diese Tage moglichst zu Ausflugen zu Wasser und zu Lande ausgenutzt. Im zoologischen Kurs werden aus der zum Schlusse mitgeteilten Programni- liste zirka 50-60 Formen durchgenommen. Es kommen nur lebcnde Objekte zur Verteilung, die je nach ihrer Beschaffenlieit rnit dem Mikroskop oder mit der Impe oder mittelst Disektion untersuclib werden. Alle Kursteilnehmer erhalteii tlann imnier das gleiche Tier beziehungsweise dieselbe Alge. Nach Moglich- lteit wird systematisch Zusammengehoriges nacheinander absolviert, aber teils zeigt sich dabei eine gewisse Ermudung, wenn zu vide Formen einer Klasse nach einander folgen und zum Teil mu13 man das Material nehmen, wie es zu bekommen ist, daher wird dieses Prinzip mitiinter durchbroclien. Tor der Untersuchung jedes Objektes wird in einem kurzen Vortrag auf die wichtigen Momente aufmerksam gemacht und eiiie Anleitung fur die Untersuchung ge- geben. Dielenigen Formen, die einen Typus reprasentieren, werden dann ein- gehender behandelt, wahrend wieder andere nur in jener Richtung studiert tverden, in welcher sie ilbweichungeii vom Typus zeigen oder irgendwie inter- essant sind. Nach lLl6glichkeit werden rolche Tiere und Algen ausgewalilt , die auch in Lehrbiichern behandelt sind. In einem Arbeitstag werden je n:ich der Natur des Gegenstandes nur 2-3 Studienobjekte durchgenommen. 9.) Uber das Auftreten yon Polyphemus pedieulus in der ,,alten Donau" bei Wien. Jeder Kursteilnehmer wird angehalten, alles Gesehene zu skizzieren und Notizen zu machen. AuBerdem sollen Besprechungen und Demonstrationen des Tici- Statioiisnachrichten und Notizen. 485 bestandes in den Stationsaquarien dazu beitragen, daR sich die Kursteilnehmer eine moglichst gro5e Formenkenntnis aneignen. g g g Im AnschluB an die Arbeiten im Laboratorium werden dann auch noch gemeinsame Studienfahrten am Meere auf dem Stationsschiffe ,Adria" unter- nommen. Diese Exkursionen haben den Zweck, den Kursteilnehmern die Fischereimethoden zu zeigen und das reiche und niaiinigfaltige Leben im Meere und die Abhangigkeit der Lebensfornien von dem Milieu vorzufiihren. Die Verschiedenartigkeit der Facies von Triest und seiner angrenzenden Ge- biete bietet eine reiche Fulle der Belehrung. Aber nicht bloD in zoologischer und botaniscber, auch in geographischer, geologischer und liistorischer Hin- sicht ist das Gebiet interessant. Im ganzen werden 6-7 Tage auf solche Studienfahrten verwendet. Davon kommen 3- 4 Tage auf Tagesausfluge im Golfe von Triest, und zwar hat eine Fahrt das Lagunengebiet von Grado und die Dune Centenara, bemerkenswert durch einen Bestand echter Pinien (Pinus pinea), zum Ziel, eine zweite das Muschelsandgebiet bei Cap Salvore und das Stadtchen Pirano und eine dritte Fahrt macht die Kursteilnehmer mit der eigentumlichen Salinenfauna und Flora in den Salinen von Capodistria (Vorkommen von Artemia salina) bekannt. Je eine halbtagige Fischereifahrt betrifft die Fauna und Flora der Zosterawiesen und die Emmersionszone an der Felskuste zur Ebbezeit. Eine dreitagige Studienfahrt fiihrt endlich von Triest entlang der Westkuste von Istrien bis in den Quarnero, wobei unter- wegs interessante Punkte wie Parenzo, Canal di Leme, Rovigno, die Insel Brioni und andere beruhrt und in Pola zwei Nachtstationen genommen werden. Diese Exkursionen werden durch entsprechende Vortrage vorbereitet. Von Nutzen ware wohl ein auf diese Exkursionen bezugliches gedrucktes Pro- gramm beziehungsweise ein kleiner illustrierter Fuhrer und sol1 ein solcher auch noeh abgefaBt werden. g Die Feridkurse werden unentgeltlich abgehalten und in gleicher Weise werden die Arbeitsplatze unentgeltlich eingpraumt. Um KUI splatze sind Ge- suche, welche mit einer Befiirwortung von Seiten eines Institutvorstandes zu versehen sind, an das Kuratorium der k. k. zoologischen Station in Triest zu richten und dem Direktor dieser Anstalt einzusenden. Der Einreichungstermin endet fur die Osterkurse mit 1. Februar und fiir die Sommerkurse mit 15. Juni. Mit Riicksicht auf den beschrankten Raum muDte ein Numerus clausus mit 20 Kursteilnehmern fur den zoologischen und mit 8 Teilnehmern fur den bo- tanischen Kurs festgestellt werden. 9.) Uber das Auftreten yon Polyphemus pedieulus in der ,,alten Donau" bei Wien. Jeder Kursteilnehmer hat nach 5 6 der Hausordnung ein Mikroskop, eine Arbeitslupe, Messer, Schere und Pinzette selbst mitzubringen; auch empfiehlt sich die Mitnahme eines Lehrbuches der Zoologie, Kiickentals Zoologisches Praktikum und eventuell des ,,Leunis" Synopsis der Tierkunde, 3. Auflage. Es eriibrigt vielleicht noch, einige Bemerkungen uber Quartier , Ver- kostigung und Kleidung fur einen Aufenthalt in Triest anzuschliefien. Die (iaste der Triester Station wohnen fast alle in Privatzimniern, die per Monst um den Betrag von 25-50 K., je nach Lage, GroBe und Einrichtung in ge- niigender Anzahl zu haben sind. An der Anstalt findet sich ein Verzeichnis empfehlenswerter Wohnungen. Das Fruhstuck stellen gewohnlich die Wirts- leute bei, wiihrend das Mittag- und Abendessen in einem Restaurant mit italienischer Kuche (Trattoria) eingenommen wird. Es gibt aber auch Speise- Shtionsnachricliten uiid Notizen. 386 liduser wit Speisen nach franzosischer bezw. deutsclier Art zubereitet. Fur Mittag- und Abendessen lrann je nach den Anspruchcn 3-5 K. veranschlagt \\.erden. Was die Kleidung anbelangt, so versehe man sich im Fruliling mit, warmen wetterfesten Kleidern , denn der geruhinte sonnige Suden ist gerade uni diese Zeit trugerisch. Fur den Sommeraufenthalt sind Linnenanzuge zu enipfehlen. Da uin diese Zeit Gelegenheit zum Bade im Meere ist, so ware ;tuch noch die Mitnahnie der Badeausrustung zu erinnern, zu welcher auch eiii paar feste Badeschuhe gehoren. Letztere sind zum Schutze gegen das Eiritreten der Nadeln von Seeigeln und beim Gelien auf den scharfkantigen 17elsriffen anllDlich des Eadens bei den Studienfalrrten notig. g Wir gebcn nachfolgrnd ein Verzeichnis der Formen fur die Kurse, uiii den Tcilnehmerii schon vorher eine Vorbereitung zu ermoglichen: g g Zoologischer Kurs: I’olystomella, Acanthonietron, Sticholonclie, Cera- tillin, Tintinnen, Podophrya, Gregarinen, Sycon, Halisarca , Kieselschwamme, l-’odocoryne, Eudendrium, Tubularia, Campanulariden, Aglaophenia, Plumulrtria, ‘riartl,, Tima, Aequorea, Sarsia, Steenstrupia, Diphycs, Dicyemiden, Trichoplax, J’i1cmq Aurelia, Chrysaora, Scyphistoma, rerimthus, Edwardsialarve, Alcyo- 1iiuIii , Cydippe, Beroe, Thysanozoon, Leptoplana, Graffilla, Anaplodiuni. Syndesmus, Mullersche Turbellarielarve , Axine, Ttkrarhynchus , Tetrasteniina, J’ilidiuin, Ecliinorhynclius , Pedicellina , Loxosoma , Polygordiiislarve , Pol>-- gordius, Ayhrodite. Syllis, Nereis , Dinophilus , Eunice, Tomopteris, Capitella, Spirographis , Serpula., Pelagische Annelidenlarven , Myzostoina , Sipunculiis. Sil)unculaslarve, Bonellia , Bugula , Zoobothryum , Actinotrocha, Evadiie. p ~ d o ~ i , Artemia, Corycaeus, Parasit. Copepoden, Lepas, Sacculina. Cirripedien- Iarven , W a g . Krebslarven , Nebalia , Mysis , Cumaceen, Syuilla Palaemon. Iirabben, Apseudes, Caprella , Pycnogoniden , Chiton, Nucula, Pholas, Teredo, Solei], Scrobicularia, Venus, Carcliuin, Arca, I’eetunculus. 9.) Uber das Auftreten yon Polyphemus pedieulus in der ,,alten Donau" bei Wien. schatzten Revue enthalt einige Ungenauig- U keiten und Differenzen gegenuber meinen Darstellungen, weshalb ich dieselben richtigstellen will. 11.) Zu dem Referat Nr* 24 auf s. 107 dieses Bandes. Von Dr. H. v. Hofer. U 11.) Zu dem Referat Nr* 24 auf s. 107 dieses Bandes. DaD icb zwei Typen des Bodenwassers, nanilich Grund- und Felswasser, aufstellte, hat einen fur die Praxis scliwerwiegenden Grund. Die ErschlieDung dieser beiden Wassertypen erfolgt &US geologischen Grunden in der Regel ganz verschieden: das Grundwasser wird mittels Schachte , das Felswasser, wie z. B. das Spaltenwasser, nieist mittels Stollen und Strecken erschlossen. Xucli die technischen Vorarbeiten , sowie die Bestimmung der entsprechrnden Schutzfelder sind aus geologischen Griinden verschieden. DaD zwischen Grund- und Felswassei &uch Ubergange vorkomnien konnen , ist doch kein Grund gegen diese beiden meist gut geschiedenen Typen, denn in allen drei Naturreichen finclet inaii verschiedene Spezies, Familien usw. durch Obergange verbunden. Die Spezies dcr hcxagonalen Karbonate Kalzit, Magnesit , Siderit u. a. m. anerkennt jeder Mineraloge als zu recht bestehend, ja er erhebt ge- wisse Uberpangsglieder, \vie z. E. Dolornit, Ankerit zur selbstandigen Art, ob- zwar es nebst diesen noch stetigc ZTbergangsreihen gibt. Auf Grund meiner langjahrigeri Erfahrungen und Studien bin ich gezwungen , die beiden Haupt- typen des Bodenwassers auch fernerhin aufrecht zu halten, um so mehr, da ltrin ausreichender Gegenbeweis vorliegt. Wenn Herr W. Halbf aR bedauert, in meinein Buche keine ,eingehende Darlegung , auf welche Weise das Bodenwasser durch Infiltration gespeist wird", gefunden zu haben, so ist mir dieses ,,Leider" unverstandlich, de icli doch auf 29 Seiten alle Faktoren der Infiltration erlautere. Es ist nicht richtig, daO icli anuehme, die Speisung der Seen erfolgc ,. ausnahmsweise" durch Grundwasser, - ini Gegenteil; denn aiif S. 69 erlautere ich diese Speisung von u n t e n und sage dann: ,,Es sei bemerkt, daD ein See auch seitlich gespeist werden kann, wie dies bereits bei Teichen erwahnt wurde. Hierher gehoren die ostpreuDischen Grundmoranenseen, deren minster Typus nach G. Bra1111 der Olrullsee ist." Es war deshalb auch der Hinweis meines Kritikers auf ,,die Seen alter Gletscherboden", die doch Grund- modnen sind, mindestens uberflussig. DaR ich inich in den Streit zwischen Katzer und Grund uber das Karstwasser ,,neutralY verhalte, ist eine subjektive Auffassung meines Herrn Kritikers , die ich ablehne; denn nicht neutral, sondern o b j ektiv bespreclic ich diese Frage auf Grund eigener Studien in Karstgebieten. 9.) Uber das Auftreten yon Polyphemus pedieulus in der ,,alten Donau" bei Wien. Mytilus, Lithodomus, l’iIltia, Anoniia, Pecten, Lima, Ostrea, Denta’liuin, Hnliotis, Fissurella, Patellit. \-errnetus, Natica , Cassidaria, Murex , Littorina, Cydostoma, Actaeon, Acera.. l’lliliiie, Aplysia, Pleurobranchus, Doris, Elysia,, Tethis, Spurilla, Creseis, Ele- ,lone, Sepia, Sepiola, Loligo, Holotliuria, Synapta, Astropecten, Asterias, Eclii- naster, Echinus , Spl~~erechiniis , Schizaster, Kunstliche Befruchtung, von See- igeltiern, Ophioderma , Antedon, Pelag. Echinodermenlarven, Balanoglossus . TOrnaiia,, Spdella, Sagitta, Sagittaentwicklung, Ciona, kunstliche Befruchtung ,\,:r Eies von Ciona, Ascidieiilarven, Styela, Ascidia, Synascidien, Copelatcn , SiL1pa, Scyllium, Sel:r cliier-Embryonen, Torpedo, Acipenser, Div. Knochenfische, I(ft11stl. Befruchtung der Eier von Knochenfischen; Jungfische. B o t s n i s c h e r Kurs: Chloropliycene : Ulva lactucn, Enteromorpha intestinalis, Chaeto- Illorpha linurn, Codium bursa, Codiuni tomentosum ~ Halimeda tuna., Udotea desfontainii, Andyoinene stellata, Bryopsis plumosa, Valonia utricularis, Dasy- cltLdus claraformis , Acetabularia meditwranra. Phacphycea,e: Cystoseira bttrbata, Fucus virsoides, Saragassum linifolium, Ectocarpus sp., Cutleria multi- fit]%, Aglaozonin reptans, Cladostephus verticillatus, Asperococcus compressus, p113;llitis Fascia , Scytosiphon lomentnrius , Dictyota dichotoma, , Dictyopteris I,Olypdioides, Padina pavonin, Zanardinia col1,zris. Rho d o p h y ceae: Bangia fi1sco-put purea , Porphyrs leucosticta, Phyllophora nervosa , Gracilaria com- ~)yessa, Gracilaria confcrvoides, Antitliamnion plumula, Ceramium sp. , Poly- sil)]ionia. sp., Laurentia paniculata, Chylocladia lraliformis, Nitopliyllum punctn- Statioiisnachricliten und Notizen. 487 tuni, Dasya elegans, Batrachospermuni attennatum, Nemalion lubricum, Geli- dium capillaceum, Corallina officinalis, Wrangelia penicillata , Polysiphonia rerticillata, Chrysimenia uvaria, Melobesia spec. Aucli wird den Teilnehmern marines Plankton zur Untersuchung gegeben , so daD ihnen die wichtigsten pelagischen Bacillariaceen- und Peridineenarten bekannt werden. Das kritische Referat des Herrn W. H a l b - f a 6 (Jena) meines kleinen Buches ,,Grund- w a s s e r u n d Quellen" in Ihrer sehr ge- Von Dr. H. v. Hofer. schatzten Revue enthalt einige Ungenauig- U keiten und Differenzen gegenuber meinen Darstellungen, weshalb ich dieselben richtigstellen will. 11.) Zu dem Referat Nr* 24 auf s. 107 dieses Bandes. Das kritische Referat des Herrn W. H a l b - f a 6 (Jena) meines kleinen Buches ,,Grund- w a s s e r u n d Quellen" in Ihrer sehr ge- schatzten Revue enthalt einige Ungenauig- U keiten und Differenzen gegenuber meinen ben richtigstellen will. Das kritische Referat des Herrn W. H a l b - f a 6 (Jena) meines kleinen Buches ,,Grund- w a s s e r u n d Quellen" in Ihrer sehr ge- Von Dr. H. v. Hofer. 9.) Uber das Auftreten yon Polyphemus pedieulus in der ,,alten Donau" bei Wien. In diesem Streite bringt jeder Autor eine oder mehrere Wahrheiten, die der Gerner zu wen& benchtet. Auf Grund meiner mehrifihrigen Erfahrungen im Karst stellte icli Stationsnachriahteii und Notizen. 488 mich in der Hauptsache auf Katz ers Seite , ohne jedocli einzelne Tatsachen zu verleugnen , welche zugunsten Gru n d s sprechen, der vielleicht manches anders aufgefaBt haben wurde, wenn er eben nicht mit HalbfaB das Hohlen- wasser als identisch mit Grundwasser angenommen hatte. g DaB ich mich in der Frage des juvenilen Wassers ,neutralK verhielt, gebe ich gern zu, und ist erklarlich, da diese Frage noch nicht endgiltig ent- schieden ist, wie dies Herr W. HalbfaD s, a. 0. selbst gesteht. DaB icli gegen das juvenile Wasser ernste Bedenken habe,' geht aus S. 120 u. 121 meines Buches hervor. Wenn mir auch Brun Y Recherches sur l'exhalation volcanique nicht vorlag , so liabe ich eine seiner fruheren Arbeiten benutzt und auf S. 108 auch zitiert, die, was das juvenile Wasser anbelangt, in1 Wesentlichen dasselbe angibt, wie die Recherches. g , Es ist ferner nicht zutreffend, daD ich bloD sage, daD nebst der juve- nilen Herkunft der Kohlens%ure diese ,,bier und da auch von anderwarts stammen kann'. Nach dieseni Satze heiDt es auf S. 114 meines Buches: Sie kann gebildet werden durch Zersetzung von Karbonatgesteinen, z. B. Kalk, entweder durch Hitze oder Siureeinwirkung , durch den KohlungsprozeD und durch Zersetzung roil anderen Organolithen, von Eisenkarbonatgesteinen. Auch durch Einwirkung von Karbonatlosungen auf Silikate kann Kohlensiiure frei werden'. Nun, das durfte wohl jene ,,prazisere Antwort" sein, welche Herr W. HalbfaD in seiner Kritik ghzlich veiscliweigt und die er von einem Fachmann gewiinscht hat. Icli ware ihm sehr dankbsr, wenn er mir nebst den genannten noch eine anclere Herkunft der Kohlensaure in den Sauerlingen angegeben hatte. g g Herr W. H a l bfaB ubersieht, daD icli nic;ht von Mineralisatoren, sondern von der Mineralisation des Wtssers spreche. Er vermiDt auch hier und da im einzelnen ein Eingehen auf die wichtigste neuere Literatur. Ich wollte in meinem L eitf a d e n (s. Vorwort) die Leser nicht mit Literaturzitaten uber- schwemmen; doch glaube ich etwas Wichtiges oder Wesentliches in der Sache dennoch nicht ubersehen zu haben, wie mich dies der Vergleich meines Leit- fadens mit dem spater erschienenen Tlehrbuch Keilhacks uberzeugte. Statt tler Zitate biete ich dem Leser vielfach eigene Erfahrungen. ) Guido Schneider, Der Obcrsee bei Reval, Arch. f. Biontologie. Bd. 11, 1908, Seite 94-97. 2, Guido Schneider, VorlBufjp Mitt. uber dieuntersuchung des Sees Wirzjejerw im Sommer 1911 (russisch), Fitzungsbr. d. Dorpater Naturf. ges. XX. Srite 25-36 und Beitrag zur Vermifauna des Wirzjenv, Korrespondenzbl. des Naturforscher-Vereins zu Riga LVI, 1913, Seite 29-34. 9.) Uber das Auftreten yon Polyphemus pedieulus in der ,,alten Donau" bei Wien. Wenn dies nicht der Fall sein sollte, so konnte ja durch Herausgabe je eines Supplementbandchens der zoologi- schen und botanischen Serie (bei der zu einem ganzeu Bgndchen allerdings bislang kaum genugend Material vorhanden sein wird) das ganze Werk dem augenblicklichen Stand unserer Kenntnis entsprechend erganzt werden. Hierzu mochten die vorstehenden Zeilen , falls der Gedanke von den Herausgebern nicht bereits in Erwagung gezogen worden sein sollte, die Anregung geben. p Selbst im botanischen Teil durfte noch da und dort eine Lucke auszu- fullen sein. So habe ich z. B. im Peridineen-Bandchen vergeblich die bei Kufstein gefundenen Arten Peridinium cunningtoni und westii gesucht, und auch die von mir bei meinen Achensee-Arbeiten gefundenen neuen odrr fur das Gebiet neum Diatomeen und Peridineen, die von Lemmermann und H u s t e d t behandelt wurden, in dam betreffenden Bhdchen nicht aufgefunden. , g So mag also durch Erganzung ubersehener Arten, durch Ausdehiiung des Untersuchungsgebietes auf ganz Mitteleuropa bei einzelnen Gruppen, denen engere Grenzen gezogen waren, ganz hauptsachlich aber durch Neuentdeckungen ein derartigrr Spezieszuwachs zu verzeichnen sein, daB die Herausgabe einer zweiten Auflage sehr wunschenswert erschiene. Ich kenne die Verhaltnisse zu wenig, um beutteilen zu konnen, ob das Erscheinen einer neuen Auflage dieses in seiner ersten Auflage noch ini Erscheinen begriffenen Werkes in naiohster Zeit zu erwarten steht. Wenn dies nicht der Fall sein sollte, so konnte ja durch Herausgabe je eines Supplementbandchens der zoologi- schen und botanischen Serie (bei der zu einem ganzeu Bgndchen allerdings bislang kaum genugend Material vorhanden sein wird) das ganze Werk dem augenblicklichen Stand unserer Kenntnis entsprechend erganzt werden. Hierzu mochten die vorstehenden Zeilen , falls der Gedanke von den Herausgebern nicht bereits in Erwagung gezogen worden sein sollte, die Anregung geben. Schon im Jake 1904 bei Gelegenheit der Erforschung des Obersees l) bei Reval (Provinz Estland in RuBland) fand ich freilrbende Nematoden bisweilen sehr zahfreich neben Tubifex, Larven undPuppen von Chironomus und Oera- t o p og o n , Trichopt errnlarven und Bodencladoceren , vermischt mit Schlamm und Sand, als Nahrung im Darm junger Brachsen (Abramis b r a m a L.). Von 43 untersuchten Brachsen hatten 18 mehr oder weniger zahlreiche Exeni- plare ( 9 und 6) von Dorylaimus s t a g n a l i s Duj. gefrrssen. Bei einigen dieser Pische bwtand die Nahrung sogar vorherrschend und fast ausschlieBlich aus den genannten Nematoden. 9.) Uber das Auftreten yon Polyphemus pedieulus in der ,,alten Donau" bei Wien. Ich muD deshalb die Kritik meines Buches .Grundwasser und Quellen" von Herrn W. Halbfafi im Einzelnen und in dem Ganzen nls vollends un- zutreffend ablehnen. 12.) Eine Anregung beziiglich der von Brauer und Pascher heraus. gegebenen Fauna und Flora. I Von Dr. v. Brehni (Eger). Das Ersclieintxi der bekanntensamm- lung hat unverkennbar eine wesent- liche Forderung der Erforschung der mittelenropaischen Gewasser zur Folge gehabt. Man lrann formliclr statistisch nachweisen, wie die Anf- findung neuer odor wenigstens fur das Gebiet neuer Arten zugenommen hat. Da leider speziell in der zoologischen Abteilung bei der Auswahl der aufzu- nehmenden Arten vielfach die politischen Grenzen des deutschen Reiches als maBgebend angesehen wurden, ergab sich ofters ein Ausfall von Arten, die tler mitteleuropiiischen Fauna angehoren und inzwischen bereits in Deutsch- 12.) Eine Anregung beziiglich der von Brauer und Pascher heraus. gegebenen Fauna und Flora. I Von Dr. v. Brehni (Eger). statistisch nachweisen, wie die Anf- findung neuer odor wenigstens fur das Gebiet neuer Arten zugenommen hat. Da leider speziell in der zoologischen Abteilung bei der Auswahl der aufzu- nehmenden Arten vielfach die politischen Grenzen des deutschen Reiches als maBgebend angesehen wurden, ergab sich ofters ein Ausfall von Arten, die tler mitteleuropiiischen Fauna angehoren und inzwischen bereits in Deutsch- Stationsnachrichten und Notizen. 489 land aufgefunden worden sind (z. B. Diaptomus amblyodon) oder voraussicht- lich noch aufgefunden werden durften. Ich habe kurzlich gezeigt, daB die Harpaktiziden einer wesentlichen Erganzung bediirfen, noch mehr gilt dies von den Hydrachniden, wie aus einer Zusammenstellung von Viet s hervorgeht. oder par von den Nematoden. p Selbst im botanischen Teil durfte noch da und dort eine Lucke auszu- fullen sein. So habe ich z. B. im Peridineen-Bandchen vergeblich die bei Kufstein gefundenen Arten Peridinium cunningtoni und westii gesucht, und auch die von mir bei meinen Achensee-Arbeiten gefundenen neuen odrr fur das Gebiet neum Diatomeen und Peridineen, die von Lemmermann und H u s t e d t behandelt wurden, in dam betreffenden Bhdchen nicht aufgefunden. So mag also durch Erganzung ubersehener Arten, durch Ausdehiiung des Untersuchungsgebietes auf ganz Mitteleuropa bei einzelnen Gruppen, denen engere Grenzen gezogen waren, ganz hauptsachlich aber durch Neuentdeckungen ein derartigrr Spezieszuwachs zu verzeichnen sein, daB die Herausgabe einer zweiten Auflage sehr wunschenswert erschiene. Ich kenne die Verhaltnisse zu wenig, um beutteilen zu konnen, ob das Erscheinen einer neuen Auflage dieses in seiner ersten Auflage noch ini Erscheinen begriffenen Werkes in naiohster Zeit zu erwarten steht. , 2, Guido Schneider, VorlBufjp Mitt. uber dieuntersuchung des Sees Wirzjejerw im Sommer 1911 (russisch), Fitzungsbr. d. Dorpater Naturf. ges. XX. Srite 25-36 und Beitrag zur Vermifauna des Wirzjenv, Korrespondenzbl. des Naturforscher-Vereins zu Riga LVI, 1913, Seite 29-34. ) Guido Schneider, Der Obcrsee bei Reval, Arch. f. Biontologie. Bd. 11, 1908, Seite 94-97. G id S h id i di h S i j j 9.) Uber das Auftreten yon Polyphemus pedieulus in der ,,alten Donau" bei Wien. Die Totallange der Nematoden freosenden Altersstufe der Brachsen im Obersee betrug nach meinen Messungen 191 bis 235 mm. Bei Untersuchung des etwa 125 km sudostlich vom Obersee in der Provinz Livland gelegenen Sees Wirzjerw'), der, obgleieh an Areal fast 30mal Stationsnachrichten und Notizen. -190 gr0Der als der Obersee, diesem hinsichtli(.h der sehr geringeii Tiefe, der Boden- beschaffenheit und der Fauna rscht ahnlich ist. finde ich wieder dieselbe Ne- matodenart, D o i y l a i m u s s t a g n a l i s Dud. in verschiedenen Stadien der Vei- dauung im Darm 97 bis 263 mm langer Braclisen. Die Menge der verzehrten Neniatoderi ist auch hier bisweilen eine sehr gro0e. Sie konnten buodel- und klumpenweis &us dem Darrninhalt isoliert werden. Von 48 utitersuchtari Brachsen hattrn aber nur 11 Dorylaimus (viele 8 $! und weriige gf$) ge- fressen. hu5er Abraniis b r a m a vertilgen gelegentlich auch Alburiius lucidus L., Gobio gobio L. und Acerina c e r n n a L. im Wirzjerw Dory- laimus s t a g n a l i s , da dieser Nematode offenbar stellenweise in groDen Massen vorkommt. Von 23 Exemplaren von Acerina r e r u n a hatte ein R9 mm langes Exemplar, von 12 Gobio gobio ein 117 mm langes und von 21 Alburnus l u c i d u s hatten zwei Exemplare von 42 und 44 mm Lange Scinatoden dieser Art gefrrssen. g DaD Fische, die sich uberhaupt vorwiegencl von Bodentieren ndhren, ditb -ie mit Schlamm und Sand verm~scht verschlingen, auch so grol3e Nematoden. \vie Dory1,~imus stagdalis, nicht verschmahen, kaun weiter nicht aufbllen, rind vermutlich wird inan auch in aiideren Fisohen des Wirzjerw und anderer Seen, die Schlamm, Algen oder Bodentiere fressen , gelegentlich freilebendc Nematoden in der Nahrung antreffan, obgleicli es mir einstweilen noch nicht gelang, im Darrninhalt der im Wirzjerw rorkonrmenden Arten Blicca bjorkna L., Leuciscus r u t i l u s L., L. i d u s L., L. leuciscus L. und L. e r y t h r o - p t h a l m u s L. Nematodrn zu finden. Vie1 merkwiirdiger ist dagegen das Vor- liommen eines anderen freilebenden Nematoden in der Nahrung der Plankton tresseriden Zwergmarane (Coregonus a l b u l a L.) des Wirzjerw. 9.) Uber das Auftreten yon Polyphemus pedieulus in der ,,alten Donau" bei Wien. Die Art dieses Xematoden liabe ich nicht genau feststellen konnen, da nur Wribchen ge- funden wurden und auch dieye meist von tler Verdauung zu stark ange- eriffen wareii. Jedenfalls handelt es sioh uni eine Art der Ciattung Trilobus, vielleicht T. gracilis Bast, Untersuchungen des Magen- nnd Darminhaltes von 70 Esemphren cler Zwergmarilne aus dem See Wirzjerw wurden von iiiir in den Monaten Juni, Juli, August, September und November der letzten drei -Jahre ausgefuhrt. Jedoch nur a m 26. August 1912 falid icli den Nematoden iintl zwar massenhaft Weibchen (kein einziges Mannchen) im Darm von 8 iintrr 15 an diesein Tage untersucliten Exemplaren der Zwergmarane , neben L e p t o d o ra Kin (1 t i , C h y d o ru s s p h ;L eri c u s , B o s m in a core g o n i t y p i D a, Cyclop i, Chioococcaceen , 1 Insektenimago und 1 Aoaride in wechselnder Mcnge. Sand und Schlaiiim wurde im Dariii dieser Fische nicht gefunden, die folglich die rematoden oberhalh des Bodens in der Planktonregion ge- tmgen hatten. L)iesei Umstsnd und zwei andere, nimlich da5 nur weiblichc Xematoden gefressen wurden, und da5 diese nur an einem Tage, dem 26. August, inassenhaft oberhalb der Bodenregion sich aufhielten, flihrt micli mr Annahme, daR die reifen Weihchen der vorliegenden Art von Trilo b u s cich im Spatsommer oder Herbst in die Regionen des Limnoylanktons erheben, u m frei schwimmend ihre Eier auszustoDen , welchen dadurch weitwe Ver- hreitung zuteil mird. Stationsnachrichten und Notimi. 49 I Wir inochten nicht versaumen, auf diese Sammlung aufmerksam zu machen, ehe. die Subskriptionsliste - it lit dem Er- scheinen des Werks- abgeschlossen wird. auf Helgoland. Es sind, soweit ich aus den vorliegenden Probetafeln ersehen habe, wohl unstrei- tig die schonsten und lebensvollsten Bilder von Seetieren, welche bisher ge- schaffen worden sind. Sie entstammen samtlich dem Aquarium der Helgo - lander Biologischen S t a t i o n , wo sie vofi dem durch seine Nordseebildw bekannten Photographen Schen s k y aufgenommen wurden, unter Beteiligung der wissenschaftlichen Beaniten der Bnstalt. Auf diese Weise ist es zu erklaren. da5 die Tafeln ebenso sehr durch kiinstlerischen Reiz als durch biologische Anschaulichkeit ausgezeichnet sind. Verantwortlicli fur die Sohriftleitmig,: Prof. R. W o l t e r e ck , Bautzscli-Leipzig. Veriag voii Dr. W e r n e r 1<1inlchsrdt, J,eipmg. Driicli von J i i l i u s X l i n k h a r d t , Leipeig. 9.) Uber das Auftreten yon Polyphemus pedieulus in der ,,alten Donau" bei Wien. g darauf hinlenlren, da13 die Postadresse der Station nicht Aneboda sondern Lamlinlt ist, unter welcher Adresse deni- gemaD alle fur die Station bestimnite Post zu senden ist. Bei der SuDwasserbiologischen Station werden hauptsachlich Fragen teich- biologischen Interesses (Nutritions- und allgemeine Produktionsbiologie ; Be- deutung des Nannoplanktons im StoffhaushaIt der Teichgewasser u.;w.) sowie betreffs der Bodenbiologie der Seen untersucht. Assistent fur Hydrobiologie ist Herr E i n a r Nauniann, Fil. Kand. drr Universitat Lund , der wahrend einiger Monate des Jahres bei der Station diese Untersuchungen im Auftrag der Fischereivereinigung fur Sudschwetlen (welche die Station leitet) ausfuhrt. Der Redakteur wird im Marz und April iii und bittet Manuskripte nur n a c h vorher- 17.) Redaktionelle Notiz. P o s it a n o (Prov. di Salerno , Italien) hein gehender a n f r a g e cinsenden zu wollen. 9.) Uber das Auftreten yon Polyphemus pedieulus in der ,,alten Donau" bei Wien. Es sind lauter charakteristische Momente aus dem reichen Leben der Helgolander Riffe und Sandgriinde zur Darstellung gelangt ; dazu kommen einige prachtige Anfnahmen von pelagischen Organismen, insbesondere Quallen. Auch die Algenflora, die bei Helgoland besonders reiz- voll ist, und die, soviel ich wei5, in keinem Aquarium der Welt so gut ver- treten ist, wie in dem der Helgolander Station, ist nicht vernachliiwigt worden. - Im ganzen werden 30 Tafeln in GroDquart (Photogravure) ausgegeben, dercii Subskriptionspreis nur 24 Mark betragt. W. auf Helgoland. lies brarin Heft 1, SeiteS, Zeile 10von oben: anstatt ,,brau" Seite9, ,, 6 ,, ,, : ,, ,,Pausen" ,, Touren Berichtigung. .l 7 >, ,, . ,, ,,planktonischen" lics p 1 an k t o n r e i c 11 e n. Tafel 111: mstatt ,,Staufalleteich" lies Stenfilleteicb. Heft 213, Seite 246, Zeile 12 von unten: anstatt ,,Bugust" lies April Seite 247, ,, 3 ,, oben: ,, ,,April" ,, August Seite 249, ,, 3 ,, unten: ,, ,,mir" ,, uns. lies brarin Heft 1, SeiteS, Zeile 10von oben: anstatt ,,brau" Seite9, ,, 6 ,, ,, : ,, ,,Pausen" ,, Touren .l 7 >, ,, . ,, ,,planktonischen" lics p 1 an k t o n r e i c 11 e n. Berichtigung. p 1 an k Tafel 111: mstatt ,,Staufalleteich" lies Stenfilleteicb. p 1 an k t Tafel 111: mstatt ,,Staufalleteich" lies Stenfilleteicb. Heft 213 Seite 246 Zeile 12 unten: anstatt Bugust" lies A p Tafel 111: mstatt ,,Staufalleteich" lies Stenfilleteicb. afel 111: mstatt ,,Staufalleteich lies Stenfilleteicb. , Seite 246, Zeile 12 von unten: anstatt ,,Bugust" lies April Seite 247, ,, 3 ,, oben: ,, ,,April" ,, August Seite 249, ,, 3 ,, unten: ,, ,,mir" ,, uns. D i e SU D w as s er bi o lo gi s che un (1 Fischereiversuchsstation Ane- b 0 d a in sidschweden u,ill hiernlit die Aufmerksamkeit der Fachgenossen darauf hinlenlren, da13 die Postadresse der Station nicht Aneboda sondern Lamlinlt ist, unter welcher Adresse deni- gemaD alle fur die Station bestimnite Post zu senden ist. b 0 d a in sidschweden u,ill hiernlit die Aufmerksamkeit der Fachgenossen darauf hinlenlren, da13 die Postadresse der Station nicht Aneboda sondern Lamlinlt ist, unter welcher Adresse deni- gemaD alle fur die Station bestimnite Post zu senden ist.
https://openalex.org/W2947161232	https://europepmc.org/articles/pmc6495649?pdf=render	English	null	A combination of omega-3 and plant sterols regulate glucose and lipid metabolism in individuals with impaired glucose regulation: a randomized and controlled clinical trial	Lipids in health and disease	2,019	cc-by	6,948	Wang et al. Lipids in Health and Disease (2019) 18:106 https://doi.org/10.1186/s12944-019-1048-x Wang et al. Lipids in Health and Disease (2019) 18:106 https://doi.org/10.1186/s12944-019-1048-x (2019) 18:106 A combination of omega-3 and plant sterols regulate glucose and lipid metabolism in individuals with impaired glucose regulation: a randomized and controlled clinical trial Ji-fang Wang1†, Hai-ming Zhang2†, Yan-yan Li1, Song Xia3, Yin Wei1, Ling Yang1, Dong Wang1, Jing-jing Ye1, Hao-xiang Li1, Jing Yuan1 and Rui-rong Pan3* Abstract Background: Lipid metabolism imbalance has been recognized as one of the major drivers of impaired glucose metabolism in the context of type 2 diabetes mellitus (T2DM), the rates of which are steadily increasing worldwide. Impaired glucose regulation (IGR) plays a vital role in the prevention and treatment of T2DM. The goal of this study was to further clarify whether the combination of plant sterols (PS) and omega-3 fatty acids yields any synergistic effect that enhances the prevention and treatment of IGR. Methods: A total of 200 participants were randomized to receive PS and omega-3 fatty acids (n = 50), PS alone (n = 50), omega-3 fatty acids alone (n = 50), or placebo soy bean powder plus placebo capsules (n = 50) for 12 weeks. Patient characteristics including body composition, blood pressure, glucose metabolism (Fasting plasma glucose (FPG), fasting insulin (FINS), glycosylated hemoglobin (HbA1c), Homeostasis Model Assessment of Insulin Resistance (HOMA-IR)), lipid metabolism (TG, TC, HDL-C, LDL-C) and inflammatory factors (Hs-CRP, IL-6) were all monitored in these IGR individuals. Results: Compared to the placebo group, the group receiving the combined intervention exhibited significantly decreased TG, HDL-C, FBG, HOMA-IR and HbA1c. Omega-3 fatty acids alone were associated with significant reductions in waistline, TG, FBG, HOMA-IR and Hs-CRP. PS alone was only associated with decreased TG and Hs-CRP. No interventions produced significant changes in body weight, BMI, blood pressure, FINS, body fat percentage, visceral fat rating, TC, LDL-C or IL-6. Conclusions: In summary, this study has demonstrated for the first time that PS, omega-3 fatty acids or the combination thereof significantly improved inflammation, insulin resistance, as well as glucose and lipid metabolism in IGR individuals. These findings may provide a scientific basis for the development of nutritional products incorporating PS and omega-3 fatty acids, and also for the development of nutritional supplement strategies aimed at preventing the development of disease in the IGR population. Keywords: Plant sterols, Omega-3 fatty acids, Impaired glucose regulation, Factorial design * Correspondence: panrr_med@126.com †Ji-fang Wang and Hai-ming Zhang contributed equally to this work. 3Department of Clinical Nutrition, Affiliated Hospital of Jiangsu University, No. 438 Jiefang Road, Zhenjiang District, Jiangsu 212000, China Full list of author information is available at the end of the article * Correspondence: panrr_med@126.com †Ji-fang Wang and Hai-ming Zhang contributed equally to this work. 3Department of Clinical Nutrition, Affiliated Hospital of Jiangsu University, No. 438 Jiefang Road, Zhenjiang District, Jiangsu 212000, China Full list of author information is available at the end of the article Introduction As many societies are undergoing rapid economic devel- opment, changes in diet, and transitioning to a sedentary lifestyle, rates of type 2 diabetes mellitus (T2DM) are gradually increasing and as such the disease has become a significant public health concern worldwide. The Inter- national Diabetes Federation (IDF) has predicted that diabetes incidence will rise from 366 million in 2011 to 552 million in 2030 [1]. In Asia, the prevalence of dia- betes is expected to further increase over the next 20 years. According to a 2010 survey conducted by profes- sor Ning Guang of Ruijin Hospital Shanghai Jiaotong Medical School, the prevalence of T2DM is as high as 11.6% [2]. In addition, there is ample evidence from epidemio- logical studies suggesting that chronic inflammation plays a substantial role in the development and progres- sion of insulin resistance, which is defined as the homeostasis model assessment of insulin resistance (HOMA-IR) [7, 8]. As a sensitive index of chronic sub- clinical inflammation, high-sensitivity C-reactive protein (Hs-CRP) may predict the development of T2DM [9]. ( ) y p p [ ] Many studies have shown that plant sterols and omega-3 fatty acids are able to regulate lipid metabolism and inflammation [10]. PS, which are not synthesized by the human body, are minimally absorbed by the gut [11] and their structures are very similar to those of choles- terol [12]. Previous studies has found that plant sterols decreased cholesterol and LDL-C. Omega-3 fatty acids decreased VLDL synthesis, while they simultaneously in- crease lipoprotein lipase expression and fatty acid oxida- tion [13]. In addition, omega-3 fatty acids have been shown inhibit inflammatory reactions [14, 15]. We therefore speculated that plant sterols and omega-3 fatty acids together might act to improve T2DM or IGR via reducing lipids and inflammatory factors. As this has not previously been studied, we designed a randomized, double-blind, placebo-controlled, 2 × 2 factorial design trial to focus on roles of PS, omega-3 fatty acids, and the combination thereof in IGR individuals, as well as to fur- ther determine whether PS and omega-3 fatty acids have synergistic activity in this context. This study will ex- plore the theoretical scientific foundation for the treat- ment of IGR and T2DM via dietary supplementation. Introduction As known as impaired glucose regulation (IGR), ab- normal glucose metabolism can be further broken down into three categories, including impaired fasting glucose (IFG), impaired glucose tolerance (IGT), and IFG com- plicated by IGT (IFG/IGT). IGR belongs to an inter- mediate state between normal glucose metabolism (NGT) and T2DM, a state commonly referred to as pre-diabetes. Epidemiological data have shown that IGR patients exhibit multiple risk factors for coronary artery disease, such as insulin resistance (IR), central obesity, hypertension, and high triglyceride (TG) levels [2]. In addition, a long-term prospective clinical study with a large sample size has found that the risk of death from cardiovascular disease or coronary heart disease in IGR patients is about two times greater than in those with normal glucose tolerance, and the overall risk of death is one point five times higher than in those with normal glucose tolerance [3]. It has been clearly shown that IGR is highly reversible, and it is possible to maintain an IGR state or even reverse diabetes in some individuals. IGR and interventions to remediate this state are therefore of major scientific interest, with positive intervention stud- ies being performed in those with IGR generally being carried out to achieve decreased blood glucose, im- proved glucose tolerance, and to ultimately prevent dia- betes progression. 1) Placebo group: placebo soybean powder (placebo 1) + placebo capsules (placebo 2) © 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. Page 2 of 9 Wang et al. Lipids in Health and Disease (2019) 18:106 Page 2 of 9 Wang et al. Lipids in Health and Disease (2019) 18:106 as decreased levels of high-density lipoprotein choles- terol (HDL-C) [6]. Materials and methods Study design This clinical trial was a double-blind, randomized, placebo-controlled study, performed in the affiliated hospital of Jiangsu University from October 2016 to June 2017. The trial has already got approval by the Ethics Committee of the affiliated hospital of Jiangsu University (JSU2016066), and conducted at the Clinical Department of Endocrinology and Metabolism, Medical University of China. This study was registered in the World Health Organization International Clinical Trials Registry Plat- form (No. ChiCTR-IOR-17013282). It has been confirmed that patients that have reached the IGR stage already exhibit insulin resistance and insu- lin secretion defects. Clinical studies strongly suggest that abnormal lipid metabolism is the root cause of glu- cose metabolism disturbance in T2DM [4]. For example, lipometabolic disturbances induce insulin resistance in peripheral tissues, cellular dysfunction, apoptosis, and necrosis, thus contributing to the progression of dia- betes. Waistline size and blood lipid content were both risk factors for abnormal glycometabolic status [5]. A study of serum lipids in rural populations with diabetes and pre-diabetes in Chengdu, China, has suggested that participants with IGR have increased levels of low- density lipoprotein cholesterol (LDL-C) and TG, as well This study was a 2 × 2 factorial design in which sub- jects were to consume the intervention foods daily over a period of 12 weeks. Of 134 participants, 69 were women (51%), and the range of age was 51–65 years. Participants were randomly assigned to the test groups as follows: Wang et al. Lipids in Health and Disease (2019) 18:106 Page 3 of 9 Page 3 of 9 Page 3 of 9 2) Omega-3 fatty acids group: placebo 1 + capsules of 2 g fish oil (bioactive components are 1000 mg EPA and 400 mg DHA) 3) PS group: daily flour with 1.7 g plant sterols + placebo 2 4) PS plus omega-3 fatty acids group: daily flour with 1.7 g plant sterols + capsules of 2 g fish oil (1000 mg EPA + 400 mg DHA) 2) Omega-3 fatty acids group: placebo 1 + capsules of 2 g fish oil (bioactive components are 1000 mg EPA and 400 mg DHA) 2) Omega-3 fatty acids group: placebo 1 + capsules of 2 g fish oil (bioactive components are 1000 mg EPA and 400 mg DHA) phase. Participants were measured both at baseline and at the 12 week endpoint for anthropometric indices in- cluding height, weight, waist circumference, systolic and diastolic blood pressure. Statistical analyses Statistical analyses included all patients who completed the 3 week trial period and from whom appropriate ma- terials were obtained. Data are presented as means ± SD. To examine differences in baseline characteristics between groups, we used the analysis of variance (ANOVA) test for differences in means for continuous data and the chi-square test for differences in proportions for categor- ical variables. A two-factor ANOVA was used to test the main effects of dietary supplementation with plant sterols, omega-3 fatty acids, and interactions between plant sterols and omega-3 fatty acids. Statistical analyses were con- ducted using the Statistical Package for the Social Sciences (SPSS) version 16 (USA). The significance threshold was set at P < 0.05. All participants were diagnosed as IGR through an oral glucose tolerance test (OGTT) and written in- formed consent before study enrollment. What’s more, baseline characteristics were collected by blood tests, such as glucose and lipid metabolism related factors, and inflammatory factors. All included participants re- ceived personal nutritional counseling on their dietary regimen for about 1 to 1.5 h, and were instructed to maintain a diet with a nutrient ratio of 50–55% carbohy- drates, 10–20% protein and < 35% fat. Importantly, participants were instructed not to take any oral antidia- betic drugs and insulin. Finally, PS and omega-3 fatty acids were randomly distributed to subjects. Participants’ compliance to taking the supplements/placebo was assessed by the quantity of surplus drugs, and subjects showed good adherence (up to 99.97%) in this study. In total, 134 IGR patients aged between 51 and 65 years old were recruited for the study. Assessment of biochemical variables Eight milliliter venous blood samples were collected after overnight fasting at baseline and 12 weeks after intervention. FPG was measured on the day of blood collection. 2 h postprandial glucose (2hPG) was also measured after consumption of 75 g glucose dissolved in water. Blood samples were immediately centrifuged at 3500 rpm (KA-1000) for 10 min to separate serum at the 0 and 12 week. Serum lipid profiles, HbA1c, FINS, and inflammatory cytokine were also quantified on the day of blood collection. TC, HDL-C, and LDL-C were de- tected by ultracentrifugation ALBK. TG, Hs-CRP, and IL-6 were measured by glycerol lipase oxidase (GPO- PAP), particle-enhanced immunophelometry, and double antibody sandwich enzyme-link immunoassay (ELISA) respectively. Moreover, the concentration of FINS was determined by isotope labeling tracer. Participant baseline characteristics In this study, a total of 134 participants were random- ized into the four intervention arms of the trial (Fig. 1). A total of 200 participants were confirmed to meet IGR criteria based on OGTT and were enrolled from October 2016 to June 2017 in the affiliated hospital of Jiangsu University. Of these, 66 participants failed subsequent analyses for reasons including: business limited availability, Materials and methods Study design Dietary habits were collected at these same times. BMI was calculated as weight in kg di- vided by height in meters squared. Body fat percentage and visceral fat rating were determined based on body composition parameters using a bioelectric impedance analysis (TBF300A human body composition analyzer, TANITA, Japan). 3) PS group: daily flour with 1.7 g plant sterols + placebo 2 4) PS plus omega-3 fatty acids group: daily flour with 1.7 g plant sterols + capsules of 2 g fish oil (1000 mg EPA + 400 mg DHA) 4) PS plus omega-3 fatty acids group: daily flour with 1.7 g plant sterols + capsules of 2 g fish oil (1000 mg EPA + 400 mg DHA) 4) PS plus omega-3 fatty acids group: daily flour with 1.7 g plant sterols + capsules of 2 g fish oil (1000 mg EPA + 400 mg DHA) In order to investigate the effects of the dietary intake of these supplements on the development of IGR dis- ease, diabetes education knowledge, dietary and nutri- tional status, dietary behaviors, and exercise habits needed to be informed and complied with in IGR indi- viduals. To avoid any influence of dietary changes, re- cruited participants were received health education weekly in the first month, and completed additional edu- cation twice a month in the next 2 months. Participants Criteria for study participation included patients with IGR, of which diagnosis was based on the 2012 American Diabetes Association diagnostic criteria [16]. Exclusion criteria included the taking of antidiabetic medication dur- ing the intervention period; acute illness within the past 2 weeks; human immunodeficiency virus infection; hepatitis or other significant liver disease (except macroprotei- nuria); untreated or inadequately treated clinically signifi- cant thyroid disease; anemia; active malignant disease; inborn or acquired bleeding disorder; pregnancy or breastfeeding. Assessment of anthropometric variables Baseline enrollment data and blood samples were col- lected before initiation of the 12-week intervention Wang et al. Lipids in Health and Disease (2019) 18:106 Page 4 of 9 Fig. 1 Consort flow diagram. ITT, intent-to-treat Fig. 1 Consort flow diagram. ITT, intent-to-treat placebo: −0.21 ± 1.19 vs. -0.06 ± 0.8, P < 0.01; PS plus omega-3 fatty acids vs. placebo: −0.94 ± 0.66 vs. -0.06 ± 0.8, P < 0.01) and HOMA-IR (omega-3 fatty acids vs. placebo: −0.14 ± 0.70 vs. -0.03 ± 1.04, P < 0.01; PS plus omega-3 fatty acids vs. placebo: −0.96 ± 0.98 vs. -0.03 ± 1.04, P < 0.05). In addition, the level of hemoglobin A1c (HbA1c) was decreased upon intervention with PS plus omega-3 fatty acids as compared to placebo (change from baseline: −0.35 ± 0.51 vs. -0.15 ± 0.42, P < 0.05) (Table 2). No interventions were associated with a sig- nificant change in fasting insulin (FINS) comparing with placebo. These results therefore demonstrate that PS had no effect on glucose metabolism in IGR individuals, while omega-3 fatty acids and the combination of these two interventions did. traffic problem, no acceptance for blood collection, diffi- culty swallowing capsules, and poor treatment compliance. Table 1 shows the baseline demographic and biomedical characteristics of the study population. Comparative ana- lysis showed no differences in characteristics of body com- position, blood pressure, glucose metabolism related parameters, lipid metabolism-related parameters, or inflam- matory factors among these four groups. Effects on lipid metabolism factors Compared with the placebo group, a decrease in TG concentration was observed in both the PS group (change from baseline: −0.11 ± 1.32 vs. -0.23 ± 0.46, P < 0.01) and the PS plus omega-3 fatty acids group (change from baseline: −0.34 ± 0.77 vs. -0.23 ± 0.46, P < 0.01). Effects on anthropometric measurements Based on records obtained after the 12-week interven- tion with PS, omega-3 fatty acids, or the combination of the two, no statistically significant differences were seen between these four groups in terms of weight, body mass index (BMI), body fat percentage, visceral fat rating, sys- tolic pressure, or diastolic pressure. Compared with the placebo group, waistline was decreased in the omega-3 fatty acids group (change from baseline: −1.90 ± 3.86 vs. -0.88 ± 5.84, P < 0.05), while no change was observed in the other two groups (Table 2). Effects on glucose metabolism factors In both the omega-3 fatty acid treatment group and the PS plus omega-3 fatty acid treatment group, comparative analysis revealed a significant decrease in the levels of fasting plasma glucose (FPG) (omega-3 fatty acids vs. The combination of PS and omega-3 fatty acids also increased HDL-C compared to placebo group (change from baseline: −0.10 ± 0.31 vs. -0.12 ± 0.12, P < 0.05) (Table 2). However, there was no significant effect of Wang et al. individuals. Unfortunately, the combined intervention had no synergistic effect on these parameters, such as HDL-C, TG, and HbA1c (Fig. 2b, d, e). either intervention on the concentrations of TC and LDL-C. These findings therefore show that PS and com- binatory interventions have positive effects on lipid me- tabolism in IGR individuals, whereas omega-3 fatty acids alone have no effect. Discussion In this randomized clinical trial performed in IGR indi- viduals, we demonstrated that a 12-week treatment with dietary supplements containing PS (daily flour with 1.7 g plant sterols), omega-3 fatty acids, or the combination thereof can significantly alter glucose and lipid metabol- ism. Specifically, PS predominantly decreased TG and Hs-CRP, whereas omega-3 fatty acids largely reduced HOMA-IR, FBG, waistline size, and Hs-CRP. Overall, these data indicate that dietary interventions, combined or alone, represent an attractive nonpharmacologic strat- egy for improving metabolic health in IGR individuals. Effects on glucose metabolism factors PS plant sterol, HT Height, BMI body mass index, FPG fasting plasma glucose, HbA1c glycated hemoglobin, TC total cholesterol, TG triglyceride, HDL-C high-density lipoprotein cholesterol, LDL-C low-density lipoprotein cholesterol, SD standard deviation a were presented as mean ± SD or n (%). PS plant sterol, HT Height, BMI body mass index, FPG fasting plasma glucose, HbA1c glycated lesterol, TG triglyceride, HDL-C high-density lipoprotein cholesterol, LDL-C low-density lipoprotein cholesterol, SD standard deviation individuals. Unfortunately, the combined intervention had no synergistic effect on these parameters, such as HDL-C, TG, and HbA1c (Fig. 2b, d, e). Effects on glucose metabolism factors Lipids in Health and Disease (2019) 18:106 Page 5 of 9 Table 1 Baseline demographic and clinical characteristics Characteristic Placebo PS Omega-3 PS + Omega-3 P value n = 28 n = 30 n = 35 n = 41 Female (%) 17 (60.71) 12 (0.40) 17 (48.57) 23 (56.10) 0.39 Age (years) 56.18 ± 4.06 56.17 ± 7.17 58.94 ± 7.21 55.63 ± 7.44 0.16 HT (cm) 162.52 ± 8.72 163.53 ± 8.41 164.23 ± 9.03 162.24 ± 8.06 0.746 Weight (kg) 69.82 ± 10.95 69.52 ± 10.48 69.54 ± 11.81 69.61 ± 10.46 1 BMI (kg/m2) 26.45 ± 3.81 26.18 ± 4.71 25.68 ± 2.98 26.43 ± 3.39 0.81 Waistline (cm) 94.84 ± 9.05 90.1 ± 6.71 90.4 ± 8.78 90.85 ± 9.69 0.136 Systolic pressure (mmHg) 132.36 ± 11.81 137.43 ± 12.91 136.69 ± 10.31 138.76 ± 14.57 0.218 Diastolic pressure (mmHg) 84.07 ± 11.15 85.63 ± 9.5 85.91 ± 10.61 84.63 ± 9.3 0.876 FPG (mmol/l) 6.49 ± 0.28 6.44 ± 0.23 6.56 ± 0.27 6.50 ± 0.26 0.369 FINS (mU/L) 10.28 ± 4.13 10.15 ± 3.14 9.85 ± 4.29 10.22 ± 5.47 0.98 HOMA-IR 2.96 ± 1.19 2.9 ± 0.89 2.86 ± 1.21 2.96 ± 1.58 0.983 HbA1c (%) 6.49 ± 0.43 6.21 ± 0.69 6.29 ± 0.74 6.36 ± 0.85 0.487 TC (mmol/l) 5.29 ± 0.82 5.42 ± 0.88 5.39 ± 0.62 5.35 ± 1.16 0.95 TG (mmol/l) 2.05 ± 0.71 2.07 ± 0.94 2.06 ± 0.84 2.07 ± 0.79 0.857 HDL-C (mmol/l) 1.22 ± 0.23 1.14 ± 0.25 1.19 ± 0.17 1.17 ± 0.23 0.632 LDL-C (mmol/l) 3.11 ± 0.90 2.56 ± 0.97 3.00 ± 0.63 2.98 ± 0.80 0.058 Hs-CRP (mg/L) 1.48 ± 0.69 1.13 ± 0.33 1.10 ± 0.85 1.11 ± 0.82 0.13 IL-6 (pg/ml) 3.03 ± 1.19 2.83 ± 1.14 2.98 ± 1.32 2.85 ± 1.36 0.9 Body fat rate (%) 32.68 ± 9.12 29.26 ± 6.97 29.65 ± 7.47 29.87 ± 8.44 0.353 Visceral fat rating 12.07 ± 4.06 11.47 ± 4.41 12.03 ± 4.27 12.07 ± 4.62 0.934 Data were presented as mean ± SD or n (%). PS plant sterol, HT Height, BMI body mass index, FPG fasting plasma glucose, HbA1c glycated hemoglobin, TC total cholesterol, TG triglyceride, HDL-C high-density lipoprotein cholesterol, LDL-C low-density lipoprotein cholesterol, SD standard deviation Data were presented as mean ± SD or n (%). Effects on inflammatory cytokines In intent-to-treat analyses, a decrease in Hs-CRP was observed with both PS (change from baseline: −0.05 ± 0.39 vs. 0.4 ± 0.89, P < 0.05) and omega-3 fatty acids (change from baseline: −0.15 ± 0.59 vs. 0.4 ± 0.89, P < 0.05) (Table 2). However, alteration of Hs-CRP was not observed after the combined intervention of PS plus omega-3 fatty acids. Similarly, there were no statistically significant changes in IL-6 among these four groups. These results suggest that a combined intervention can- not reduce inflammation in the IGR population. Plant sterols are comprised of a group of sterols that enter the human body only from dietary sources. Rela- tively large quantities of plant sterols are present in plant oils, nuts, and avocados. Plant sterols have many import- ant physiological functions; for example, they block the absorption of dietary and endogenously-derived choles- terol in the gut [11]. Researchers have found that plant sterols (0.7–3.2 g/d) can reduce TC by 5.0–13.0%, and LDL-C by 5.6–26.8% from baseline in both normo- and Interaction of combinatory intervention in IGR individuals Comparative analyses showed that the combinatory intervention facilitated a more significant reduction in FBG and HOMA-IR than either PS or omega-3 fatty acids alone (Fig. 2a, c). These results suggest that PS and omega-3 fatty acids have a synergistic effect on the improvement of insulin resistance and FBG in IGR Wang et al. Effects on inflammatory cytokines Lipids in Health and Disease (2019) 18:106 Page 6 of 9 Table 2 Effects of plant sterol and Omega-3 supplementation on variables Variables Placebo PS Omega-3 PS + Omega-3 Main effect of PS P value Main effect of Omega-3 P value Interaction P value Post Difference Post Difference Post Difference Post Difference Weight (kg) 68.97 ± 10.89 −0.85 ± 3.62 68.24 ± 8.14 −1.28 ± 9.14 68.13 ± 11.17 −1.41 ± 1.91 67.35 ± 10.34 −2.26 ± 2.81 −0.64 0.47 −0.77 0.38 −0.21 0.81 BMI (kg/m2) 26.13 ± 3.8 −0.33 ± 1.33 25.74 ± 4.24 −0.45 ± 3.43 25.19 ± 2.89 −0.50 ± 0.71 25.59 ± 3.55 −0.84 ± 1.02 −0.23 0.47 −0.28 0.39 −0.11 0.73 Waistline (cm) 93.96 ± 9.99 −0.88 ± 5.84 90.77 ± 9.30 0.67 ± 6.04 88.50 ± 9.22 −1.90 ± 3.86 88.88 ± 10.32 −1.97 ± 4.21 0.74 0.40 −1.83 0.04* −0.81 0.35 Systolic pressure (mmHg) 130.57 ± 13.22 −1.79 ± 14.71 140.23 ± 18.33 2.80 ± 20.50 135.49 ± 12.87 −1.20 ± 16.82 139.44 ± 20.99 0.68 ± 20.85 3.235 0.32 −0.77 0.81 −1.36 0.68 Diastolic pressure (mmHg) 82.5 ± 12.08 −1.57 ± 7.52 82.33 ± 9.44 −3.3 ± 14.66 79.94 ± 8.86 −5.97 ± 8.23 81.54 ± 10.87 −3.1 ± 8.76 0.57 0.75 −2.10 0.23 2.30 0.19 FPG (mmol/l) 6.43 ± 0.86 −0.06 ± 0.8 6.92 ± 1.01 0.47 ± 0.96 6.34 ± 1.06 −0.21 ± 1.19 5.56 ± 0.62 −0.94 ± 0.66 −0.1 0.55 −0.78 0.01 −0.63 0.01 FINS (mU/L) 10.25 ± 3.9 −0.02 ± 3.67 9.57 ± 2.96 −0.58 ± 4.46 9.45 ± 3.87 −0.40 ± 2.25 8.12 ± 3.45 −2.09 ± 2.95 −1.125 0.06 −0.95 0.11 −0.57 0.33 HOMA-IR 2.93 ± 1.17 −0.03 ± 1.04 2.96 ± 1.18 0.06 ± 1.45 2.72 ± 1.34 −0.14 ± 0.70 2.00 ± 0.89 −0.96 ± 0.98 −0.365 0.05* −0.57 0.00 −0.46 0.01 HbA1c (%) 6.35 ± 0.68 −0.15 ± 0.42 6.34 ± 0.92 0.13 ± 1.11 6.15 ± 0.78 −0.14 ± 0.55 6.01 ± 0.73 −0.35 ± 0.51 0.035 0.78 −0.24 0.05* −0.25 0.05* TC (mmol/l) 5.23 ± 0.89 −1.73 ± 2.07 5.61 ± 0.95 0.18 ± 1.35 5.05 ± 0.70 −0.33 ± 0.48 4.96 ± 1.16 −0.39 ± 0.87 0.925 0.24 0.42 0.06 −0.99 0.06 TG (mmol/l) 1.82 ± 0.6 −0.23 ± 0.46 1.96 ± 1.27 −0.11 ± 1.32 1.95 ± 0.81 0.16 ± 0.95 1.73 ± 0.64 −0.34 ± 0.77 −0.19 0.01 0.08 0.61 −0.31 0.01 HDL-C (mmol/l) 1.10 ± 0.22 −0.12 ± 0.12 1.16 ± 0.19 0.01 ± 0.30 1.21 ± 0.18 0.02 ± 0.19 1.27 ± 0.29 −0.1 ± 0.31 0.005 0.90 0.02 0.73 −0.13 0.05* LDL-C (mmol/l) 3.02 ± 0.77 −0.09 ± 0.45 2.79 ± 0.78 0.22 ± 1.34 2.92 ± 0.55 −0.08 ± 0.78 2.83 ± 0.88 −0.14 ± 0.75 0.125 0.42 −0.18 0.25 −0.19 0.22 Hs-CRP (mg/L) 1.88 ± 0.53 0.4 ± 0.89 1.08 ± 0.46 −0.05 ± 0.39 0.95 ± 0.43 −0.15 ± 0.59 0.93 ± 0.38 −0.18 ± 0.59 −0.24 0.03* −0.34 0.03* 0.21 0.06 IL-6 (pg/ml) 3.05 ± 0.87 0.02 ± 1.61 2.49 ± 1.41 −0.33 ± 1.36 2.53 ± 1.37 −0.45 ± 1.7 2.45 ± 1.38 −0.4 ± 1.76 −0.15 0.59 −0.27 0.35 0.20 0.48 Body fat rate (%) 32.28 ± 8.84 −0.39 ± 3.9 28.43 ± 7.13 −0.83 ± 4.74 27.68 ± 5.82 −1.12 ± 4.75 28.11 ± 8.40 −1.76 ± 2.34 −0.54 0.44 −0.83 0.23 −0.1 0.89 Visceral fat rating 11.86 ± 4.49 −0.21 ± 1.55 11.03 ± 4.17 −0.43 ± 3.62 11.14 ± 4.38 −0.83 ± 1.25 11.27 ± 4.49 −0.8 ± 1.47 −0.095 0.79 −0.495 0.19 0.125 0.74 Data were presented as mean ± SD or n (%). Effects on inflammatory cytokines PS plant sterol, HT Height, BMI body mass index, FPG fasting plasma glucose, HbA1c glycated hemoglobin, TC total cholesterol, TG triglyceride, HDL-C high-density lipoprotein cholesterol, LDL-C low-density lipoprotein cholesterol, SD standard deviation. P* ≤0.05; P** ≤0.01 Wang et al. Lipids in Health and Disease (2019) 18:106 Page 7 of 9 Fig. 2 The interaction of PS and omega-3 fatty acids on indicators. a interaction diagram of FPG; b interaction diagram of HbA1c; c interaction diagram of HOMA-IR; d interaction diagram of TG; e interaction diagram of HDL-C. PS: plant sterol; FPG: fasting plasma glucose; HbA1c: hemoglobin A1c; TG: triglyceride; HDL-C: high-density lipoprotein cholesterol. Data were presented as mean ± SD or n (%). P < 0.05 was considered as statistically significant. Fig. 2 The interaction of PS and omega-3 fatty acids on indicators. a interaction diagram of FPG; b interaction diagram of HbA1c; c interaction diagram of HOMA-IR; d interaction diagram of TG; e interaction diagram of HDL-C. PS: plant sterol; FPG: fasting plasma glucose; HbA1c: hemoglobin A1c; TG: triglyceride; HDL-C: high-density lipoprotein cholesterol. Data were presented as mean ± SD or n (%). P < 0.05 was considered as statistically significant. (HNF-4a) activity. In patients with T2DM, omega-3 fatty acids did not improve glucose metabolism, but high doses reduced the levels of TG and LDL-C [13]. There is ample evidence from clinical trials [26, 29] and animal studies [30] suggesting that omega-3 fatty acids inhibit inflamma- tory reactions. Therefore, we designed a randomized and placebo-control study to observe whether omega-3 fatty acids can affect inflammatory factors, lipid metabolism, and glucose metabolism in IGR individuals. Our results demonstrated that omega-3 fatty acids improved glucose metabolism, insulin resistance, and inflammation via de- creases in FBG, HOMA-IR, waistline size, and Hs-CRP. Our results support studies that indicate omega-3 fatty acids may contribute to IGR treatment. hypercholesterolemic individuals with [17–19] and with- out [20–24] T2DM. In addition, plant sterols signifi- cantly reduced LDL-C in both nondiabetic (by 15.1%) and diabetic (by 26.8%) patients [25]. In a randomized, double-blind, placebo-controlled study [26], plant sterols significantly lowered fasting LDL-C (by 4.6%), TC (by 4.2%), and TG (by 8.3%) in dyslipidemic individuals with or at risk of developing T2DM. In our present study, we have demonstrated for the first time that a significant re- duction in TG and Hs-CRP can be achieved in IGR pa- tients via dietary supplementation with plant sterols (2 g/d). Competing interests Competing interests The authors declare that they have no competing interests. p g The authors declare that they have no competing interests. Funding g This study was supported by the Newtrition Asia Research Grant by BASF Company (SH2016034, SH2018071, SHW2016003, SWHY201604). The funding agencies had no involvement with the design, implementation, analysis, and interpretation of the study. Availability of data and materials The datasets supporting the conclusions of this article are included within the article. Authors’ contributions JFW and RRP conceived of the study, and drafted the manuscript. YYL, SX and GYY participated in the design of the study and performed the statistical analysis. YW, LY, DW, JJY, HXL and JY participated in data curation and helped to draft the manuscript. All authors read and approved the final manuscript. Effects on inflammatory cytokines Thus, we speculate that PS might prevent IGR pro- gression to T2DM via improving lipid metabolism and inflammation. Espinosa et al. [31] has demonstrated that the combin- ation of omega-3 fatty acids and plant sterols has a posi- tive effect on antioxidant activity. In addition, a study by Bitzur et al. [7] found that treatment with n-3-PSE, which contained 1.3 g omega-3 fatty acids and 1.6 g plant sterols, significantly decreased TG (by 19%) and Hs-CRP (by 7.8%), while there were no significant changes in LDL-C levels. However, there have been only a few studies of the combined interaction of PS and omega-3 fatty acids on diabetic dyslipidemia, and what role the combinatory interaction plays in IGR patients is yet unclear. Our results indicate for the first time that Omega-3 fatty acids contain a number of double bonds of unsaturated fatty acid polymers. The most common type of omega-3 fatty acids is alpha-linoleic acid (ALA), which is primarily derived from plant seeds, as well as eicosapentaenoic acid (EPA), and docosahex- aenoic acid (DHA), which mainly come from deep sea fish [27]. It has been demonstrated that omega-3 fatty acids can improve peripheral tissue insulin sensitivity by increasing the expression of GLUT4 in the skeletal muscle [28]. Moreover, omega-3 fatty acids also enhance peroxisome proliferator-activated receptor alpha (PPARa) activity, while reducing hepatocyte nuclear factor-4a Wang et al. Lipids in Health and Disease (2019) 18:106 Page 8 of 9 supplement. This study is of great scientific import- ance, as it provides a theoretical basis for the diet-based prevention of diabetes and its complications. Nutritional products including PS and omega-3 fatty acids thus have broad application potential for delaying the development of diabetes. the combinatory interaction of PS and omega-3 fatty acids improves diabetic dyslipidemia, glucose metabol- ism, and insulin resistance via regulating TG, HDL-C, FBG, HbA1c, and HOMA-IR. In particular, this combin- atory interaction had better effects on the improvement of FBG and HOMA-IR than did either single dietary supplement. Interestingly, this combinatory interaction did not decrease waistline size or Hs-CRP, even though omega-3 fatty acids alone significantly decreased these two parameters. This may simply be a consequence of the relatively short observation period utilized in the present study. Ethics approval and consent to participate The trial was approved by the Ethics Committee of the affiliated hospital of Jiangsu University and conducted in accordance with Helsinki’s Declaration. All the patients gave their written information consent. Consent for publication Not applicable. N-3 fatty acids decrease TG levels, as well as amelior- ate inflammation and endothelial dysfunction [32]. In our study, we haven’t collected N-3 fatty acids levels in participants because of shortage in samples, but benefi- cial effect of N-3 fatty acids on the metabolic profile of patients with T2DM has been reported. It has been in- vestigated that N-3 fatty acids could decrease cardiomet- abolic complications of T2DM [33]. Therefore, we speculated that the levels of N-3 fatty acids might be in- creased in IGR individuals which were received omega-3 fatty acids and the combination of PS and omega-3 fatty acids. In addition, the role and mechanism of N-3 fatty acids on glucose and lipid metabolism in IGR individuals might be a potential research area, also is the direction of our further study. Received: 24 January 2019 Accepted: 8 April 2019 Received: 24 January 2019 Accepted: 8 April 2019 Publisher’s Note 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. Author details 1 1Division of Endocrinology, Affiliated Hospital of Jiangsu University, Zhenjiang, Jiangsu 212000, China. 2Department of General Surgery, Affiliated Hospital of Jiangsu University, Zhenjiang, Jiangsu 212000, China. 3Department of Clinical Nutrition, Affiliated Hospital of Jiangsu University, No. 438 Jiefang Road, Zhenjiang District, Jiangsu 212000, China. 1Division of Endocrinology, Affiliated Hospital of Jiangsu University, Zhenjiang, Jiangsu 212000, China. 2Department of General Surgery, Affiliated Hospital of Jiangsu University, Zhenjiang, Jiangsu 212000, China. 3Department of Clinical Nutrition, Affiliated Hospital of Jiangsu University, No. 438 Jiefang Road, Zhenjiang District, Jiangsu 212000, China. j g, g , p g y, Hospital of Jiangsu University, Zhenjiang, Jiangsu 212000, China. 3Department of Clinical Nutrition, Affiliated Hospital of Jiangsu University, No. 438 Jiefang Road, Zhenjiang District, Jiangsu 212000, China. Abbreviations FINS: Fasting insulin; FPG: Fasting plasma glucose; HDL-C: High-density lipoprotein cholesterol; HOMA-IR: Homeostasis model assessment of insulin resistance; Hs-CRP: High-sensitivity C-reactive protein; IDF: International Diabetes Federation; IFG: Impaired fasting glucose; IFG/IGT: IFG complicated by IGT; IGR: Impaired glucose regulation; IGT: Impaired glucose tolerance; IR: Insulin resistance; LDL-C: Low-density lipoprotein cholesterol; NGT: Normal glucose metabolism; PS: Plant sterols; T2DM: Type 2 diabetes mellitus; TG: Triglyceride In summary, this is the first study to our knowledge that has focused on diabetic dyslipidemia, inflammation, and glucose metabolism by dietary supplementation with PS and omega-3 fatty acids in a randomized, double- blind, placebo-controlled manner in IGR individuals. Based on these results, we believe that this intervention can weaken the inherent lipotoxicity of insulin resist- ance, promoting positive IGR outcomes. The combined interaction of PS and omega-3 fatty acids, which have therapeutic potential, may offer a safe and effective therapeutic approach in IGR patients. There is a need for additional innovative studies to further investigate in- flammatory markers and blood pressure in IGR individ- uals who take PS and omega-3 fatty acids. For example, the studies need enlarge samples, lengthen observation period, and detect more inflammatory factors. Therefore, PS and omega-3 fatty acids play an important role in the prevention and treatment of IGR progression to T2DM. This study provides support for a scientific approach to using plant sterols and omega-3 fatty acids for the treat- ment of IGR, as well as T2DM. References In summary, we have demonstrated for the first time that omega-3 fatty acids and the combination of PS and omega-3 fatty acids can correct imbalances in glucose and lipid metabolism, and can additionally improve inflammation in IGR individuals. 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https://openalex.org/W2141907279	https://figshare.com/articles/journal_contribution/Supplementary_Figure_3_from_GSK3_Inhibitors_Regulate_i_MYCN_i_mRNA_Levels_and_Reduce_Neuroblastoma_Cell_Viability_through_Multiple_Mechanisms_Including_p53_and_Wnt_Signaling/22500357/1/files/39959628.pdf	German	null	GSK3 Inhibitors Regulate <i>MYCN</i> mRNA Levels and Reduce Neuroblastoma Cell Viability through Multiple Mechanisms, Including p53 and Wnt Signaling	Molecular cancer therapeutics	2,014	cc-by	17	Figure S3 A Predicted Upstream Regulator Level Overlap B A Predicted Upstream Regulator Level Overlap B B
https://openalex.org/W3203346592	https://economyandsociety.in.ua/index.php/journal/article/download/514/492	Ukrainian	null	ІННОВАЦІЙНІ ПІДХОДИ ДО ВИКОРИСТАННЯ РЕКЛАМИ ТА PR-ТЕХНОЛОГІЙ В ГОТЕЛЬНО-РЕСТОРАННОМУ БІЗНЕСІ	Ekonomìka ta suspìlʹstvo	2,021	cc-by	3,261	ІННОВАЦІЙНІ ПІДХОДИ ДО ВИКОРИСТАННЯ РЕКЛАМИ ТА PR-ТЕХНОЛОГІЙ В ГОТЕЛЬНО-РЕСТОРАННОМУ БІЗНЕСІ INNOVATIVE APPROACHES TO THE USE OF ADVERTISINGAND PR-TECHNOLOGIES IN THE HOTEL AND RESTAURANT BUSINESS Лисюк Тетяна Василівна кандидат педагогічних наук, доцент, Волинський національний університет імені Лесі Українки ORCID: https://orcid.org/0000-0003-1629-9652 Терещук Оксана Степанівна кандидат географічних наук, доцент, Волинський національний університет імені Лесі Українки ORCID: https://orcid.org/0000-0001-8131-1270 Д О А ії Терещук Оксана Степанівна кандидат географічних наук, доцент, Волинський національний університет імені Лесі Українки ORCID: https://orcid.org/0000-0001-8131-1270 Демчук Ольга Андріївна студентка, Волинський національний університет імені Лесі Українки ORCID: https://orcid.org/0000-0001-5524-4633 Демчук Ольга Андріївна студентка, Волинський національний університет імені Лесі Українки ORCID: https://orcid.org/0000-0001-5524-4633 Випуск # 28 / 2021 Випуск # 28 / 2021 ЕКОНОМІКА ТА СУСПІЛЬСТВО УДК 659.44:640.43 Lysiuk Tetiana, Tereshchuk Oksana, Demchuk Olga Lesya Ukrainka Volyn National University У статті розглянуто вплив реклами та PR-технологій на готельно-ресторанну індустрію. Проаналізовано ефективність використання соціальної мережі Instagram для просування готельно-ресторанного бізнесу. Роз глянуто основні переваги та недоліки рекламної кампанії у мережі. Зважаючи на розвиток індустрії гостин ності, реклама та PR-технології є основними засобами просування ресторанного бізнесу. Адже сучасна го тельно-ресторанна сфера дуже динамічна, щодня відкриваються різноманітні заклади, яким з урахуванням зростаючої конкуренції потрібно просувати власний заклад за допомогою реклами і PR-акцій. Також, важли во, що за допомогою Інтернету і соціальних мереж можна розробляти різні види інтернет-реклами. Вельми актуальним на сьогодні у розвитку та популяризації ресторану чи готелю є створення контенту для закладу, або ж як це називають Social Media Marketing (SMM). ц g ( ) Ключові слова: реклама, PR-технології, соціальні мережі, готельно-ресторанний бізнес, Social Media Marketing. В статье рассмотрено влияние рекламы и PR-технологий в гостинично-ресторанной индустрии. Проана лизирована эффективность использования социальной сети Instagram для продвижения гостинично-ресто ранного бизнеса. Рассмотрены основные приемущества и недостатки рекламной кампании в сети. Несмотря на развитие индустрии гостеприимства, реклама и PR-технологии являются основными средствами про движения ресторанного бизнеса. Ведь современная гостинично-ресторанная индустрия очень динамичная, ежедневно открываются различные заведения, которым с учетом растущей конкуренции нужно продвигать собственное заведение с помощью различных реклам и PR-акций. Также, важно, что с помощью Интернета и социальных сетей можно разрабатывать различные виды интернет-рекламы. Весьма актуальным на сегодня в развитии и популяризации ресторана или гостиницы является создание контента для заведения, или как это называют Social Media Marketing (SMM). ГОТЕЛЬНО-РЕСТОРАННА СПРАВА g ( ) Ключевые слова: реклама, PR-технологии, социальные сети, гостинично-ресторанний бизнес, Social Media Marketing. The article considers the impact of advertising and PR-technologies on the hotel and restaurant industry. The effectiveness of using the social network Instagram to promote the hotel and restaurant business is analyzed. 242 ©Лисюк Т.В., Терещук О.С., Демчук О.А. ЕКОНОМІКА ТА СУСПІЛЬСТВО Випуск # 28 / 2021 Випуск # 28 / 2021 The main advantages and disadvantages of an online advertising campaign are considered. Due to the develop ment of the hospitality industry, advertising and PR-technologies are the main means of promoting the restaurant business. After all, the modern hotel and restaurant industry is very dynamic, every day various establishments are opened, which, taking into account the growing competition, need to promote their own establishments and PR-pro motions. Also, it is important that with the help of the Internet and social networks you can develop different types of online advertising. Lysiuk Tetiana, Tereshchuk Oksana, Demchuk Olga Lesya Ukrainka Volyn National University A very important aspect in the development and promotion of a restaurant or hotel is the creation of content for the institution or as it is called Social Media Marketing (SMM). In addition, competition in the market of hotel and restaurant services forces management to resort to various PR-technologies. As a rule, these are serious PR-actions and creation of the concept of the institution that meets the requirements of the modern market. Thus, in the article, we considered the effectiveness of advertising in the hotel and restaurant industry. However, the article raises the question of how to promote the restaurant in the service market. After all, how attractive and popular a restaurant is depends on its attendance and the number of regular customers. Therefore, the image and concept of the institution depends on advertising and PR-technologies. The main tasks of advertising activity and sequence of actions at the organization of advertising campaign are investigated. The article examines the main trends in the hotel and restaurant industry, which is one of the highly profitable industries of the tertiary sector of the economy, which ultimately requires successful promotion in the modern market. That is why advertising and PR-technologies in the hotel and restaurant business are a multifaceted and promising area. Its task is to promote the institution and build the trust of the guest in the long run. Keywords: advertising, PR-technologies, social networks, hotel and restaurant business, Social Medi Постановка проблеми. На сьогоднішній день, готельно-ресторанна сфера – це одна з найбільш швидкозростаючих галузей. Міжна родний досвід свідчить, що необхідною пере думовою активного та успішного просування цієї галузі на ринок держави є сучасна турис тична інфраструктура. З кожним роком зрос тає кількість готелів та ресторанів, які вимага ють успішного просування. Зрештою, жорстка конкуренція на ринку готельно-ресторанних послуг змушує керівництво застосовувати спеціальні заходи, які можуть звернути увагу потенційного гостя. Адже просування закладу залежить від індивідуальних особливостей гостя. Від того, наскільки привабливий рес торан, залежить його відвідуваність та обсяг продажів ресторанних послуг. що сприяють залученню нових потенційних гостей. що сприяють залученню нових потенційних гостей. Формулювання цілей статті. Основним завданням даної статті є визначення особли востей PR-технологій, механізмів створення реклами та їх вплив на готельно-ресторанну сферу. Виклад основного матеріалу, дослі дження. На сьогоднішній день готельно-рес торанна індустрія є однією з найбільш кон курентоспроможних, проте Covid-пандемія стала причиною консервації багатьох об’єктів розміщення. Щоб зберегти частку ринку, не кажучи вже про зростання, готелям потрібні стратегічні зв’язки з громадськістю. Lysiuk Tetiana, Tereshchuk Oksana, Demchuk Olga Lesya Ukrainka Volyn National University Готелі та ресторани повинні мати проду ману комунікаційну стратегію і постійно під тримувати зв’язки з контактними аудиторіями і громадськістю. Комунікаційні процеси пови нні бути безперервними і ефективними. Аналіз останніх досліджень та публікацій. Дослідженню аспектів реклами та PR-тех- нологій в готельно-ресторанному бізнесі при свячені праці таких вчених Балабанов Л.В. [1], Балабанов А.В. [2], Слісаренко І.Ю. [6], Почепцов Г.Г. [5], Тихомирова Є.Б. [7]. Найчастіше характер здійснення PR діяль ності в готельно-ресторанному бізнесі зале жить від специфіки, рівня і характеру надання послуг. Завдання PR полягає в тому, щоб налагодити взаєморозуміння, позитивне від ношення і довіру клієнта до пропозицій під приємств готельно-ресторанного бізнесу на перспективу. Йдеться про формування в очах громадськості позитивного іміджу, хорошої репутації і поваги до підприємств [2]. У зазначених працях авторів аналізується стан індустрії гостинності, висвітлюються про блеми застосування рекламних концепцій управління, а також проаналізовано питання застосування PR-технологій в готельно-рес торанному бізнесі. ГОТЕЛЬНО-РЕСТОРАННА СПРАВА Виділення невирішених раніше частин загальної проблеми. Більша частина нау ковців у своїх працях акцентує увагу на ана ліз конкретних ситуацій при формуванні та здійсненні рекламної діяльності. Необхідне ретельне дослідження цільової аудиторії, партнерів, клієнтів та споживачів, з метою роз робки загальних методів просування закладу, З кожним роком зростає кількість готелів та ресторанів як у нашій країні, так і за кордоном. Велика кількість ресторанів та готелів сти каються із жорсткою конкуренцією на ринку готельно-ресторанних послуг, і зрештою зму шує менеджерів вдаватися до різних неорди нарних рекламних ідей. Більшість менеджерів ЕКОНОМІКА ТА СУСПІЛЬСТВО Випуск # 28 / 2021 Випуск # 28 / 2021 ємств сфери послуг. І для того щоб створити такий імідж, потрібен фахівець який займа ється контентом для закладу. SMM-менеджер вирішує ряд складних завдань, пов’язаних із залученням потенційних споживачів з соці альних мереж, підвищенням іміджу закладу, а також популяризує послуги в закладі. Він ство рює та оформляє корпоративні сторінки в різ них соціальних мережах, зацікавлює цільову аудиторію, відповідає на питання і коментарі потенційних гостей, займається просуванням закладу. ще просто не встигли опанувати цю незвичну область, адже зорієнтувати потенційного гостя на такому великому та різноманітному ринку послуг дуже важко. Проте, вплив реклами та PR-технологій на готельно-ресторанну сферу очевидна. На сьо годнішній день ефективність реклами почала знижуватись і виникла потреба у чомусь новому. Відбулося так зване перенасичення споживача рекламою взагалі і недовіра до неї призвела до того, що заклади розміщення та харчування постали перед проблемою пошуку нових шляхів приваблювання гостей. На сучасному етапі ефективним інструмен том просування готельно-ресторанного біз несу є соціальна мережа Instagram. Lysiuk Tetiana, Tereshchuk Oksana, Demchuk Olga Lesya Ukrainka Volyn National University Справжній SMM-спеціаліст повинен розу міти, що рекламна інформація яка публіку ється, має бути цікавою та доступною. Однією з основних цілей використання соціальних мереж у маркетингу є комунікація, яка робить індустрію гостинності доступною для тих, хто зацікавлений у видах послуг, які надаються закладом, і робить їх доступними для тих, хто не знає про ці послуги. Instagram – це потужний інструмент для просування. Завдяки цій соціальній мережі у менеджерів підприємств сфери послуг є доступ до величезної аудиторії гостей, з якими можна спілкуватися, залучати, озна йомлювати з послугами, що надаються [3]. Реклама в Інтернеті сьогодні набуває над звичайно великого значення, проте має пере ваги і недоліки. Просування в соціальних мережах сприяє охопленню максимально широкої аудиторії. За допомогою реклами в соціальних мере жах, підприємства можуть знаходити партне рів, співробітників, і таким чином сприяти роз витку свого бізнесу. Сьогодні майже всі рекламні агентства пропо нують своїм клієнтам таку послугу, як Інтернет – реклама. Основна мета реклами в мережі – це створення позитивного іміджу готельно-ресто ранних підприємств, залучити потенційних гос тей і перетворити їх на постійних. Загалом, зареєструвавши аккаунт закладу в Instagram, можна підвищити впізнава ність ресторану чи готелю, дізнатися про те, що думають користувачі про якість надава них послуг у закладі. Що подобається, що потрібно покращити або виключити. Для забезпечення стабільного ефектив ного функціонування підприємств індустрії гостинності необхідно розробити стратегію їх розвитку, метою якої буде досягнення макси мально ефективних результатів, які безпосе редньо вплинуть на діяльність готельно-рес торанних підприємств [4]. Таким чином, спілкуючись зі своєю аудито рією і поступово додаючи нову інформацію, можна створити вигідний імідж для підпри ГОТЕЛЬНО-РЕСТОРАННА СПРАВА Рис. 1. Структура просування готельно-ресторанного бізнесу за допомогою рекламної кампанії в соціальній мережі Instagram Джерело: складено авторами Просування підприємств сфери послуг за допомогою рекламної кампанії Збільшення відвідуваності Підвищення впізнаваності закладу Просування послуг, що надаються Підвищення довіри до підприємств Залучення нових працівників Ознайомлення гостей з новинками закладу Просування підприємств сфери послуг за допомогою рекламної кампанії ГОТЕЛЬНО-РЕСТОРАННА СПРАВА Ознайомлення гостей з новинками закладу Збільшення відвідуваності Залучення нових працівників Просування послуг, що надаються Підвищення впізнаваності закладу Підвищення довіри до підприємств Рис. 1. Lysiuk Tetiana, Tereshchuk Oksana, Demchuk Olga Lesya Ukrainka Volyn National University Структура просування готельно-ресторанного бізнесу за допомогою рекламної кампанії в соціальній мережі Instagram Джерело: складено авторами Випуск # 28 / 2021 ЕКОНОМІКА ТА СУСПІЛЬСТВО Таблиця 1 Основні переваги та недоліки рекламної кампанії у мережі Переваги Недоліки Легке розташування реклами для споживача У мережі велика конкуренція, і перш ніж розміщувати рекламу, варто обміркувати концепцію закладу Доступна ціна Важко знайти надійний сайт Більш за будь-який інший засіб розміщення реклами привертає увагу потенційних гостей Працює тільки тоді, коли фінансується Висока довіра користувачів Вимагає довгострокових інвестицій Величезний простір для креативності Особлива обережність клієнтів до реклами, які раніше не мали доступу до мережі, і відповідно не могли оцінити її ефективності Можна скорегувати рекламу під свою цільову аудиторію Можна відслідкувувати ефективність реклами після запуску, і відразу вносити коригування, якщо результат незадовільний Джерело: складено авторами Джерело: складено авторами Слід зазначити, що ефективність реклами – це невизначений і мінливий критерій. Адже, можна визначати ефективність розміром при бутку або підвищеним інтересом до закладу. клієнтів і, отже, обсяг продажів ресторанних послуг. Серед методів просування закладів інду стрії гостинності є проведення спеціальних заходів. Найбільш використовуваними у прак тиці готельно-ресторанної індустрії є наступні PR-акції [8]: Можна виділити наступні найпоширеніші види реклами для підвищення ефективності підприємств індустрії гостинності: – благодійні заходи; – білборди; – благодійні заходи; – реклама на радіо; – проведення дитячих свят, національ них подій і ін.; – проведення дитячих свят, національ них подій і ін.; – реклама на телебаченні; – тижні кухонь різних регіонів; й – реклама на моніторах; – реклама в Інтернеті. – майстер-класи від шеф-кухаря; – дегустації. і ій Таким чином, оперативне використання різних видів реклами, допомагає підвищити ефективність та продуктивність роботи готельно-ресторанних комплексів. Адже реклама впливає на економічну та соці альну складові роботи готельно-ресторанної індустрії. На сьогоднішній день ні одне підприємство не може існувати без належного просування. PR-технології допомагають створити пози тивний імідж закладу, привести багато нових гостей та стати впізнаваним місцем, до якого завжди хочеться повернутись. Головною умовою для успішного готельно- ресторанного бізнесу є професійне просу вання закладу на сучасному ринку послуг. Заклади індустрії гостинності повинні постійно впроваджувати інновації, щоб залишатись провідними у своєму сегменті та бути попе реду свої конкурентів. Основні завдання, які реалізуються під час здійснення реклами та PR-діяльності ресто рану [8]: ГОТЕЛЬНО-РЕСТОРАННА СПРАВА – інформування потенційних гостей про ресторан; – формування позитивного іміджу; і і – проведення спеціальних заходів; Просування закладів індустрії гостинності на ринку послуг, залежить від їх індивідуаль них особливостей: напрямку кухні, цінової політики, розташування, якості обслугову вання та інших факторів. СПИСОК ВИКОРИСТАНИХ ДЖЕРЕЛ: 1. Балабанов А.В. Цікаве медіапланування. Москва : РВП-Холдинг, 2000. 104 с. 1. Балабанов А.В. Цікаве медіапланування. Москва : РВП-Холдинг, 2000. 104 с. медіапланування. Москва : РВП-Холдинг, 2000. 1 2. Балабанов Л.В. Основи паблік рілейшнз : Навчальний посібник, Київ, 2001. 2. Балабанов Л.В. Основи паблік рілейшнз : Навчальний посібник, Київ, 2001. 3. Гвозденко Є.М., Чекштуріна В.М. Instagram як ефективний інструмент просування бізнесу. Розвиток європейського простору очима молоді: економічні, соціальні та правові аспекти. м. Харків, 2019 року. Харків : ХНЕУ ім. С. Кузнеця, 2019. 3. Гвозденко Є.М., Чекштуріна В.М. Instagram як ефективний інструмент просування бізнесу. Розвиток європейського простору очима молоді: економічні, соціальні та правові аспекти. м. Харків, 2019 року. Харків : ХНЕУ ім. С. Кузнеця, 2019. 3. Гвозденко Є.М., Чекштуріна В.М. Instagram як ефективний інструмент просування бізнесу. Розвиток європейського простору очима молоді: економічні, соціальні та правові аспекти. м. Харків, 2019 року. Харків : ХНЕУ ім. С. Кузнеця, 2019. 4. Миронова М.І., Миронов Ю.Б. Показники ефективності діяльності підприємств індустрії гостинності: Матеріали Всеукраїнської науково-практичної конференції (м. Черкаси, 16–17 квітня 2020 р.). Черкаси, 2020. С. 517–520. 5. Почепцов Г.Г. Теорія комунікацій. Київ : ВЦ «Київськийуніверситет», 2015. 308 с. 5. Почепцов Г.Г. Теорія комунікацій. Київ : ВЦ «Київськийуніверситет», 2015. 308 с. 6. Слісаренко І.Ю. Паблік рилейшнз у системі комунікації та управління: Навч. посіб. Київ : МАУП, 2017. 104 с. 6. Слісаренко І.Ю. Паблік рилейшнз у системі комунікації та управління: Навч. посіб. Київ : МАУП, 2017. 104 с. 6. Слісаренко І.Ю. Паблік рилейшнз у системі комунікації та управління: Навч. посіб. Київ 104 с. ГОТЕЛЬНО-РЕСТОРАННА СПРАВА 7. Тихомирова Є.Б. Зв’язки з громадськістю : Навч. посіб. Київ : НМЦВО, 2011. 560 с. 8. Шутенко В.П., Голуб М. О. PR-технології у ресторанному бізнесі. Розвиток європейського простору очима молоді: економічні, соціальні та правові аспекти. м. Харків, 2019 року. Харків : ХНЕУ ім. С. Кузнеця, 2019. 8. Шутенко В.П., Голуб М. О. PR-технології у ресторанному бізнесі. Розвиток європейського простору очима молоді: економічні, соціальні та правові аспекти. м. Харків, 2019 року. Харків : ХНЕУ ім. С. Кузнеця, 2019. Lysiuk Tetiana, Tereshchuk Oksana, Demchuk Olga Lesya Ukrainka Volyn National University Від того, наскільки привабливий ресторан для відвідувачів, зале жить його відвідуваність, кількість постійних – підвищення лояльності клієнтів. При організації рекламної кампанії у готельно-ресторанному бізнесі рекоменду ється наступна послідовність дій [8]: При організації рекламної кампанії у готельно-ресторанному бізнесі рекоменду ється наступна послідовність дій [8]: – вивчити маркетингову ситуацію, про аналізувати конкурентні умови на ринку або його сегменті; ЕКОНОМІКА ТА СУСПІЛЬСТВО Випуск # 28 / 2021 Випуск # 28 / 2021 – визначити цільову аудиторію, портрет потенційного гостя і перелік рекламованих послуг; для просування послуг ресторану та готелю. Однак, забезпечення бажаного результату можливе лише в тому випадку, якщо реклама орієнтована на цільовий сегмент споживача. у – сформувати цілі рекламної кампанії; у – сформувати цілі рекламної кампанії; Проте, засоби комунікації зі зв’язками з громадськістю, які були ефективними мину лого року або навіть на початку цього року, дуже ймовірно, сьогодні не будуть практич ними. Визнання ситуації є надзвичайно важ ливим, але наступним кроком є переоцінка цих зусиль щодо PR та з’ясування того, як саме їх можна максимізувати, навіть серед поточних подій, що склалися. – розробити рекламну стратегію: кон цепцію основної ідеї проведення рекламної кампанії; – вибрати кошти на поширення реклами, періодичність та строки розміщення реклами; р д р р щ р – розрахувати кошторис витрат на рекламні заходи; – визначити реальні розміри грошових коштів, які можна використовувати на рекламу і в залежності від цього провести коригування плану рекламної кампанії; Сучасний медіа-пейзаж та мінливий світ поставили готельно-ресторанний бізнес у скрутне становище, але впровадження сучас них методів PR може сприяти підвищенню довіри до бренду та поінформованості, навіть в умовах усього хаосу. Зараз, як ніколи, готельно-ресторанній індустрії потрібні ефек тивні стратегічні комунікації, щоб мати мож ливість відійти від пандемії та економічного спаду. – остаточно узгодити потреби в рекламі з реальними можливостями ресторану на певний період (квартал, рік); р р р – розробити рекламні повідомлення і тексти; – скласти поетапний план розміщення акцій і видання реклами; – організувати роботу підприємств під час рекламної кампанії; Отож, на сучасному етапі важливо визнати, що застарілі методи у сфері зв’язків з громад ськістю зараз не будуть результативними, а інноваційні методи та стратегії спілкування мають силу залучити більше гостей і, в свою чергу, принести значний прибуток готельно- ресторанним підприємствам. – оцінити ефективність рекламної кам панії після її проведення. Висновки. Ефективність застосування реклами та PR-діяльності залишається акту альною і вимагає постійної уваги. Реклама та PR-технології є важливим інструментом 4. Myronova M.I., Myronov Ju.B. (2020) Pokaznyky efektyvnosti dijaljnosti pidpryjemstv industriji ghostynnosti [Indicators of efficiency of enterprises of the hospitality industry]. Cherkasy. (in Ukrainian) 5. Pochepcov Gh.Gh. (2015) Teorija komunikacij [Communication theory]. Kyiv: Kyjivsjkyj'universytet. (in Ukrainian) 8. Shutenko V.P., Gholub M.O. (2019) PR-tekhnologhiji u restorannomu biznesi [PR-technologies in the restaurant business]. Kharkiv: KhNEU im. S. Kuznecja. (in Ukrainian) ( ) 6. Slisarenko I.Ju. (2017) Pablik rylejshnz u systemi komunikaciji ta upravlinnja [PR in the system of communication and management]. Kyiv: MAUP. (in Ukrainian) 4. Myronova M.I., Myronov Ju.B. (2020) Pokaznyky efektyvnosti dijaljnosti pidpryjemstv industriji ghostynnosti [Indicators of efficiency of enterprises of the hospitality industry]. Cherkasy. (in Ukrainian) 5. Pochepcov Gh.Gh. (2015) Teorija komunikacij [Communication theory]. Kyiv: Kyjivsjkyj'universytet. (in Ukrainian) 6. Slisarenko I.Ju. (2017) Pablik rylejshnz u systemi komunikaciji ta upravlinnja [PR in the system of communication and management]. Kyiv: MAUP. (in Ukrainian) 7. Tykhomyrova Je.B. (2011) Zv'jazky z ghromadsjkistju [Public relations]. Kyiv: NMCVO. (in Ukrainian) 8. Shutenko V.P., Gholub M.O. (2019) PR-tekhnologhiji u restorannomu biznesi [PR-technologies in the restaurant business]. Kharkiv: KhNEU im. S. Kuznecja. (in Ukrainian) g ] y ( ) 7. Tykhomyrova Je.B. (2011) Zv'jazky z ghromadsjkistju [Public relations]. Kyiv: NMCVO. (in Ukraini g ] y ( ) 7. Tykhomyrova Je.B. (2011) Zv'jazky z ghrom REFERENCES: 1. Balabanov A.V. (2000) Cikave mediaplanuvannja [Interesting media planning]. Moskow: RVP-Kholdyngh. (in Russian) 1. Balabanov A.V. (2000) Cikave mediaplanuvannja [Interesting media planning]. Moskow: RVP-Kholdyngh. (in Russian) 2. Balabanov L.V. (2001) Osnovy pablik rilejshnz [Basic of PR]. Kyiv. (in Ukrainian) 2. Balabanov L.V. (2001) Osnovy pablik rilejshnz [Basic of PR]. Kyiv. (in Ukrainian) 3. Ghvozdenko Je.M., Chekshturina V.M. (2019) Instagram jak efektyvnyj instrument prosuvannja biznesu [Instagram as an effective tool for business promotion]. Kharkiv: KhNEU im. S. Kuznecja. (in Ukrainian) ( ) y p j [ ] y ( ) 3. Ghvozdenko Je.M., Chekshturina V.M. (2019) Instagram jak efektyvnyj instrument prosuvannja biznesu [Instagram as an effective tool for business promotion]. Kharkiv: KhNEU im. S. Kuznecja. (in Ukrainian) Випуск # 28 / 2021 ЕКОНОМІКА ТА СУСПІЛЬСТВО 4. Myronova M.I., Myronov Ju.B. (2020) Pokaznyky efektyvnosti dijaljnosti pidpryjemstv industriji ghostynnosti [Indicators of efficiency of enterprises of the hospitality industry]. Cherkasy. (in Ukrainian) ( ) 6. Slisarenko I.Ju. (2017) Pablik rylejshnz u systemi komunikaciji ta upravlinnja [PR in the system of communication and management]. Kyiv: MAUP. (in Ukrainian) 7. Tykhomyrova Je.B. (2011) Zv'jazky z ghromadsjkistju [Public relations]. Kyiv: NMCVO. (in Ukrainian) 8. Shutenko V.P., Gholub M.O. (2019) PR-tekhnologhiji u restorannomu biznesi [PR-technologies in the restaurant business]. Kharkiv: KhNEU im. S. Kuznecja. (in Ukrainian) 7. Tykhomyrova Je.B. (2011) Zv jazky z ghromadsjkistju [Public relations]. Kyiv: NMCVO. (in Ukrainian) 8. Shutenko V.P., Gholub M.O. (2019) PR-tekhnologhiji u restorannomu biznesi [PR-technologies in the restaurant business]. Kharkiv: KhNEU im. S. Kuznecja. (in Ukrainian) ГОТЕЛЬНО-РЕСТОРАННА СПРАВА
https://openalex.org/W2601750885	https://europepmc.org/articles/pmc5359962?pdf=render	English	null	Quantification of silver nanoparticle uptake and distribution within individual human macrophages by FIB/SEM slice and view	Journal of nanobiotechnology	2,017	cc-by	9,596	© The Author(s) 2017. 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. Quantification of silver nanoparticle uptake and distribution within individual human macrophages by FIB/SEM slice and view Erik Guehrs1, Michael Schneider1,2, Christian M. Günther1, Piet Hessing2, Karen Heitz1, Doreen Wittke4, Ana López‑Serrano Oliver5, Norbert Jakubowski5, Johanna Plendl3, Stefan Eisebitt1,2† and Andrea Haase4† Abstract Background: Quantification of nanoparticle (NP) uptake in cells or tissues is very important for safety assessment. Often, electron microscopy based approaches are used for this purpose, which allow imaging at very high resolution. However, precise quantification of NP numbers in cells and tissues remains challenging. The aim of this study was to present a novel approach, that combines precise quantification of NPs in individual cells together with high resolution imaging of their intracellular distribution based on focused ion beam/ scanning electron microscopy (FIB/SEM) slice and view approaches. Results: We quantified cellular uptake of 75 nm diameter citrate stabilized silver NPs (Ag 75 Cit) into an individual human macrophage derived from monocytic THP-1 cells using a FIB/SEM slice and view approach. Cells were treated with 10 μg/ml for 24 h. We investigated a single cell and found in total 3138 ± 722 silver NPs inside this cell. Most of the silver NPs were located in large agglomerates, only a few were found in clusters of fewer than five NPs. Further‑ more, we cross-checked our results by using inductively coupled plasma mass spectrometry and could confirm the FIB/SEM results. Conclusions: Our approach based on FIB/SEM slice and view is currently the only one that allows the quantification of the absolute dose of silver NPs in individual cells and at the same time to assess their intracellular distribution at high resolution. We therefore propose to use FIB/SEM slice and view to systematically analyse the cellular uptake of various NPs as a function of size, concentration and incubation time. Keywords: Nanoparticles, FIB/SEM slice and view, Absolute dose, Cellular internalization, Macrophag Journal of Nanobiotechnology Journal of Nanobiotechnology Guehrs et al. J Nanobiotechnol (2017) 15:21 DOI 10.1186/s12951-017-0255-8 Correspondence: andrea.haase@bfr.bund.de †Stefan Eisebitt and Andrea Haase contributed equally to this work 4 Department of Chemical and Product Safety, German Federal Institute for Risk Assessment (BfR), Max‑Dohrn‑Str. 8‑10, 10589 Berlin, Germany Full list of author information is available at the end of the article Background One being that uni- form labelling of all NPs is typically not achieved, which results in differences in fluorescence yield from one NP to another. Furthermore, the determination of absolute labelling efficacy of individual particles is difficult. l The lateral spatial image resolution of conventional SEM is about 1 nm and the depth of the consecutive FIB slices can be decreased to a few tens of nm. Thus, FIB/SEM slice and view is a suitable method to image a large number of different types of NPs in cells and to determine the abso- lute dose and the intracellular distribution. Hence, FIB/ SEM is an ideal combination of a high resolution intracel- lular imaging and a precise quantification approach for intracellular dose providing single cell resolution. As for TEM, an EDX detector can add spectroscopic information to each single-slice image and, thus, to the complete model of the cell. A major drawback for widespread application of this technique for this purpose, however, is the fact that NP numbers per cell are not readily available from the images. NPs numbers per cell may be obtained by manu- ally counting the particles, which is time-consuming and prone to artefacts in particular when many agglomerates are present. Automated evaluation, however, is in practice a prerequisite for systematic studies involving many sam- ples. Beyond image detection and accounting for shading effects, this also involves taking the physics of contrast generation into account, as given by the escape depth of secondary electrons within an organic matrix. Commer- cial software applications are not available for this pur- pose. Therefore, we have developed a model allowing for automatic analysis of FIB/SEM images to retrieves infor- mation on the NP numbers per cell. With our approach it is now possible to determine absolute doses as a func- tion of particle sizes, exposure times or concentrations for various types of NPs. These data are expected to give addi- tional impact to toxicological in vitro studies. fifi A standard technique to quantify uptake of NPs is induc- tively coupled plasma mass spectrometry (ICP-MS). ICP- MS allows for elemental analysis and can quantify the total mass of different elements in cells or tissues. Not every ele- ment can be quantified using ICP-MS, typically this tech- nique is applied for metals or metal oxides. Importantly, only the average dose of NPs is determined in standard measurement mode on larger cell ensembles [8, 9]. Background Doses per cell can be estimated by taking the total numbers of cells into account. Furthermore, standard ICP-MS does not allow to discriminate between individual NPs, agglom- erates or dissolved ions. Modification of ICP-MS such as single nanoparticle ICP-MS are available that allow some quantification of individual NPs [10]. Other modifications such as laser-ablation-ICP-MS allow imaging of the sub- cellular localization of NPs with a μm-resolution [11]. In order to investigate intracellular distribution of NPs with very high spatial resolution below 1 nm, elec- tron microscopy based techniques are required. Fre- quently, transmission electron microscopy (TEM) has been applied to record high resolution images of cell slices which are exposed to NPs [10, 12, 13]. In combina- tion with spectroscopic methods (EDX), this allows the identification of the chemical composition of the parti- cles. The major disadvantage of TEM is that only single slices and not the complete cell volume can be imaged. As a result, the absolute dose of NPs per cell is not acces- sible using TEM imaging. In contrast, a combination of a focused ion beam (FIB) with a scanning electron micros- copy (SEM) allows the 3D imaging of NPs in a single cell, albeit at lower spatial resolution than in TEM [14–16]. This approach is known as FIB/SEM slice and view. A focused Ga+ ion beam is used to expose a plane cutting through the cell for SEM imaging. Consecutive thin slices of the cell are removed perpendicular to the cell sub- strate. An SEM image of this exposed cross-section of the cell is recorded after each slicing step [14–19]. In this fashion, one can slice through the entire cell volume. The resultant image stack can then be assembled into a 3D image of the whole cell. Sample preparation is rather sim- ple: the cells are fixed chemically and then dried for SEM imaging [19]. The contrast of metallic NPs embedded in an organic matrix such as a cell is intrinsically high when imaging by SEM, due to the high secondary electron yield Background gas barrier capabilities in beverage packaging, or block ultraviolet radiation from human skin in sunscreens [1–4]. The increasing use of these materials demands proper risk assessment, which includes hazard as well as exposure assessment. Therefore, it is very important to quantify precisely the amounts of NPs, which are actually taken up into different tissues and into individual cells. Nanotechnology is becoming a mainstream technology in modern product design. Due to their unique physico- chemical properties, nanoparticles (NPs) are increasingly used worldwide in diverse applications ranging from composites in construction, fuel additives, nanomedi- cine or consumer products like cosmetics or textiles. NPs can enhance mechanical properties for instance in con- crete, facilitate cleaning of surfaces in paints, enhance f Several techniques are available for this purpose. Fluo- rescence based techniques such as flow cytometry and/or fluorescence microscopy are often applied. They allow for relative quantification of NP uptake while microscopy- based approaches allow insight into intracellular NP distribution, at least at medium resolution of 200 nm or larger. They do, however, require fluorescent-labelled NPs *Correspondence: andrea.haase@bfr.bund.de †Stefan Eisebitt and Andrea Haase contributed equally to this work 4 Department of Chemical and Product Safety, German Federal Institute for Risk Assessment (BfR), Max‑Dohrn‑Str. 8‑10, 10589 Berlin, Germany Full list of author information is available at the end of the article Page 2 of 11 Guehrs et al. J Nanobiotechnol (2017) 15:21 of metallic particles [20]. Thus, for metallic NPs coun- ter-staining is not required, in contrast to e.g. the use of osmium in conventional TEM or the use of specific labels in fluorescence microscopy.h [5, 6]. Possible disadvantages may arise from dye leakage or from possible changes to the NP surface from fluo- rescence labelling, which may also alter uptake behav- iour [7]. Furthermore, absolute quantification is typically not possible for several reasons. One being that uni- form labelling of all NPs is typically not achieved, which results in differences in fluorescence yield from one NP to another. Furthermore, the determination of absolute labelling efficacy of individual particles is difficult. [5, 6]. Possible disadvantages may arise from dye leakage or from possible changes to the NP surface from fluo- rescence labelling, which may also alter uptake behav- iour [7]. Furthermore, absolute quantification is typically not possible for several reasons. Characterization of silver NPs We used citrate-coated silver NPs with a diameter of 75 nm, herein referred to as Ag 75 Cit. We have already used this type of nanosilver in a previous study to quantify cellular uptake using different ICP-MS based approaches [21]. This allows us to compare the results of both studies. NP characterization is described in detail in [21] and is summarized in Table 1. In pure water, the NP diameter was determined via dynamical light scattering (DLS) to be 79 ± 0.5 nm and in cell culture medium (CCM) to be 71 ± 0.2 nm. This is in good agreement with data from TEM measurements (74 ± 8 nm) provided by the Manufacturer (NanoCom- posix) [22]. Furthermore, we have analysed the cell viability using the WST-1 assay in THP-1 derived macrophages that Page 3 of 11 Guehrs et al. J Nanobiotechnol (2017) 15:21 Table 1 Characterization of Ag 75 Cit. Data are taken from [21] TEM value of Ag 75 Cit was reported by NanoComposix, Prague, Czech Republic [22]. DLS data are presented as mean and standard deviation. Dissolution was determined at 2 and 10 μg/ml, at lower concentrations higher dissolution rates were observed Dispersion in water Dispersion in CCM Dissolution in water (%) TEM (nm) DLS (nm) DLS (nm) Ag 75 Cit 74 ± 8 79 ± 0.5 71 ± 0.2 1 ± 0.03 to 3.1 ± 0.7 Table 1 Characterization of Ag 75 Cit. Data are taken from [21] TEM value of Ag 75 Cit was reported by NanoComposix, Prague, Czech Republic [22]. DLS data are presented as mean and standard deviation. Dissolution was determined at 2 and 10 μg/ml, at lower concentrations higher dissolution rates were observed Dispersion in water Dispersion in CCM Dissolution in water (%) TEM (nm) DLS (nm) DLS (nm) Ag 75 Cit 74 ± 8 79 ± 0.5 71 ± 0.2 1 ± 0.03 to 3.1 ± 0.7 Table 1 Characterization of Ag 75 Cit. Data are taken from [21] During the chemical fixation process of the sample the structure of the intracellular cell compartments is not preserved. As a result, the inner part of the cell is visible as a homogeneous grey area in the SEM images and the Ag 75 Cit NPs can be readily identified within the cells as bright spots within a background of this cell matrix. Characterization of silver NPs In this situation the NPs show up bright as the second- ary electron yield of silver is approximately twice as high as that of organic carbon [20, 23]. Note, that Carbon NPs do not show such a high contrast as the secondary elec- tron yields of an organic matrix and Carbon are similar to each other. TEM value of Ag 75 Cit was reported by NanoComposix, Prague, Czech Republic [22]. DLS data are presented as mean and standard deviation. Dissolution was determined at 2 and 10 μg/ml, at lower concentrations higher dissolution rates were observed Both, the homogeneous intensity distribution of the cross-section of the cell and the high contrast of the sil- ver NPs within the cell simplifies the detection of the cell shape and of the incorporated silver NPs during the segmentation process. The shape of the cell and the sil- ver NPs are detected using threshold and edge detection algorithms. Details about the procedure can be found in the methods section. were incubated with Ag 75 Cit in concentrations up to 100 μg/ml. We did not observe any significant reduction in cell viability after 24 h. In our experiment we used a much lower concentration of 10 μg/ml Ag 75 Cit with an incubation time of 24 h, which is clearly non-toxic to the cells. Silver NP detection using FIB/SEM slice and view In b the segmented shape of the cell as well as the detected Ag 75 Cit NPs (highlighted in red) are presented. Based on the segmentation, a 3D model (c) of the cell (blue) and the silver NPs (red) is calculated. In the left panel, two orthogonal cross-sections through the cell are shown below the surface. This is due to the fact that the escape depth of secondary electrons can be larger than the slic- ing interval of 40 nm in our case [24]. As a result, the sil- ver NPs appear larger than they actually are. of the inner cell matrix is not high enough for threshold- ing and these areas of the cell are not segmented. As no silver NPs are located in this very small remaining part of the cell as verified by visual inspection of the SEM images of the slices, we decided not to adjust the algorithm. To quantify this effect, the escape depth of electrons in the cell matrix has to be determined. In a second step, this information can be used to correct the apparent volume of a single silver NP. For quantification, 16 sin- gle silver NPs were selected by hand and then analysed. The average escape depth is determined from consecu- tive slices as shown in Fig. 3 and amounts to 89 ± 17 nm. Without this correction the total voxel size for a single silver NP would be 230 ± 69 voxels (each voxel has a size of 6.3 nm × 6.3 nm × 40 nm). However, after correc- tion the voxel size was determined to be 134 ± 23 voxels, which corresponds to ~210,000 ± 36,000 nm3 and thus, fits very well to the volume of a 74 nm silver NP (calcu- lated to be 212,000 nm3). We note that this correction is significant in order to obtain reliable quantifications. From our data, the average projected NP size can also be extracted and was calculated to be 109 ± 16 pixel. This value is equivalent to a particle diameter of 74 ± 10 nm and in good agreement with the values of the manufac- turer in Table 2. In a second step, we segmented the silver NPs within the cell (Fig. 2b). The segmentation process is applied to each NP cluster individually. Silver NP detection using FIB/SEM slice and view In Fig. 2 a sample image of cell is shown before (a) and after segmentation (b). To ensure that the complete cell is segmented, the shape of the segmented cell is slightly increased. This region appears as a light grey area around the cell. The segmentation process comes to a limit at cross-sections where the cell height above the substrate is very low. This is the case for the rear part of the cell, which can be seen in Fig. 1 at slice 502. Here, the contrast A single macrophage cell was imaged by the FIB/SEM slice and view technique. In each slicing step 40 nm of the cell was removed and the cross section was imaged by SEM. The 40 nm slice separation ensured that each sil- ver NP was cut at least once in the process. In total 625 images of the cell were recorded. Six slices of this cell are shown in Fig. 1. Fig. 1 FIB/SEM slice and view. Gradual degradation of a single THP-1 macrophage using FIB/SEM slice and view. Here, six sample images of the slicing process are shown. In total, 625 images were taken to record the complete cell. A single slicing step removes 40 nm of the cell. The Ag 75 Cit NPs are visible as bright spots in the cell Fig. 1 FIB/SEM slice and view. Gradual degradation of a single THP-1 macrophage using FIB/SEM slice and view. Here, six sample images of the slicing process are shown. In total, 625 images were taken to record the complete cell. A single slicing step removes 40 nm of the cell. The Ag 75 Cit NPs are visible as bright spots in the cell Guehrs et al. J Nanobiotechnol (2017) 15:21 Page 4 of 11 Fig. 2 Cell and silver NP segmentation. In a one single slice of the cell (slice 238) is shown before segmentation. In b the segmented shape of the cell as well as the detected Ag 75 Cit NPs (highlighted in red) are presented. Based on the segmentation, a 3D model (c) of the cell (blue) and the silver NPs (red) is calculated. In the left panel, two orthogonal cross-sections through the cell are shown Fig. 2 Cell and silver NP segmentation. In a one single slice of the cell (slice 238) is shown before segmentation. Silver NP detection using FIB/SEM slice and view In this way, intensity fluc- tuations of the SEM signal generated by the silver NPs relative to the cell matrix can be compensated. Based on this segmentation a 3D surface model of the cell as well as of the silver NPs is calculated. This is depicted in Fig. 2c. Characterization of single silver NPs In the simplest model, the total number of silver NPs could be calculated from the segmentation data, if the number of voxels corresponding to a single NP is known and then used for calibration. In principle, such calibra- tion data can be extracted from the present data set, as it contains several isolated NPs. However, in order to determine the absolute dose of silver NPs within the cell, the image formation process of SEM imaging needs to be taken into account to avoid quantification artefacts from this simple model. Such artefacts come about as silver NPs are not only visible if they are exposed in the sur- face layer defined by the respective cross-section slice, but can already be detected in the SEM if they are located We conclude that the parameters of a single Ag 75 Cit NP extracted from the SEM images are in close agree- ment with the values provided by the manufacturer and complementary characterization. Furthermore, this Guehrs et al. J Nanobiotechnol (2017) 15:21 Page 5 of 11 Fig. 3 Analysis of a single silver NP. Nine slices in the immediate vicinity of a single silver NP at a distance of 40 nm to each other are shown above the x-axis. The maximum intensity of each image is taken to calculate the escape depth of electrons. The escape depth for the particle shown is ~79 nm. The average escape depth for all 16 single silver NPs investigated in this fashion is 89 ± 17 nm. The escape depth is used to determine total normalized intensity of a segmented single SNP in our sample to be (89 ± 17) of each cluster (which is proportional to its volume) needs to be calculated taking into account the escape depth of the electrons for correction. Before performing this analysis, two cases depicted in Fig. 4 have to be investigated in more detail. After slic- ing, when two adjacent silver NPs are imaged with the SEM and when they are located next to each other within the slicing plane, they are imaged like two individual NPs (see Fig. 4). The situation is different, if the silver NPs are located in different depth along SEM optical axis, spe- cifically, if they are located behind each other in differ- ent slices within the electron escape depth. Characterization of single silver NPs As the escape depth of electrons within the NPs is much smaller than the escape within the cell matrix, all electrons escaping from the posterior NP (further away from the slice sur- face) are absorbed by the anterior NP (closer to or at the surface). Thus, the posterior NP will only be detected after the anterior NP is removed by the FIB. As a result, the apparent elongation due to the escape depth has to be considered only for one silver NP to calculate the correct voxel size of this kind of cluster (see Fig. 4). Fig. 3 Analysis of a single silver NP. Nine slices in the immediate vicinity of a single silver NP at a distance of 40 nm to each other are shown above the x-axis. The maximum intensity of each image is taken to calculate the escape depth of electrons. The escape depth for the particle shown is ~79 nm. The average escape depth for all 16 single silver NPs investigated in this fashion is 89 ± 17 nm. The escape depth is used to determine total normalized intensity of a segmented single SNP in our sample to be (89 ± 17) Fig. 3 Analysis of a single silver NP. Nine slices in the immediate vicinity of a single silver NP at a distance of 40 nm to each other are shown above the x-axis. The maximum intensity of each image is taken to calculate the escape depth of electrons. The escape depth for the particle shown is ~79 nm. The average escape depth for all 16 single silver NPs investigated in this fashion is 89 ± 17 nm. The escape depth is used to determine total normalized intensity of a segmented single SNP in our sample to be (89 ± 17) Therefore, to determine the corrected voxel size for larger clusters, the projected area of the cluster with respect to the cross-section plane needs to be calcu- lated. Then the escape depth can be used for correction and thus the volume of each cluster can be calculated. Details about this procedure can be found in the methods section. result also proves that our algorithm are able to segment the silver NPs within the cell. The results are summarized in Table 2. Characterization of single silver NPs Quantification of silver NP uptake using ICP‑MS To further cross-check our results, we also determined the amount of silver NPs taken up by using the well established ICP-MS approach with the same experimen- tal treatment conditions. In total three and six different replicates for the control and the samples incubated with silver NPs respectively were measured by ICP-MS. We found 2613 ± 271 silver NPs within the cells. This coin- cides very nicely with the results obtained using FIB/SEM slice and view and therefore validates our new approach to determine the absolute dose in single cells. Most of the silver NPs are found in large agglomerates. This can be caused by (a) the formation of agglomerates outside the cell, which are then taken up by phagocytosis, or (b) by the uptake of single silver NPs or small agglom- erates that once inside the cell create larger clusters, e.g. after fusion of endocytotic vesicles to phagolysosomes. From our FIB/SEM data alone, this cannot be clarified. In addition, our data do not allow to discriminate whether the silver NPs are located inside organelles or are located within the cytoplasm. To clarify this point, we performed a TEM analysis of cells grown in the same culture and found that the silver NPs are always detected inside membrane-enclosed structures, which most likely are phagolysosomes (Fig. 6).i g Our result is also in good agreement with other stud- ies. For the same type of silver NPs also treating the cells (Neuro-2a cells) with a silver NP concentration of 10 μg/ml for 24 h, Hsiao et al. [21] determined the abso- lute dose of silver NPs as 1474–1740 silver NPs by SP- IPC-MS. They used the same type of silver NPs and also treated the cells with a silver NP concentration of 10 μg/ ml for 24 h. The difference to our results can be explained as follows. Firstly, Hsiao et al. used a neural cell type, Neuro-2a cells. It may be expected that macrophages accumulate more silver NPs than other cell types, given their biological function is to incorporate foreign par- ticles and to remove them. Secondly, when when Hsiao et al. determined the absolute dose by complementary analysis using laser ablation ICP-MS, they find that the cell-to-cell variation of silver NPs uptake is very high. In contrast, we analyzed only a single cell. Characterization of single silver NPs To analyse all segmented silver NPs within a single cell and thus to get the absolute internal dose, the voxel size After taking this correction into consideration a total of 3138 ± 722 Ag 75 Cit NPs were detected within this Table 2 Size determination of the silver NPs from the SEM images Experimental data Calculated using manufacturer’s data [22] Escape depth of electrons within the cell matrix (nm) 89 ± 17 Volume of a single silver NP (nm3) 210,000 ± 36,000 212,000 ± 2400 Diameter of a single silver NP (nm) 74 ± 10 74 ± 8 Fig. 4 Simple model of the imaging process of silver NPs by SEM. Due to the escape depth of the electrons within the cell matrix, the detected silver NP volume appears elongated along the optical axis of the SEM. To correct for this effect, the escape depth of the electrons and the shape of the NP clusters needs to be taken into account Table 2 Size determination of the silver NPs from the SEM images Calculated using manufacturer’s data [22] Fig. 4 Simple model of the imaging process of silver NPs by SEM. Due to the escape depth of the electrons within the cell matrix, the detected silver NP volume appears elongated along the optical axis of the SEM. To correct for this effect, the escape depth of the electrons and the shape of the NP clusters needs to be taken into account Guehrs et al. J Nanobiotechnol (2017) 15:21 Page 6 of 11 single macrophage cell. Most of the NPs were found in large agglomerates. 53% of all NP were located in clus- ters with a size of at least 20 NPs. Only 9% of all silver NPs were found in clusters of one to five particles. Only 34 single silver NPs were detected within the single mac- rophage (Fig. 5). single macrophage cell. Most of the NPs were found in large agglomerates. 53% of all NP were located in clus- ters with a size of at least 20 NPs. Only 9% of all silver NPs were found in clusters of one to five particles. Only 34 single silver NPs were detected within the single mac- rophage (Fig. 5). Characterization of single silver NPs Others studies confirm that NPs are located inside membrane-bound structures in macrophage cells [25–27]. However, FIB/SEM slice and view may also be applied to unravel more details on uptake mechanisms. To this end it would be necessary to improve the sample preparation protocol such that the cell organelles are pre- served and to analyse cells after several different incuba- tion times to follow and quantify cells being present in different organelles over time. y y g We show that FIB/SEM slice and view can be applied to quantify silver NPs within a single cell as well as to image silver NP distribution inside the fixed cell. As the silver NPs are imaged directly by SEM, the number of sil- ver NPs per agglomerate and thus the absolute dose can be determined. Inclusion of the effects of the electron escape depth as well as shading of particles were found to be crucial for quantitative and automatable analysis. To our knowledge, this is the first time that the absolute dose of silver NPs could be determined within a single cell, i.e. without resorting to averaging over a large cell ensemble. Here, we found that 3138 ± 722 silver NPs were located inside the cell investigated. The cluster size distribution was determined and most of the NPs were found to be agglomerated in clusters with a size larger than 20 NPs. Fig. 5 Cluster size distribution of uptaken silver NPs. Histogram and cumulative distribution function of all silver NPs within the cell. The absolute dose of silver NPs within the cell was 3138 ± 722. Although only a few clusters with a size larger than 20 NPs were present in the cell, most NPs were located in these larger clusters (~53%). Only 9% of all silver NPs were found in very small clusters (cluster size 1–5, corre‑ sponding histogram not shown). The binning width for the histogram is 10 silver NPs. The left y-axis corresponds to the histogram and the right y-axis to the cumulative distribution function Conclusion As a proof-of-principle experiment we determined the absolute dose of uptaken silver NPs in a single cell by FIB/SEM slice and view. This method can be used to investigate other types of NPs that are readily detected by SEM provided that the interslice distance of the FIB is smaller than the NP diameter. In fact, even NPs some- what smaller than the slice spacing should be detectable taking the electron escape depth into account. We see Fig. 5 Cluster size distribution of uptaken silver NPs. Histogram and cumulative distribution function of all silver NPs within the cell. The absolute dose of silver NPs within the cell was 3138 ± 722. Although only a few clusters with a size larger than 20 NPs were present in the cell, most NPs were located in these larger clusters (~53%). Only 9% of all silver NPs were found in very small clusters (cluster size 1–5, corre‑ sponding histogram not shown). The binning width for the histogram is 10 silver NPs. The left y-axis corresponds to the histogram and the right y-axis to the cumulative distribution function Fig. 5 Cluster size distribution of uptaken silver NPs. Histogram and cumulative distribution function of all silver NPs within the cell. The absolute dose of silver NPs within the cell was 3138 ± 722. Although only a few clusters with a size larger than 20 NPs were present in the cell, most NPs were located in these larger clusters (~53%). Only 9% of all silver NPs were found in very small clusters (cluster size 1–5, corre‑ sponding histogram not shown). The binning width for the histogram is 10 silver NPs. The left y-axis corresponds to the histogram and the right y-axis to the cumulative distribution function Guehrs et al. J Nanobiotechnol (2017) 15:21 Page 7 of 11 Fig. 6 TEM images of slices through THP-1 cells with uptaken silver NPs. In the TEM images, the silver NPs appear as dark spots in a–d. From all images it can be seen that most silver NPs are found in loosely- or densely-packed agglomerates. The larger magnification b reveals that silver NPs are located inside membrane-enclosed structures, which likely represent phagolysosomes. In c and d exemplary sizes of several agglomerates were determined. The agglomerate sizes are A = 336 nm; B = 344 nm; C = 224 nm; D = 425 nm; E = 525 nm Fig. Nanoparticles Commercially available 75 nm Ag nanospheres (Bio- pure quality), carrying a citrate modification, were used (NanoComposix, Prague, Czech Republic) [22]. The stock solution of silver NPs was first ultrasonicated for 5 min. Then the NPs were diluted directly into the indicated cell culture medium (see below) at the desired final con- centration and applied to the cells. NP suspensions were freshly prepared for each experiment. Characterization of nanoparticlesh potential for our approach to analyse the uptake mech- anisms of NPs as a function of size, concentration and incubation time. Without having to resort to averaging over many cells, the variance of these processes in larger cell ensembles could be investigated. Furthermore, the approach presented here can be used as a complemen- tary method for toxicological studies and as a calibration tool for methods that can only determine the relative dose of NPs in cells or observe other parameters depend- ing on NP uptake and toxicity. The hydrodynamic size of the Ag 75 Cit NPs in water and in cell culture medium was monitored using a Zetasizer Nano ZS apparatus (Malvern Instruments GmbH, Her- renberg, Germany). Conclusion 6 TEM images of slices through THP-1 cells with uptaken silver NPs. In the TEM images, the silver NPs appear as dark spots in a–d. From all images it can be seen that most silver NPs are found in loosely- or densely-packed agglomerates. The larger magnification b reveals that silver NPs are located inside membrane-enclosed structures, which likely represent phagolysosomes. In c and d exemplary sizes of several agglomerates were determined. The agglomerate sizes are A = 336 nm; B = 344 nm; C = 224 nm; D = 425 nm; E = 525 nm Cell culture THP-1 cells (ACC 16 from DSMZ, Braunschweig, Ger- many) were cultured in RPMI 1640 medium supple- mented with 10% fetal calf serum (FCS), 1% L-glutamine, 1% penicillin/streptomycin, 1% Hepes and 1 sodium pyruvate. Phorbol-12-myristate-13-acetate (PMA) at 100 ng/ml for 24 h was used to differentiate THP-1 cells into macrophages. Cells were cultivated at 37 °C, 5% CO2 and 95% relative humidity on silicon wafers from crystec (Berlin, Germany). Cytotoxicityh The WST-1 cell viability assay was used to evaluate the toxicity of Ag 75 Cit NPs according to manufacturer instructions (Roche Diagnostics; Mannheim, Germany). Guehrs et al. J Nanobiotechnol (2017) 15:21 Page 8 of 11 Table 3 Operating parameters for the ICP-MS element XR Parameter Values Rf power 1550 W Ar cooling gas flow rate 15 l/min Ar auxiliary gas flow rate 1 l/min Sample and skimmer cone Nickel Micronebulizer Micromist 200 μl Data acquisition mode Time resolved analysis (TRA) Isotope Ag107, In115 Uptake rate 0.4 ml/min Dwell time 0.1 ms Acquisition time 65 s Table 3 Operating parameters for the ICP-MS element XR Cells were treated 24 h after seeding in 96-well plates and had been treated with 5, 10, 20, 30, 50 and 100 μg/ ml silver NP for 24 h using three technical replicates per dose. As a positive control known to to be toxic to the cells, 10 μl DMSO was used. Interfering NP and cells were removed in a table top centrifuge by centrifugation with maximum speed prior to spectrophotometric read- out. Supernatants were analyzed using a plate reader (TECAN, Switzerland) at 450 nm. The experiment was performed using three independent biological repeats. TEM analysis TEM processing was performed as previously described [30]. Cells in the culture dish were washed with phos- phate buffered saline (PBS) and fixed overnight by immersion with Karnovsky’s fixative at 4 °C. After three washes in 0.1 M cacodylate buffer, postfixation was per- formed with 2% osmium tetroxide in 0.1 M cacodylate buffer for 1 h at 4 °C. After another three washes in 0.1 M cacodylate buffer, cells were removed from the culture dish and centrifuged at 2000×g for 5 min. The resultant pellet was then coated with 1.5% agar (Merck Eurolab, Darmstadt, Germany) for 30 min at 4 °C. Subsequently the agar with the attached cell layer was removed from the wells. The samples were dehydrated in an ascending ethanol series (30–100% alcohol v/v) and embedded in Epon using beem capsules (Plano, Marburg, Germany). Polymerisation was carried out at 60 °C for 24 h. Semi- thin sections (1 μm) were cut on an Ultracut E ultrami- crotome (Reichert-Jung, Vienna, Austria) with a diamond knife, stained as published elsewhere [30] and analyzed by light microscopy. Ultrathin sections (60 nm) were cut with a diamond knife, mounted on copper grids (Plano, Marburg, Germany) and examined with a Zeiss 10CR electron microscope (Jena, Germany). Cell incubation and sample preparation For analysis using FIB/SEM slice and view, the cells were exposed to 10 μg/ml Ag 75 Cit NPs for 24 h. This dose has been selected based on comparisons to doses used in in vivo inhalation studies. In inhalation studies cellular overload corresponds to a cellular dose of approximately 90–120 pg NP per macrophage cell [28, 29]. Therefore, the dose chosen here is a realistic dose likely to occur dur- ing in vivo inhalation studies but should be well below overload conditions und is clearly a non-toxic dose. Cells were washed three times with DPBS before being fixed with paraformaldehyde (4% in DPBS, Carl Roth GmbH, Karlsruhe, Germany). The citrate coating of the silver NPs make sure that their position is preserved while chemi- cal fixation. After that the cells were washed again three times with DPBS. The medium was replaced by serial dilution with acetone (Carl Roth, Karlsruhe, Germany): 30% acetone, 50% acetone, 70% acetone, 90% acetone, two times 95% acetone and three times 100% acetone. Finally, the cells were dried using critical point drying. For ICP-MS analysis the THP-1 cells were cultivated and treated as for the FIB/SEM analysis, i.e. incubated 10 μg/ml Ag 75 Cit NPs for 24 h. The average concentra- tion of NPs in THP-1 cells was determined by ICP-MS after cell digestion. An aliquot of the washed cells con- taining approximately 105 cell/ml was digested overnight with 0.15 ml concentrated nitric acid and 0.05 ml hydro- gen peroxide at room temperature. The digested solution was freshly diluted and the total Ag content uptake per single cell was determined by calibration with dissolved silver standards. Indium was added to the digested sam- ples as internal standard in order to correct the instru- mental variations in sensitivity during the analysis. FIB/SEM slice and view close to the boundaries of the cell shape by threshold- ing and removing the notches by stepwise increasing the boundary shape of the cell shape at this position until it was almost at the same intensity level as the surround- ing area. Finally, the detected boundary of the cell was smoothed by a convolution kernel to remove any remain- ing edges and notches. For FIB/SEM slice and view a FEI Helios NanoLab 600 system (FEI Eindhoven, Netherlands) was used. To increase the electric conductivity of the samples they were coated with platinum (about 5 nm) using ion beam induced deposition (30 kV, 146 pA). After that a single cell was sequentially sliced by the FIB (Ga+ ions using 30 kV, 146 pA). In each step, a slice of the cell (thickness 40 nm) was removed perpendicular to the silicon wafer surface. The remaining cell was imaged by the SEM (5 kV, detecting secondary electrons, magnification 5000×). This process was repeated until the complete cell was consumed and imaged. As SEM amplification and contrast settings remained unchanged during the stack of slice images, the intensity per pixel, and hence also per resulting voxel reflects the amount of second- ary electrons generated with a constant proportionality factor throughout the sliced volume. We denote these units as "arbitrary intensity units" (a.i.u.). Integrating this intensity over space after suitable segmentation results in a measure of particle volume, which is for convenience also measured in a.i.u. Note, that the SEM images were taken under an angle of 52° to the surface normal as FIB and SEM columns are by design not collinear to each other. The resulting distortion of the image can be corrected automatically using computer algorithms. The silver NPs within the cell were detected in a two- step approach. First, the images were low pass filtered for contrast enhancement of the edges of the silver NPs. The rough position of the silver NPs was found by apply- ing an edge detection algorithm on the low-pass filtered images (Roberts edge detection) [31]. In the second step the shape of the silver NPs was refined individually for each cluster in 3D. For that, the mean grey level intensity of each cluster and of the surrounding cell matrix was determined using Otsu’s multi threshold method [32]. Data analysis: cell and silver NP segmentation y g To correct for image drift during the slice and view pro- cess, cross-correlation was used to determine the shift between pairs of images. Afterwards, the distortion caused by the different working angle between FIB and SEM image was corrected by undistorting the image. Due to the preparation process the inner cell matrix was homogeneous which allowed the use of a threshold algorithms to detect the shape of the cell in each image. Before applying this algorithm, a Wiener filter was used to reduce the noise level of the images. As the detected electrons have a specific escape depth l within the cell matrix, silver NPs which are located in deeper layers of the cell will be detected before they are sliced using the FIB [20, 23, 24]. As a consequence the detected volume Vs = (Vs0 + V ′ s) = 160 ± 42 a.i.u. of a single silver NPs appears larger than the actual volume Vs0 (see Fig. 4). Here, V ′ s is the offset volume caused by electrons escaping from deeper layers. The FIBing process produced a trench in the substrate at the bottom of the cell. As this trench moves along with the position of the slicing plane, it can be used to calculate the lower boundary of the cell shape. After- wards the threshold algorithm can be applied. The upper boundary of the cell does not have sharp edges but had a smooth transition. To make sure that the complete cell is segmented from the image, the shape of the cell was extended by 10 pixels via a convolution. To determine the escape depth, the maximum inten- sity (which is normalized to the surrounding noise) of each slice is used for each of the 16 single silver NPs and an exponential function is fitted to the data points (see Fig. 3). For the silver NP shown in Fig. 3, the escape depth is l = 79 nm and the average escape depth for all 16 sil- ver NPs is l = 89 ± 17 nm. V ′ s can now be calculated by multiplying Vs with exp(−1/l × 40 nm) (40 nm is used as each slice has a depth of 40 nm). The result is shown in Table 4. ICP‑MS analysis An ICP-MS Sector Field instrument, ICP-SFMS (Element XR, Thermo Fisher Scientific GmbH, Bremen Germany) equipped with a concentric nebulizer (Micromist 0.2 ml, L90350, AHF) and a conical spray chamber (ML145026, Meinhard) with an impact bead was used for the experi- ment. The ICP-MS instrumental and operational param- eters are given in Table 3. Before analysis, the ICP-MS was tuned using an aqueous multi-element standard solution (1 ng/ml each of Li, In and U) for consistent sensitivity and minimum levels of doubly charged ions and oxide species of Ce. The time-resolved analysis (TRA) mode was used and thus intensities were collected as a function of time (counts per second). The data of this experiment were recorded using Thermo Plasma Lab software. h Silver and Indium ICP Standard, 65% w/w ultrapure grade nitric acid and hydrogen peroxide were purchased from Merck (Darmstadt, Germany). Milli Q water from purification system Millipore gradient, (Merck MilliPore, Darmstadt, Germany) was used for dilution of concen- trated nitric acid. Guehrs et al. J Nanobiotechnol (2017) 15:21 Page 9 of 11 Data analysis: voxel size of a single silver NP l l h l b f d To calculate the total number of NPs and the size of the clusters, the segmented boundary of the silver NPs were used. First, the voxel size of a single NP was analysed. To this end 16 single NPs were identified “manually” without automation. In Fig. 3 the slices through one of these silver NP are shown. In the segmentation process, this silver NP is detected in slices 3, 4 and 5. For better compari- son and to minimize background effects, the intensity of the segmented silver NPs is normalized and then accu- mulated. The average total normalized intensity for all 16 single silver NPs is 160 ± 42 a.i.u. FIB/SEM slice and view This was necessary as the intensity levels of the silver NPs and of the cell can fluctuate with respect to the position of the cluster in the cell. The determined values were then used to segment each cluster of silver NPs within the cell shape. CCM: cell culture medium; CCT: collision cell technique; DLS: dynamic light scattering; DPBS: Dulbecco’s phosphate buffered saline; FCS: fetal calf serum; FIB: focused ion beam; ICP-MS: inductively coupled plasma mass spectrome‑ try; NPs: nanoparticles; MEM: minimal eagle’s medium; SEM: scanning electron microscopy; TEM: transmission electron microscopy. References 1. Kessler R. Engineered nanoparticles in consumer products: understand‑ ing a new ingredient. Environ Health Perspect. 2011;119(3):120–5. c s To analyse larger clusters, the projected clus- ter area Ac normal to the plane of the cross-section needs to be determined to get V ′ c. From Ac the num- ber of projected silver NPs np can be calculated by np = Ac/As. Here, As is the projected area of a single silver NP which is As = 109 ± 16 pixels. This can be used to get V ′ c = np × V ′ s. As a result, the total num- ber of silver NPs within a cluster can be calculated by nc = (Vc −V ′ s × Ac/As)/Vs0. To analyse larger clusters, the projected clus- ter area Ac normal to the plane of the cross-section needs to be determined to get V ′ c. From Ac the num- ber of projected silver NPs np can be calculated by np = Ac/As. Here, As is the projected area of a single silver NP which is As = 109 ± 16 pixels. This can be used to get V ′ c = np × V ′ s. As a result, the total num- ber of silver NPs within a cluster can be calculated by nc = (Vc −V ′ s × Ac/As)/Vs0. 2. Li W-R, Xie X-B, Shi Q-S, Zeng H-Y, Ou-Yang Y-S, Chen Y-B. Antibacterial activity and mechanism of silver nanoparticles on Escherichia coli. Appl Microbiol Biotechnol. 2010;85(4):1115–22. 3. Wilkinson LJ, White RJ, Chipman JK. Silver and nanoparticles of silver in wound dressings: a review of efficacy and safety. J Wound Care. 2011;20(11):543–9. 4. Larguinho M, Baptista PV. Gold and silver nanoparticles for clinical diag‑ nostics—from genomics to proteomics. J Proteom. 2012;75(10):2811–23. 5. Thurn KT, Arora H, Paunesku T, Wu A, Brown EMB, Doty C, Kremer J, Woloschak G. Endocytosis of titanium dioxide nanoparticles in prostate cancer PC-3M cells. Nanomed Nanotechnol Biol Med. 2011;7(2):123–30. 6. Braun GB, Friman T, Pang H-B, Pallaoro A, Hurtado de Mendoza T, Willmore AM, Kotamraju VR, Mann AP, She Z-G, Sugahara KN, Reich NO, Teesalu T, Ruoslahti E. Etchable plasmonic nanoparticle probes to image and quantify cellular internalization. Nat Mater. 2014;13(9):904–11. Competing interests p g The authors declare that they have no competing interests. The authors declare that they have no competing interests. Received: 3 November 2016 Accepted: 8 March 2017 Received: 3 November 2016 Accepted: 8 March 2017 Authors’ contributions The study was planned by AH, SE and EG. Cell were cultured by DW. EG and KH prepared the samples for FIB/SEM. FIB/SEM slice and view was realized by MS and CMG. ICP-MS was done by ALO and NJ. Data analysis was done by EG with input of PH (silver NP segmentation process) and KH. The manuscript was written by EG, SE and AH with input of all co-authors. All authors read and approved the final manuscript. Acknowledgements The project was funded by the German Federal Institute for Risk Assessment (1329-517). In addition, the authors acknowledge funding from their institutes. Data analysis: cluster analysis and total number of silver NPs To calculate the number of silver NPs in a cluster nc , the shape of the cluster needs to be considered to cor- rect for the offset volume of the cluster V ′ c. If the actual volume for a cluster Vc0 can be extracted from the detected volume Vc for each cluster, the number of silver NPs in this cluster can be determined by nc = Vc0/Vs0 . Assume a cluster of two silver NPs. If the silver NPs are located next to each other within the plane of the cross-section, the detected total normalized intensity of the cluster is Vc = V ′ c + Vc0 = 2 × (Vs0 + V ′ s) (see Fig. 4). If both silver NPs are located behind each other in different slices, the secondary electrons emerging from the posterior NP are absorbed by the anterior NP. As a result, the detected total normalized intensity is Vc = V ′ c + Vc0 = 2 × Vs0 + V ′ s (see Fig. 4). References Note, that it is not necessary to correct for the angle between the normal to the plane of the cross-section and the SEM column as the escape of the secondary electrons from the cell matrix is related to the shortest path length within the matrix (which is orthogonal to the plane of the cross section). In the case of two silver NPs located above each other in different slices, it is theoretically possible, that the secondary electrons from the posterior silver NP escape by moving around the anterior silver NP through the cell matrix. But this effect can be neglected as the escape probability decays exponentially with increasing path length. 7. Salvati A, Aberg C, dos Santos T, Varela J, Pinto P, Lynch I, Dawson KA. Experimental and theoretical comparison of intracellular import of polymeric nanoparticles and small molecules: toward models of uptake kinetics. Nanomed Nanotechnol Biol Med. 2011;7(6):818–26. 8. Rashkow JT, Patel SC, Tappero R, Sitharaman B. Quantification of single- cell nanoparticle concentrations and the distribution of these concentra‑ tions in cell population. J R Soc Interface. 2014;11(94):20131152. 8. Rashkow JT, Patel SC, Tappero R, Sitharaman B. 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Data analysis: cell and silver NP segmentation When silver NPs were located close to the boundaries of the cell, the threshold algorithm produced a notch in the outer boundary as the intensity level of the silver NPs in the image was much higher than the intensity level of the cell. This was corrected by detecting the silver NPs Page 10 of 11 Page 10 of 11 Guehrs et al. J Nanobiotechnol (2017) 15:21 Table 4 Intensity parameter for single silver NPs Total intensity Detected volume Vs 160 ± 42 Offset volume V′ s 62 ± 26 Actual volume Vs0 98 ± 18 Table 4 Intensity parameter for single silver NPs Author details 1 1 Institute for Optics and Atomic Physics, Technical University of Berlin, Straße des 17. Juni 135, 10623 Berlin, Germany. 2 Max-Born-Institute for Nonlinear Optics and Short Pulse Spectroscopy, Max‑Born‑Straße 2A, 12489 Berlin, Germany. 3 Institute of Veterinary Anatomy, Free University Berlin, Koserstr. 20, 14195 Berlin, Germany. 4 Department of Chemical and Product Safety, German Federal Institute for Risk Assessment (BfR), Max‑Dohrn‑Str. 8‑10, 10589 Berlin, Germany. 5 Division 1.1 Inorganic Trace Analysis, Federal Institute for Materials Research and Testing (BAM), Richard‑Willstätter‑Str. 11, 12489 Berlin, Germany. 12. Brown AP, Brydson RMD, Hondow NS. Measuring in vitro cellular uptake of nanoparticles by transmission electron microscopy. J Phys Conf Ser. 2014;522(1):012058. 10. Klingberg H, Oddershede L, Loeschner K, Larsen EH, Loft S, Møller P. Uptake of gold nanoparticles in primary human endothelial cells. Toxicol Res. 2015;4(3):655–66. 11. Büchner T, Drescher D, Traub H, Schrade P, Bachmann S, Jakubowski N, Kneipp J. 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https://openalex.org/W3128805201	https://www.nature.com/articles/s41598-021-82288-z.pdf	English	null	Effects of marathon race on selected myokines and sclerostin in middle-aged male amateur runners	Scientific reports	2,021	cc-by	8,737	amateur runners Ewa Śliwicka 1, Tomasz Cisoń 2, Łucja Pilaczyńska‑Szcześniak 3, Andrzej Ziemba 4 & Anna Straburzyńska‑Lupa 5 In recent years, there has been increasing interest in the homeostatic response to extreme exercises, especially in the integrated function of muscle and bone. The aim of this study was to evaluate the effects of a marathon race on selected myokines and sclerostin in 10 male recreational runners (mean age 41 ± 7.7 years). Body composition, bone mineral density (BMD), and the serum concentration of myostatin, irisin, sclerostin, osteoprotegerin (OPG), 25-hydroxyvitamin D (25(OH)D), parathyroid hormone (PTH), high-sensitivity interleukin-6 (hsIL-6), tumor necrosis factor α (TNFα), high-sensitivity C-reactive protein (hsCRP) and myoglobin, were determined 24 h before and 24 h and 72 h after a marathon race. Post-marathon increases were observed in the levels of myostatin (1.2-fold), OPG (1.5-fold), and PTH (1.3-fold), hsIL-6 (1.9-fold), myoglobin (4.1-fold), hsCRP (fivefold), TNFα (2.6-fold), after 24 h; and in myostatin (1.2-fold), irisin (1.1-fold), sclerostin (1.3-fold), OPG (1.3-fold), and PTH (1.4-fold), hsIL-6 (1.4-fold), TNFα (1.9-fold), after 72 h compared to the baseline level. The results show that in response to the marathon run, a complex network of endocrine interactions is initiated. Further research is needed to fully elucidate the long-term impact of prolonged high intensity exercise on the human body. In recent years, long-distance running has become increasingly popular among people of different ages1. Although physical exercise is an important component of a healthy lifestyle, still little is known how homeo- static response to long-lasting exercises performed during a marathon run influences muscle-bone crosstalk2.f In recent years, long-distance running has become increasingly popular among people of different ages1. Although physical exercise is an important component of a healthy lifestyle, still little is known how homeo- static response to long-lasting exercises performed during a marathon run influences muscle-bone crosstalk2.f l A large component of long-distance running involves eccentric muscle contractions, which can lead to differ- ent levels of damage in the muscles and connective and bone tissue3,4. Exercise-induced muscle damage (EIMD) is associated with delayed-onset-muscle-soreness (DOMS), muscle weakness and a decrease range of motion5. EIMD has been associated with inflammatory response, which is crucial for the repair of damaged tissue6. The inflammatory cascade is characterized by an initial proinflammatory response (1.5–24 h after exercise) and anti- inflammatory muscle regenerative response (24–72 h after exercise)6,7. www.nature.com/scientificreports www.nature.com/scientificreports Scientific Reports \| (2021) 11:2813 www.nature.com/scientificreports/ Namely, while myostatin reduces protein synthesis and increases protein degradation in skeletal muscles11, irisin is a pro-myogenic factor that induces skeletal muscle hypertrophy12. Namely, while myostatin reduces protein synthesis and increases protein degradation in skeletal muscles11, irisin is a pro-myogenic factor that induces skeletal muscle hypertrophy12. p y g yp p y Myostatin belongs to the transforming growth factor-β superfamily (TGF β). It is mainly expressed in skeletal muscle as negative regulator of its mass2. Myostatin has also negative impact on bone remodelling, enhancing catabolic and resorptive state trough increased osteoclastogenesis and reduced bone formation2. p g g Irisin is produced primarily by muscles and is released into the circulation during physical activity, result- ing in an increased energy expenditure, oxidative metabolism, and improved glucose metabolism13. Studies on animal models showed that irisin can improve osteoblastogenesis and bone mass14,15.h g The main source of IL-6 release into the circulating blood is the contracting skeletal muscle in response to exercise. IL-6 participates not only in inflammatory response, but also in glucose uptake, fatty acid oxidation and bone metabolism via increasing osteoclast formation and osteoblast differentiation16. f Sclerostin, glycoprotein mainly secreted by osteocytes, acts as an antagonist of bone formation through the canonical Wnt/β-catenin signaling pathway17. The Wnt/β-catenin signaling pathway plays a role in insulin resist- ance, inflammation, metabolic disturbance17, and skeletal muscle regeneration18. Recent studies indicate that sclerostin can also acts as a bone regulator17,18. Robling et al.19 demonstrated that sclerostin expression decreased by loading, leading to increased bone formation. These results suggesting that sclerostin may be a key protein involved in mechanical loading. Moreover, bone forming cells possess nuclear receptors for 1,25(OH)2D and membrane receptors for PTH20. Vitamin D is a hormone that acts and integrates bone and muscle function. Its indirect action is related to calcium and phosphate levels, while direct effects is connected to local activity of the vitamin D receptor in some tissues (e.g., skeletal muscle, adipose tissue). Vitamin D may also indirectly affecting muscle-bone crosstalk via regulation of muscle and bone-derived hormones, like myostatin, IL-6 and sclerostin21. g y PTH is a key hormone in the metabolism of calcium and phosphates, playing an important role in neuro- muscular signaling, muscle contraction and the biosynthesis of adenosine triphosphate (ATP) and other energy substrates. Results One day (24 h) after finishing the marathon, the blood levels of myostatin (1.2-fold), OPG (1.5-fold), and PTH (1.3-fold) (Fig. 1) as well as of increased of myoglobin (4.1-fold), hsCRP (fivefold), TNFα (2.6-fold) and hsIL-6 (1.9-fold) (Fig. 2), compared to the baseline level. After 72 h, the levels of myostatin (1.2-fold), irisin (1.1-fold), sclerostin (1.3-fold), OPG (1.3-fold), and PTH (1.4-fold) (Fig. 1) and also increased TNFα (1.9-fold) and hsIL-6 (1.4-fold) (Fig. 2) compared to the baseline level. In the preliminary study, we observed a significant correlation between TNF-α and myostatin (r = 0.64, p = 0.049). y As shown in Table 1, we found a positive correlation between changes (Δ1-2) in the concentration of myostatin and hsCRP, as well as sclerostin and hsIL-6, and a negative correlation between irisin and TNF-α. The correlation analysis of these changes (Δ1-3) indicated that sclerostin was positively correlated with hsIL-6. Similar correla- tions were observed for the 25(OH)D and irisin levels. A negative relationship was observed between sclerostin and myostatin, as well as PTH and OPG. www.nature.com/scientificreports/ Therefore, exercise can affect the expression and secretion of PTH22.ii hf Although regular physical activity has benefit on muscle and bone health23, a scientific evidence indicates that long distance running can also have adverse effects24,25. Among others, it has been observed that men who run long distances (> 100 km per week) have a decreased bone mass and increased bone turnover compared to controls, which is indicative of an acceleration of bone loss26. Further research into the molecular physiology of physical exercise is need to better understand the mechanisms of exercise-induced health effects and prevent its potentially negative influence. p y gl In this study, we investigated the impact of extreme exertion experienced during a marathon race on selected myokines and sclerostin in middle-aged male amateur runners during the Visegrad Marathon, one of the most difficult marathons in Europe due to changes in altitude. amateur runners l y g pt Skeletal muscles and bones are closely related and play a key role in the physical health of humans8. In response to exercise, these organs communicate not only via mechanotransduction, but also through the endo- crine system via myokines (e.g., myostatin, irisin, IL-6) and osteokines (e.g., sclerostin). In addition, it is nec- essary to take into account the effect of adipokines (e.g., TNF-α released from adipose tissue) on both these secretory organs2. y g Myokines participate in the autocrine regulation of metabolism, angiogenesis, and muscle growth, as well as in the paracrine and endocrine regulation of other tissues and organs, such as bone, liver, brain, and adipose tissue9. Exercise-induced myokines can have an anti-inflammatory effect in acute inflammation (during the course of infection) and chronic low-grade inflammation (due to aging or metabolic disorders)10. gl g g Myostatin and irisin play opposing roles in the functional bone-muscle unit8. Moreover, studies have indic that both myokines have contrasting effects on muscle mass and strength via the IGF-1/Akt/mTOR pathway 1Department of Physiology and Biochemistry, Poznan University of Physical Education, Królowej Jadwigi Str. 27/39, 61‑871 Poznań, Poland. 2Department of Physiotherapy, State University of Applied Science in Nowy Sącz, Nowy Sącz, Poland. 3Faculty of Rehabilitation and Sport, The President Stanisław Wojciechowski State University of Applied Sciences in Kalisz, Kalisz, Poland. 4Department of Applied Physiology, Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland. 5Department of Physical Therapy and Sports Recovery, Poznan University of Physical Education, Poznań, Poland. email: sliwicka@awf.poznan.pl \| https://doi.org/10.1038/s41598-021-82288-z Scientific Reports \| (2021) 11:2813 www.nature.com/scientificreports/ Discussionhi The main findings of our study clearly indicate that the marathon race contributed to a significant increase in the concentrations of myokines (myostatin, irisin, hsIL-6) and sclerostin. At the same time, changes in other bio- chemical parameters associated with the inflammatory response and muscle and bone metabolism were observed.t l Irisin and myostatin are believed to be secreted from skeletal muscles inversely after physical activity27, wherein increasing the amount of irisin could inhibit myostatin synthesis8,28. Our results appear to confirm this relationship: the levels of irisin grew significantly, reaching their highest concentration at 72 h post-marathon compared to the baseline level, while a significant increase in myostatin was observed 24 h post- marathon (the highest value), and plateau at 72 h post-marathon.i Furthermore, when analyzing our baseline data, we identified a positive correlation between myostatin and TNFα, as well as a correlation between the changes (Δ1-2) in the concentration of myostatin and hsCRP. Our results suggest that myostatin has a negative effect on muscle tissue via its involvement in inflammation29.i fl Our results are in agreement with those of previous studies29,30, in which a significant increase in the myostatin levels was observed up to 48–72 h after a single bout of exercise. Several factors may influence the expression of myostatin, including the duration31 or intensity of the physical activity. Pereirea et al.32 used an animal model to show that overtraining leads to inflammation and the upregulation of myostatin. l Research conducted by Kraemer et al.33 showed that prolonged exercise moderate intensity on a treadmill leads to a temporary increase in the amount of circulating irisin, which occurs in the first hour of exercise and decreases after 90 min of exercise and 20 min of recovery. Some authors have pointed out that acute exercise Scientific Reports \| (2021) 11:2813 \| https://doi.org/10.1038/s41598-021-82288-z www.nature.com/scientificreports/ 60.0 80.0 100.0 120.0 140.0 160.0 1 2 3 PTH [pg/mL] 22.0 24.0 26.0 28.0 30.0 32.0 34.0 36.0 1 2 3 25(OH)D [ng/mL] 2.2 2.7 3.2 3.7 4.2 4.7 5.2 5.7 6.2 1 2 3 OPG [pmol/L] 18.0 23.0 28.0 33.0 38.0 1 2 3 Sclerostin [pmol/L] 20.0 22.0 24.0 26.0 28.0 30.0 32.0 34.0 36.0 1 2 3 Myostatin [ng/mL] 6.2 6.7 7.2 7.7 8.2 8.7 9.2 1 2 3 Irisin [ng/mL] a b c d § §§ §§ §§ † e f * §§ Figure 1. Discussionhi Blood concentrations of irisin, myostatin, sclerostin, OPG, 25(OH)D and PTH in male amateur runners before and after the Visegrad Marathon. (a) irisin, (b) myostatin, (c) sclerostin, (d) OPG, (e) 25(OH) D, (f) PTH. 1: before marathon; 2: 24 h after the marathon; 3: 72 h after the marathon. ** p < 0.01 significant differences between measurements before and 24 h after the marathon. * p < 0.01 significant differences between measurements before and 24 h after the marathon. † p < 0.05 significant differences between measurements 24 h and 72 h after the marathon. §§ p < 0.01 significant differences between measurements before and 72 h after the marathon. § p < 0.05 significant differences between measurements before and 72 h after the marathon. 6.2 6.7 7.2 7.7 8.2 8.7 9.2 1 2 3 Irisin [ng/mL] a § 20.0 22.0 24.0 26.0 28.0 30.0 32.0 34.0 36.0 1 2 3 Myostatin [ng/mL] b §§ Myostatin [ng/mL] 2.2 2.7 3.2 3.7 4.2 4.7 5.2 5.7 6.2 1 2 3 OPG [pmol/L] d §§ † f d c 18.0 23.0 28.0 33.0 38.0 1 2 3 Sclerostin [pmol/L] §§ 60.0 80.0 100.0 120.0 140.0 160.0 1 2 3 PTH [pg/mL] 2.2 2.7 3.2 3.7 4.2 4.7 5.2 1 2 3 OPG [pmol/L] f * §§ e 60.0 80.0 100.0 120.0 140.0 160.0 1 2 3 PTH [pg/mL] f * §§ Figure 1. Blood concentrations of irisin, myostatin, sclerostin, OPG, 25(OH)D and PTH in male amateur runners before and after the Visegrad Marathon. (a) irisin, (b) myostatin, (c) sclerostin, (d) OPG, (e) 25(OH) D, (f) PTH. 1: before marathon; 2: 24 h after the marathon; 3: 72 h after the marathon. ** p < 0.01 significant differences between measurements before and 24 h after the marathon. * p < 0.01 significant differences between measurements before and 24 h after the marathon. † p < 0.05 significant differences between measurements 24 h and 72 h after the marathon. §§ p < 0.01 significant differences between measurements before and 72 h after the marathon. § p < 0.05 significant differences between measurements before and 72 h after the marathon. transiently increases the circulating levels of irisin, while chronic exercise training does not cause changes or decreases in the basic level of irisin33,34.f Despite an increasing number of studies, the biological effects of irisin in humans remains largely unknown. Discussionhi Therefore, further research is needed to determine how irisin acts beyond 72 h post-exercise. According to Mazur-Biały et al.35, irisin has potential anti-inflammatory properties, which supports the negative correlation between the changes (Δ1-2) in irisin and TNFα observed in our study. High concentrations of irisin decreased the levels of pro-inflammatory cytokines by suppressing certain signaling pathways, resulting in a reduction of the levels of RANKL35. Research carried out in recent years has demonstrated that sclerostin is important for bone homeostas and its expression in osteocytes has been found to be negatively regulated by mechanical loading37.i esearch carried out in recent years has demonstrated that sclerostin is important for bone home ts expression in osteocytes has been found to be negatively regulated by mechanical loading37.i We observed a significant increase in the levels of sclerostin only 72 h post-marathon compared to the base- line level. In contrast, other studies have reported a range of changes. For example, Kerschan-Schnidl et al.29 observed that the sclerostin levels did not change immediately or 72 h post-run in ultramarathon runners. Phil- lipou et al.38 observed a gradual decrease in the levels of circulating sclerostin up to 48 h after a single bout of eccentric exercise, followed by a slight increase at 120 h post-exercise. On the other hand, a study carried out by Kouvelioti et al.39 on young men reported an increase in sclerostin 5 min after exercise, similar to that observed Scientific Reports \| (2021) 11:2813 \| https://doi.org/10.1038/s41598-021-82288-z www.nature.com/scientificreports/ 2.0 4.0 6.0 8.0 10.0 12.0 14.0 1 2 3 hsIL-6 [pg/mL] 1.5 2.5 3.5 4.5 5.5 6.5 7.5 1 2 3 TNFα [pg/mL] 0 0.02 0.04 0.06 0.08 0.10 0.12 0.14 1 2 3 hsCRP [mg/L] 25.0 75.0 125.0 175.0 225.0 275.0 325.0 1 2 3 Myoglobin [ng/mL] †† a b c d § §§ † †† †† Figure 2. Blood concentrations of myoglobin, hsCRP, TNFα, and hsIL-6 in male amateur runners before and after the Visegrad Marathon. (a) myoglobin, (b) hsCRP, (c) TNFα, (d) hsIL-6. 1: before marathon; 2: 24 h after the marathon; 3: 72 h after the marathon. p < 0.01 significant differences between measurements before and 24 h after the marathon. †† p < 0.01 significant differences between measurements 24 h and 72 h after the marathon. † p < 0.05 significant differences between measurements 24 h and 72 h after the marathon. Discussionhi §§ p < 0.01 significant differences between measurements before and 72 h after the marathon. § p < 0.05 significant differences between measurements before and 72 h after the marathon. 25.0 75.0 125.0 175.0 225.0 275.0 325.0 1 2 3 Myoglobin [ng/mL] a † 0 0.02 0.04 0.06 0.08 0.10 0.12 0.14 1 2 3 hsCRP [mg/L] b †† b a 2.0 4.0 6.0 8.0 10.0 12.0 14.0 1 2 3 hsIL-6 [pg/mL] 1 2 3 d § †† 1.5 2.5 3.5 4.5 5.5 6.5 7.5 1 2 3 TNFα [pg/mL] †† c §§ d c 2 3 Figure 2. Blood concentrations of myoglobin, hsCRP, TNFα, and hsIL-6 in male amateur runners before d ft th Vi d M th ( ) l bi (b) h CRP ( ) TNF (d) h IL 6 1 b f th 2 24 Figure 2. Blood concentrations of myoglobin, hsCRP, TNFα, and hsIL-6 in male amateur runners before and after the Visegrad Marathon. (a) myoglobin, (b) hsCRP, (c) TNFα, (d) hsIL-6. 1: before marathon; 2: 24 h after the marathon; 3: 72 h after the marathon. p < 0.01 significant differences between measurements before and 24 h after the marathon. †† p < 0.01 significant differences between measurements 24 h and 72 h after the marathon. † p < 0.05 significant differences between measurements 24 h and 72 h after the marathon. §§ p < 0.01 significant differences between measurements before and 72 h after the marathon. § p < 0.05 significant differences between measurements before and 72 h after the marathon. igure 2. Blood concentrations of myoglobin, hsCRP, TNFα, and hsIL-6 in male amateur runners before nd after the Visegrad Marathon. (a) myoglobin, (b) hsCRP, (c) TNFα, (d) hsIL-6. 1: before marathon; 2: 24 h t g y g after the marathon; 3: 72 h after the marathon. ** p < 0.01 significant differences between measurements before and 24 h after the marathon. †† p < 0.01 significant differences between measurements 24 h and 72 h after he marathon. † p < 0.05 significant differences between measurements 24 h and 72 h after the marathon. §§ p < 0.01 significant differences between measurements before and 72 h after the marathon. § p < 0.05 significant differences between measurements before and 72 h after the marathon. Discussionhi after high intensity and high impact exercise (running) or without impact exercise (cycling), and no changes 1, 24, and 48 h post-exercise.i p Our findings regarding the levels of sclerostin post-marathon are partly consistent with the results obtained by Kouvelioti et al.39, who studied the changes in the sclerostin levels for up to 48 h after exercise. These observa- tions may be related to the intensity of catabolic processes induced by marathon running. However, the level of sclerostin after 72 h was not measured in their study. Thus, we cannot dismiss the possibility that the increased levels of sclerostin in our study, lasting 72 h, are related to catabolic processes induced by strenuous exercise. In fact, this was confirmed by the positive correlation between the changes (Δ1-2 and Δ1-3) in the sclerostin and hsIL-6 levels observed in our study. y Research has shown that sclerostin acts as a negative regulator of bone mass and strength by inhibiting bone formation37. Myostatin has also been described as a negative regulator of bone metabolism, increasing bone resorption by activating the RANKL signaling pathway8. Irisin, on the other hand, plays a major role in the regu- lation of the bone mass, positively influencing cortical mineral density and geometry14. However, no association was found between irisin and total body and regional BMD in our study. It has been reported that osteogenic exercise inhibits sclerostin and myostatin, but induces irisin40. However, our research has shown that running a marathon with the differences in altitude along the route induces an upregulation of sclerostin, with a steady increase observed 72 h post-run. Our results suggest that such an intense weight-bearing exercise does not have an osteogenic effect and may lead to disturbances in bone metabolism.if f Moreover, the changes in myostatin levels demonstrated in our study suggest a beneficial effect of irisin on myostatin. Recent studies have reported that irisin activates the core-binding factor α-1 and Wnt/β-catenin bone formation pathways40, as well as stimulating new bone formation by increasing the osteoblast activity, thus improving bone mass and strength8. Thus, the inhibition of myostatin by irisin supports the negative correlation observed between the changes (Δ1-3) in the sclerostin and myostatin levels. g y Vitamin D plays an important role in bone and muscle metabolism. In our previous study, vitamin D was found to be involved in the inflammatory response41,42. Discussionhi In our study, a negative correlation was observed between the changes (Δ1-3) in PTH and OPG, confirming that PTH downregulates OPG expression51. The inhibition of OPG expression stimulates osteoclastogenesis and the resorption of osteoclasts, which in turn leads to an increase in the osteoblast pool in the body and stimulates osteoblast activity and bone anabolism22.h observed between the changes (Δ1-3) in PTH and OPG, confirming that PTH downregulates OPG expression51. The inhibition of OPG expression stimulates osteoclastogenesis and the resorption of osteoclasts, which in turn leads to an increase in the osteoblast pool in the body and stimulates osteoblast activity and bone anabolism22. The marathon race with dominance of eccentric muscle contractions induces mechanical damage to muscle fibers and the corresponding biochemical changes in the blood, as confirmed by previous studies38,52,53.i p y y The marathon race with dominance of eccentric muscle contractions induces mechanical damage to mu fibers and the corresponding biochemical changes in the blood, as confirmed by previous studies38,52,53.i ii In our study, a significant increase in the levels of myoglobin was observed 24 h post-marathon, followed by a significant decrease after 72 h. However, the levels did not return to the basal value. Myoglobin is released as a result of the degradation of muscle protein structures after strenuous exercise. Its levels can rise within 30 min of the start of exercise, and can remain high for 5 days, most likely due to low-grade inflammation54.i l We observed a significant increase in the hsIL-6 levels compared to the baseline level 24 h post-marathon, which remained high 72 h after the race. According to Philippou et al.53, the increase in IL-6 concentration may indicate its involvement in the acute phase response and inflammatory state regulation. These authors reported a correlation between IL-6 and OPG levels in the blood after high-intensity exercise with a dominant eccentric contraction component. OPG is a key player in suppressing the RANKL/RANK system and may work together with anti-inflammatory cytokines to inhibit inflammation55.il l y yl In the present study a significant parallel increase in the OPG levels and another classic proinflammatory cytokine, TNFα, were observed 24 and 72 h after the race compared with the baseline level. Some authors have previously suggested that various cytokines (e.g. TNFα) induce the synthesis of OPG by immune cells56. Discussionhi In the current study, no significant changes in the serum levels of 25(OH)D were observed post-exercise, and only a slight decrease was found 72 h post-run. Moreover, Scientific Reports \| (2021) 11:2813 \| https://doi.org/10.1038/s41598-021-82288-z www.nature.com/scientificreports/ Table 1. Spearman’s rank correlation coefficients of tested variables. 1 = before marathon race, 2 = 24 h after marathon race, 3 = 72 h after marathon race. Variables R p-value Δ1-2 Myostatin/Δ1-2 hsCRP 0.64 0.0479 Δ1-2 Irisin/Δ1-2 TNFα − 0.79 0.0061 Δ1-2 Sclerostin/Δ1-2 hsIL-6 0.66 0.0376 Δ1-3 Irisin/Δ1-3 25(OH)D 0.72 0.0190 Δ1-3 TNFα/Δ1-3 Myoglobin 0.79 0.0061 Δ1-3 Sclerostin/Δ1-3 hsIL-6 0.73 0.0158 Δ1-3 Sclerostin/ Δ1-3 Myostatin − 0.66 0.0376 Δ1-3 PTH/ Δ1-3 OPG − 0.82 0.0032 Table 1. Spearman’s rank correlation coefficients of tested variables. 1 = before marathon race, 2 = 24 h after marathon race, 3 = 72 h after marathon race. the positive correlation observed between the changes (Δ1-3) in 25(OH)D and irisin suggest that vitamin D has anti-inflammatory properties, confirmed by the results of previous studies41–45. Research using animal models demonstrated that a decrease in the levels of 25(OH)D in the blood serum depended on the damage to myocytes and were associated with an increase in the PTH levels46,47.it the positive correlation observed between the changes (Δ1-3) in 25(OH)D and irisin suggest that vitamin D has anti-inflammatory properties, confirmed by the results of previous studies41–45. Research using animal models demonstrated that a decrease in the levels of 25(OH)D in the blood serum depended on the damage to myocytes and were associated with an increase in the PTH levels46,47.it In our study, we observed a significant increase in the levels of PTH 24 and 72 h after the marathon compared to the basal value. These results are consistent previous studies, in which an increase in PTH was reported in the late phase of long-lasting exercise, as well as during recovery48,49. p g g g y PTH may contribute to skeletal muscle function. Studies on animal models have shown that PTH receptors are expressed on the cell membrane of skeletal muscle fibers, and PTH has been found to modulate the uptake and release of 25(OH)D by muscle cells50. No association between irisin or myostatin and PTH was observed in the present study. p y PTH stimulates bone turnover. However, depending on other factors, the direction of this stimulus may be towards the formation (anabolic) or resorption (catabolic) of bone. Table 1. Spearman’s rank correlation coefficients of tested variables. 1 = before marathon race, 2 = 24 h after marathon race, 3 = 72 h after marathon race. Discussionhi These changes are consistent with the results of our previous study, in which we suggested that OPG was involved in inhibiting the inflammatory response41. These results are supported by the significant positive correlation observed between the changes (Δ1-3) in myoglobin and TNFα and the tendency in the changes (Δ1-2) in myoglobin and OPG (r = 0.64, p = 0.0537). ( p ) Although TNFα is not thought to rise with exercise, its moderate plasma increase may occur57. Our results confirm the findings of previous studies, wherein TNFα was stimulated by intense endurance exercise (more than 1 h)58,59, suggesting the activation of an inflammatory reaction in response to local damage to active muscles60.l p Although TNFα is not thought to rise with exercise, its moderate plasma increase may occur57. Our results confirm the findings of previous studies, wherein TNFα was stimulated by intense endurance exercise (more than 1 h)58,59, suggesting the activation of an inflammatory reaction in response to local damage to active muscles60.l gg gl y g CRP plays a role in the induction of anti-inflammatory cytokines from circulating monocytes, and it sup- presses the synthesis of pro-inflammatory cytokines from tissue macrophages61. We observed a significant increase in the levels of hsCRP 24 h after marathon, followed by a decrease and the tendency to maintain an elevated level during the 72 h recovery period. In contrast, Niemelä et al.52 reported a steady increase in the CRP levels even 48 h after exercise. These authors found high levels of CRP, a marker of acute phase response and systemic inflammation, in the subjects with symptoms of post-race fatigue, with the highest values of IL-6 and TNFα52. In our study we did not observe osteopenia in any of the study participants, indicating that they were ama runners and did not run more than 70 km/week.i In their review, Scofield and Hecht25 concluded that, despite weight-bearing exercise being widely recognized as beneficial for long-term bone health, recent research suggests that runners and cyclist often have a lower bone mineral density than athletes participating in power and ball sports. This is most likely due to differences in the degree of physical exertion, such as differences in the loading forces and energy expenditure, during exercise24,25. Fredericson et al.24 observed a higher BMD in long-distance runners only at directly loaded sites (e.g. Discussionhi calcaneus), Scientific Reports \| (2021) 11:2813 \| https://doi.org/10.1038/s41598-021-82288-z www.nature.com/scientificreports/ Table 2 Baseline characteristics of the study participants Data are presented as mean±SD Variables Marathon runners (n = 10) Age (years) 40.6 ± 7.68 Body height (cm) 176.3 ± 4.85 Body mass (kg) 74.7 ± 9.52 Fat (%) 22.1 ± 5.67 Fat mass (kg) 16.1 ± 5.79 Lean mass (kg) 55.6 ± 5.68 Total BMD (g/cm2) 1.224 ± 0.090 Total BMD T-score 0.21 ± 0.877 Lumbar spine BMD (g/m2) 1.131 ± 0.078 Lumbar spine BMD T-score − 0.81 ± 0.706 Total femur BMD (g/m2) 1.077 ± 0.138 Total femur BMD T-score − 0.31 ± 0.768 Femoral neck BMD (g/m2) 1.014 ± 0.165 VO2 max (ml kg−1 min−1) 48.8 ± 1.83 Table 2. Baseline characteristics of the study participants. Data are presented as mean ± SD. but not the rest of the skeleton, in comparison to sedentary men. Additionally, 40% of the runners in their study were found to have lower lumbar spine T-scores consistent with osteopenia, according to the criteria of the World Health Organization (WHO). According to the authors, this may be attributed to increased levels of stress hormones, lower testosterone levels, or increased levels of inflammation markers, but may also be explained by an artificially high reference ranges.hi but not the rest of the skeleton, in comparison to sedentary men. Additionally, 40% of the runners in their study were found to have lower lumbar spine T-scores consistent with osteopenia, according to the criteria of the World Health Organization (WHO). According to the authors, this may be attributed to increased levels of stress hormones, lower testosterone levels, or increased levels of inflammation markers, but may also be explained by an artificially high reference ranges. i y g g Our study has some potential limitations that are worth noting. The first is the relatively small sample size. The Visegrad Marathon takes place once a year and is popular among recreational runners. Data was obtained from all of the men who responded positively to our invitation to participate in the study, as placed in the race’s announcement. The second limitation is the fact that any changes in biochemical indicators immediately after the marathon race were not assessed, and these could represent a potential narrow window of changes in the meas- ured variables that was missed. Lastly, the mass spectrometry is recognized as a gold standard of determination of serum irisin levels. Discussionhi However, in our laboratory we were able to perform the measurements of irisin levels using the ELISA kit by Aviscera Bioscience with standard range: 0.8–51.2 ng/ml and sensitivity: 100 pg/ml. The pro- ducer stated that this ELISA kit was formulated by human irisin derived human cells and monoclonal antibody. y y To conclude, our results suggest that in response to a single bout of strenuous exercise lasting more than 3 h, a complex network of endocrine interactions is initiated. Thus, this study provides a basis for further research to fully elucidate the long-term effects of repeated bouts of exercise of high intensity and a long duration. Methods S bj t All measurements were performed one week before the start of the marathon. Physical fitness measurement. Two weeks before the start of the marathon, the study participants per- formed a treadmill exercise test (HP Cosmos Saturn, Germany), as described previously62. The test was initiated at a baseline speed of 6 km/h and then continuously increased. At 3-min intervals, the speed of the treadmill was increased by 2 km/h, up to the maximal speed for a given subject characterized by the lack of increase in minute oxygen uptake despite increased exercise intensity. The test was not preceded by a warm-up session. The cardio- vascular and respiratory parameters were monitored continuously using an ergospirometer (VO2 max Finder; MES, Poland). The heart rate was recorded every 5 s using a Polar Accurex Plus device (Polar Elektro, Finland). Biochemical analyses. Three samples of blood in fasting conditions (between 08:00 AM and 10:00 AM), were obtained from the antecubital vein for biochemical analyses (1) 24 h before the marathon, (2) 24 h after the marathon, (3) 72 h after marathon. The samples were collected with all safety standards into serum-separating tubes (9 ml, S-Monovette, SARSTEDT, Nümbrecht, Germany) and centrifuged at 2000 × g for 10 min at 4 °C. The serum was separated from the sample and stored at − 70 °C. p p Circulating levels of myoglobin, hsCRP, and PTH levels were determined by immunoenzymatic assay using commercially available kits (DRG International Inc., Springfield Township, NJ, USA; test sensitivity: 5 ng/mL, 0.1 mg/L, and 1.57 pg/mL, respectively; intra-assay coefficients of variations (CV): 5.4%, 4.4%, 4.9%, respec- tively; inter-assay CV: 8.3%, 3.3%, 3.2%, respectively), as well as the levels of hsIL-6 and TNFα (R&D Systems Inc., Minneapolis, MN, USA; test sensitivity: 0.039 pg/mL and 5.5 pg/mL, respectively; intra-assay CV: 4.1%, 4.7%, respectively; inter-assay CV: 6.5%, 5.8%, respectively), irisin (Aviscera Bioscience Inc., Santa Clara, CA, USA; test sensitivity: 100 pg/mL; intra-assay CV: 4.2%; inter-assay CV: 5.6%), myostatin (Immundiagostic AG, Bensheim, Germany; test sensitivity: 0.37 ng/mL; intra-assay CV: 9.1%; inter-assay CV: 13.1%), osteoprotegerin (OPG) and sclerostin (Biomedica GmbH & Co KG, Wien, Austria; test sensitivity: 0.07 pmol/L and 3.2 pmol/L, respectively intra-assay precision CV: 2.5%, 6.0%, respectively; inter-assay precision CV: 4.0%, 6.5%, respectively). The serum concentration of 25(OH)D was determined by chemiluminescent immunoassay (CLIA, DiaSorin Liaison, Stillwater, USA; test sensitivity: 4 ng/mL intra-assay precision CV: 5.4%; inter-assay precision CV: 4.9%). Statistical analysis. Methods S bj t Subjects. The study group consisted of 10 male was comprised of participants of the Visegrad Marathon race who responded to the invitation to participate in the study. They regularly trained 4–5 times/week, and ran an average of 58.5 km/week. g All study participants (aged 32–51) were characterized by a good state of physical health, recreational level of physical activity, no injuries or chronic conditions, non-smoking, and not taking any medications or dietary sup- plements that might affect bone health (e.g. vitamin D). The anthropometric, body composition, and bone densi- tometry characteristics of both groups are shown in Table 2. All of the subjects were informed both verbally and in writing of the experimental protocol prior to signing their written consent to participate. The study protocol was approved by the Ethics Committee for Human Research at the Poznań University of Medical Sciences (refer- ence no. 150/14). All experimental procedures were performed in accordance with the Declaration of Helsinki. Marathon race. The running trial took place during the Visegrad Marathon in June (length: 42.195 km, total level difference: 1161 m and total level gain: 491 m). The study participants completed the marathon in times ranging from 3:16:25 to 4:25:20 (mean 3:44:03). The route runs through the Vabec massif in Slovakia and Beskid Sądecki in Poland. The finish line is located in Rytro, at the foot of the Radziejowa Mountain. The route was certified by the Polish Athletics Association (PZLA). On the day of the run, the weather was partly cloudy with a temperature ranging from 10 °C at the start to 23 °C at the finish line, and a relative humidity between 40 and 46%. During the marathon, liberal amounts of fluids and exogenous carbohydrates were available to the runners. Anthropometric, body composition, and bone densitometry measurements. Body mass and height were measured using a certified digital medical scale (model WPT 80/150.O; Radwag, Radom, Poland), with an accuracy of 0.01 kg, and a mechanical measuring rod to measure body height, with an accuracy of 0.5 cm. Scientific Reports \| (2021) 11:2813 \| https://doi.org/10.1038/s41598-021-82288-z www.nature.com/scientificreports/ Dual-energy X-ray absorptiometry (DXA) body composition and BMD measurements were performed on an empty stomach using a GE Lunar Prodigy Primo Full Densitometer with the enCore Body Composition option (GE Healthcare Technologies, USA). The assessment of BMD were acquired for the total body, lumbar spine (L1–L4) and left hip (total femur and femoral neck). Methods S bj t The data are presented as the mean ± standard deviation (SD). The Shapiro–Wilk test was used to check the normality of the data distribution. The assumption on sphericity was tested using Mauch- ley’s test (verifying if variances of certain variables were identical and equal to respective co-variances). One-way analysis of variance (ANOVA) with repeated measures was used to compare one quantitative variable with a normal distribution at three points in time. When ANOVA showed significance, the post-hoc Tukey’s honestly significant different test was used to indicate the measurements tested in three time points of the study that were significantly different. 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E.Ś. and T.C. conducted the experiments. E.Ś. and A.S.L. analyzed the data. E.Ś. and A.S.L. wrote the manuscript. E.Ś., A.Z., and A.S.L. designed, drafted, and critically revised the manuscript. All authors have read and approved the final version of the manuscript and agree with the order of presentation of the authors. p 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/s41598-021-82288-z. Correspondence and requests for materials should be addressed to E.Ś. 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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Additional information Supplementary Information The online version contains supplementary material available at https://doi. org/10.1038/s41598-021-82288-z. Correspondence and requests for materials should be addressed to E.Ś. Reprints and permissions information is available at www.nature.com/reprints. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and nstitutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. 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 Author(s) 2021 https://doi.org/10.1038/s41598-021-82288-z Scientific Reports \| (2021) 11:2813 \|
https://openalex.org/W1696170920	https://europepmc.org/articles/pmc4489206?pdf=render	English	null	Tracking Human Mobility Using WiFi Signals	PloS one	2,015	cc-by	7,650	Piotr Sapiezynski1, Arkadiusz Stopczynski1,2, Radu Gatej3, Sune Lehmann1,4 Piotr Sapiezynski1, Arkadiusz Stopczynski1,2, Radu Gatej3, Sune Lehmann1,4 1 Department of Applied Mathematics and Computer Science, Technical University of Denmark, Kongens Lyngby, Denmark, 2 Media Lab, Massachusetts Institute of Technology, Cambridge, MA, United States of America, 3 Department of Economics, University of Copenhagen, Copenhagen, Denmark, 4 Niels Bohr Institute, University of Copenhagen, Copenhagen, Denmark * pisa@dtu.dk * pisa@dtu.dk a1111 OPEN ACCESS Citation: Sapiezynski P, Stopczynski A, Gatej R, Lehmann S (2015) Tracking Human Mobility Using WiFi Signals. PLoS ONE 10(7): e0130824. doi:10.1371/journal.pone.0130824 Citation: Sapiezynski P, Stopczynski A, Gatej R, Lehmann S (2015) Tracking Human Mobility Using WiFi Signals. PLoS ONE 10(7): e0130824. doi:10.1371/journal.pone.0130824 Academic Editor: Ye Wu, Beijing University of Posts and Telecommunications, CHINA Academic Editor: Ye Wu, Beijing University of Posts and Telecommunications, CHINA Academic Editor: Ye Wu, Beijing University of Posts and Telecommunications, CHINA Received: January 20, 2015 Accepted: May 26, 2015 Published: July 1, 2015 Received: January 20, 2015 Accepted: May 26, 2015 Published: July 1, 2015 Received: January 20, 2015 Accepted: May 26, 2015 Published: July 1, 2015 Abstract We study six months of human mobility data, including WiFi and GPS traces recorded with high temporal resolution, and find that time series of WiFi scans contain a strong latent loca- tion signal. In fact, due to inherent stability and low entropy of human mobility, it is possible to assign location to WiFi access points based on a very small number of GPS samples and then use these access points as location beacons. Using just one GPS observation per day per person allows us to estimate the location of, and subsequently use, WiFi access points to account for 80% of mobility across a population. These results reveal a great opportunity for using ubiquitous WiFi routers for high-resolution outdoor positioning, but also significant privacy implications of such side-channel location tracking. RESEARCH ARTICLE Introduction Copyright: © 2015 Sapiezynski 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. Due to the ubiquity of mobile devices, the collection of large-scale, longitudinal data about human mobility is now commonplace [1]. High-resolution mobility of individuals and entire social systems can be captured through a multitude of sensors available on modern smart- phones, including GPS and sensing of nearby WiFi APs (access points or routers) and cell tow- ers. Similarly, mobility data may be collected from systems designed to enable communication and connectivity, such as mobile phone networks or WiFi systems (e.g. at airports or on com- pany campuses) [2, 3]. Additionally, large companies such as Google, Apple, Microsoft, or Sky- hook, combine WiFi access points with GPS data to improve positioning [4], a practice known as ‘wardriving’. While widely used, the exact utility and mechanics of wardriving are largely unknown, with only narrow and non-systematic studies reported in the literature [5, 6]. As a consequence, it is generally not known how WiFi networks can be used for sensing mobility on a societal scale; this knowledge is proprietary to large companies. The dataset Out of the 130+ participants of the study [20], we selected 63 for which at least 50% of the expected data points are available. The methods of collection, anonymization, and storage of data were approved by the Danish Data Protection Agency, and complies both with local and EU regulations. Written informed consent was obtained via electronic means, where all invited participants read and digitally signed the form with their university credentials. The median period of WiFi scans for these users was 16 seconds, and the median period of GPS sampling was 10 minutes. The data spans a period of 200 days from October 1st, 2012 to April 27th, 2013. Tracking Human Mobility Using WiFi Signals assistants; for example Google Now [16] is a mobile application, which learns users’ habits to, among other services, conveniently provide directions to the next inferred location. Copenhagen. Please direct your queries to Sune Lehmann, the Principal Investigator of the study, at sljo@dtu.dk. Mobility traces are highly unique and identify individuals with high accuracy [17]. Sensitive features can be extracted from mobility data, including home and work locations, visited places, or personality traits [18]. Moreover, location data are considered the most sensitive of all the commonly discussed personal data collected from or via mobile phones [19]. Funding: This work was supported by Villum Foundation, http://villumfoundation.dk/ C12576AB0041F11B/0/ 4F7615B6F43A8EA5C1257AEF003D9930? OpenDocument, Young Investigator programme 2012, High Resolution Networks (SL) and University of Copenhagen, http://dsin.ku.dk/news/ucph_funds/, through the UCPH2016 Social Fabric grant (SL). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Funding: This work was supported by Villum Foundation, http://villumfoundation.dk/ C12576AB0041F11B/0/ Here, we show that a time sequence of WiFi access points is effectively equal to location data. Specifically, having collected both GPS and WiFi data with high temporal resolution (median of 5 minutes for GPS and 16 seconds for WiFi) in a large study [20], we use six months of data for 63 participants to model how lowering the rate of location sampling influences our ability to infer mobility. The study participants are students with heterogeneous mobility pat- terns. They all attend lectures on campus located outside of the city center, but live in dormito- ries and apartments scattered across the metro area at various distances from the university. Competing Interests: The authors have declared that no competing interests exist. By mapping the WiFi data, we are able to quantify details of WiFi-based location tracking, which are usually not available to the general public. We find that the geo-positioning inferred from WiFi access points (APs or routers) could boost efficacy in other data collection contexts, such as research studies. In addition, our findings have significant privacy implications, indi- cating that for practical purposes WiFi data should be considered location data. As we argue in the following sections, this finding is not recognized in current practices of data collection and handling. Data Availability Statement: Data are from Data Availability Statement: Data are from Copenhagen Networks study (http://journals.plos.org/ plosone/article?id=10.1371/journal.pone.0095978). Due to privacy consideration regarding subjects in our dataset, including European Union regulations and Danish Data Protection Agency rules, we cannot make our data publicly available. The data contains detailed information on mobility and daily habits of 63 individuals at a high spatio-temporal resolution. We understand and appreciate the need for transparency in research and are ready to make the data available to researchers who meet the criteria for access to confidential data, sign a confidentiality agreement, and agree to work under our supervision in In the scientific realm, the mobility patterns of entire social systems are important for modeling spreading of epidemics on multiple scales: metropolitan networks [7–9] and global air traffic networks [10, 11]; traffic forecasting [12]; understanding fundamental laws govern- ing our lives, such as regularity [13], stability [14], and predictability [15]. Predictability and stability of human mobility are also exploited by commercial applications such as intelligent PLOS ONE \| DOI:10.1371/journal.pone.0130824 July 1, 2015 1 / 11 PLOS ONE \| DOI:10.1371/journal.pone.0130824 July 1, 2015 Tracking Human Mobility Using WiFi Signals Fig 1. The time coverage provided by the routers with known position depends on who collects the corresponding location data and when it happens. In each subplot the orange line describes the scenario where each individual collects data about themselves and does not share it with others; the blue line corresponds to a system in which the location of routers discovered by one person is made known to other users; the green line presents a situation where each individual can use the common pool of known routers but does not discover access points herself. a. Stability of location. Learning the location of APs seen during the first seven (left panel) or 28 (right panel) days, leads to performance gradually decreasing with time in the personal case (orange line). The histograms of time coverage distribution for day 190 show that this decline is driven by a growing number of people who spend only 10% of time in the locations they visited in the beginning of the observation. The global approach (blue line) does not show this tendency, which indicates that people rotate between common locations rather than moving to entirely new places. b, c. Representativeness of randomly selected locations. Random subsampling with an average period of 24 hours (less than 1% of all available location estimations) is sufficient to find the most important locations in which people spend more than 80% of their time; using an average period of 4 hours (2.5% percent of all available location data) results in 85% coverage. The personal database does not expire since the location is sampled throughout the experiment, not only in the beginning. d. Limited number of important locations. Although querying commercial services for WiFi geolocation is costly, knowing the location of only the 20 most prevalent routers per person in the dataset results in an average coverage of 90%. Since people’s mobility overlaps, there is a benefit of using a global database rather than treating all mobility disjointly. doi:10.1371/journal.pone.0130824.g001 by the routers with known position depends on who collects the corresponding location data and when it Fig 1. The time coverage provided by the routers with known position depends on who collects the corresponding location data and when it happens. In each subplot the orange line describes the scenario where each individual collects data about themselves and does not share it with others; the blue line corresponds to a system in which the location of routers discovered by one person is made known to other users; the green line presents a situation where each individual can use the common pool of known routers but does not discover access points herself. a. Stability of location. Learning the location of APs seen during the first seven (left panel) or 28 (right panel) days, leads to performance gradually decreasing with time in the personal case (orange line). The histograms of time coverage distribution for day 190 show that this decline is driven by a growing number of people who spend only 10% of time in the locations they visited in the beginning of the observation. The global approach (blue line) does not show this tendency, which indicates that people rotate between common locations rather than moving to entirely new places. b, c. Representativeness of randomly selected locations. Random subsampling with an average period of 24 hours (less than 1% of all available location estimations) is sufficient to find the most important locations in which people spend more than 80% of their time; using an average period of 4 hours (2.5% percent of all available location data) results in 85% coverage. The personal database does not expire since the location is sampled throughout the experiment, not only in the beginning. d. Limited number of important locations. Although querying commercial services for WiFi geolocation is costly, knowing the location of only the 20 most prevalent routers per person in the dataset results in an average coverage of 90%. Since people’s mobility overlaps, there is a benefit of using a global database rather than treating all mobility disjointly. doi:10.1371/journal.pone.0130824.g001 subsampling scenario we select a set fraction of available GPS location estimations, each paired with a WiFi scan. Each GPS estimation provides information on the position of all routers seen in the paired scan. This scenario can be realized after the data collection is finished, as the loca- tion estimations are used to locate the WiFi scans which happened both before and after said esti- mations. The results are presented in Fig 1b. Top routers. We select the top routers in a greedy fashion after the data collection is finished. Known routers and coverage In the article we use a simple model of locating the WiFi routers. We consider an access point as known if it occurred in a WiFi scan within one second of a GPS location estimation. The shortcomings of this approach and possible remedies are described in more detail in S1 File. We define time coverage as a fraction of ten-minute bins containing WiFi data in which at least one known router was scanned. For example, let us assume that the user has data in 100 out of 144 timebins during a day, and in 80 of these timebins there is a known router visible. Therefore, that user’s coverage for that day is 80%. The average time coverage for a day is the mean coverage of all users who had any WiFi information in that day. This way our results are independent from missing data caused by imperfections in data collection system deployed in the study. In Fig 1 we present three different approaches to sampling, which we describe here in detail. Initial-period sampling. As presented in Fig 1a, we learn the location of the routers sequentially. With each GPS location estimation accompanied with a WiFi scan, we add the visible access points to the list of known routers. The learning curve can be observed for the first seven days (Fig 1a, left panel) or the first 28 days (Fig 1a, right panel). Random subsampling. In the random PLOS ONE \| DOI:10.1371/journal.pone.0130824 July 1, 2015 2 / 11 Results Ubiquitously available WiFi access points can be used as location beacons, identifying locations based on BSSID (basic service set identifier, uniquely identifying every router) broadcast by APs. These locations are not intrinsically geographical, as the APs do not have geographical coordinates attached. However, since the placement of APs tends to remain fixed, mapping an AP to a location where it was seen once is sufficient to associate all the subsequent scans from the user device with geographical coordinates. See S1 File for details on inferring the geographi- cal locations of routers, as well as identifying (and discarding data from) mobile access points. WiFi networks are ubiquitous. In our population, 92% of all WiFi scans detect at least one access point, and 33% detect more than 10 APs, as shown in Fig 2c. In densely-populated areas, an average of 25 APs are visible in every scan, with population density explaining 50% of the variance of the number of APs, as shown in Fig 2b. WiFi scans containing at least one visible AP can be used for discovering the location of the user, with a typical spatial resolution on the order of tens of meters. We investigate three approaches to using access points as location beacons, all of which enable WiFi-based location tracking even with limited resources: (1) recovering APs’ locations from mobility traces collected during an initial training period (exploiting the long-term stabil- ity of human mobility), (2) recovering APs’ locations from randomly sampled GPS updates (exploiting low entropy of human mobility, see S1 File for distinction between stability and low entropy), and (3) using only the most frequently observed APs for which location can be feasi- bly obtained from external databases. The task is to efficiently assign geographical coordinates (latitude and longitude) to particular APs, so they can be used as beacons for tracking user’s location. In the following sections, we refer to time coverage as the fraction of ten-minute time- bins, in which at least one router with a known location is observed. We sort the routers in descending order by the subsampling scenario we select a set fraction of available GPS location estimations, each paired with a WiFi scan. Each GPS estimation provides information on the position of all routers seen in the paired scan. This scenario can be realized after the data collection is finished, as the loca- tion estimations are used to locate the WiFi scans which happened both before and after said esti- mations. The results are presented in Fig 1b. Top routers. We select the top routers in a greedy fashion after the data collection is finished. We sort the routers in descending order by the 3 / 11 PLOS ONE \| DOI:10.1371/journal.pone.0130824 July 1, 2015 Tracking Human Mobility Using WiFi Signals number of user timebins they occur in. We choose the top one router, and then we select the routers which provide the biggest increase in the number of user timbebins covered. Due to high density of access points, each semantic place is described by presence of several routers, but loca- tion of only one of them has to be established to find the geographic position of the place. In this sampling method we do not rely on our own GPS data—top routers are found purely based on their occurrence in the WiFi scans, regardless of availability of GPS scans within the one second time delta. The results of such sampling are presented in Fig 1e. Data collection scenarios Each subplot in Fig 1 contains series coming from three different simulated collection scenar- ios. In the global scenario, there is a pool of WiFi routers locations estimations coming from all users, and a router is considered known if at least one person has found its location. This scenario simulates the function of such services as for example mobile Google Maps. In the personal scenario each user can only use their own data, a router can be known to them only if they found its location themselves. It simulates collecting data in a disjoint society, where each person frequents different locations. Finally, in the global with no personal data scenario, each user can exploit estimations created by everybody else, but without contributing their own data. Stability of human mobility allows for efficient WiFi-based positioning This decline is evi- dent both when a week (Fig 1a, left panel) and four weeks (right panel) are used for training, with the time coverage dropping 18 percentage points between days 60 and 160. The histo- grams above each plot show the distribution of time coverage in selected points in time (at 7, 80, 190 days respectively). The distribution for day 190 reveals that the expiry of the personal database validity is driven by individuals who significantly altered routines, with 40% of partic- ipants spending only around 10% of time in locations they have visited in the first week. In contrast, when the inferred locations of routers are shared among people (the global database case, represented by a blue line) the information does not expire and shows no decreasing trend during the observation period. This implies that rather than moving to entirely new PLOS ONE \| DOI:10.1371/journal.pone.0130824 July 1, 2015 5 / 11 routers seen during the first seven days (corresponding to 3.5% of the observations, shown in Fig 1a, left panel) provides APs’ locations throughout the rest of the experiment sufficient for recovering 55% of users mobility until the Christmas break around days 75–90. When the location of routers seen by each person is inferred using only this person’s data (the per- sonal-only WiFi database case, shown using an orange line in Fig 1), the information expires with time: there is a stable decrease in time coverage after Christmas break. This decline is evi- dent both when a week (Fig 1a, left panel) and four weeks (right panel) are used for training, with the time coverage dropping 18 percentage points between days 60 and 160. The histo- grams above each plot show the distribution of time coverage in selected points in time (at 7, 80, 190 days respectively). The distribution for day 190 reveals that the expiry of the personal database validity is driven by individuals who significantly altered routines, with 40% of partic- ipants spending only around 10% of time in locations they have visited in the first week. In contrast, when the inferred locations of routers are shared among people (the global database case, represented by a blue line) the information does not expire and shows no decreasing trend during the observation period. PLOS ONE \| DOI:10.1371/journal.pone.0130824 July 1, 2015 Stability of human mobility allows for efficient WiFi-based positioning Human mobility has been shown to remain stable over long periods of time [13]. We find that participants in our study have stable routines, with locations visited in the first one, two, three, and four weeks of the study still visited frequently six months later. Learning the locations of PLOS ONE \| DOI:10.1371/journal.pone.0130824 July 1, 2015 4 / 11 Tracking Human Mobility Using WiFi Signals Fig 2. WiFi routers are located where people live. a: Map of Greater Copenhagen Area, divided into parishes with color indicating average number of routers discovered per scan; rectangle overlay indicates the city center. b: The number of access points visible in each scan is correlated with the population density (r2 = 0.5). Even in low population density areas there are several routers visible on average in each scan. Therefore, knowing the positions of only a subset of routers is enough for precise location sensing. c: Distribution of number of routers per scan. In our dataset 92% of scans contain at least one router. d i 10 1371/j l 0130824 002 Fig 2. WiFi routers are located where people live. a: Map of Greater Copenhagen Area, divided into parishes with color indicating average number of routers discovered per scan; rectangle overlay indicates the city center. b: The number of access points visible in each scan is correlated with the population density (r2 = 0.5). Even in low population density areas there are several routers visible on average in each scan. Therefore, knowing the positions of only a subset of routers is enough for precise location sensing. c: Distribution of number of routers per scan. In our dataset 92% of scans contain at least one router. doi:10.1371/journal.pone.0130824.g002 doi:10.1371/journal.pone.0130824.g002 routers seen during the first seven days (corresponding to 3.5% of the observations, shown in Fig 1a, left panel) provides APs’ locations throughout the rest of the experiment sufficient for recovering 55% of users mobility until the Christmas break around days 75–90. When the location of routers seen by each person is inferred using only this person’s data (the per- sonal-only WiFi database case, shown using an orange line in Fig 1), the information expires with time: there is a stable decrease in time coverage after Christmas break. Stability of human mobility allows for efficient WiFi-based positioning This implies that rather than moving to entirely new routers seen during the first seven days (corresponding to 3.5% of the observations, shown in Fig 1a, left panel) provides APs’ locations throughout the rest of the experiment sufficient for recovering 55% of users mobility until the Christmas break around days 75–90. When the location of routers seen by each person is inferred using only this person’s data (the per- sonal-only WiFi database case, shown using an orange line in Fig 1), the information expires with time: there is a stable decrease in time coverage after Christmas break. This decline is evi- dent both when a week (Fig 1a, left panel) and four weeks (right panel) are used for training, with the time coverage dropping *18 percentage points between days 60 and 160. The histo- grams above each plot show the distribution of time coverage in selected points in time (at 7, 80, 190 days respectively). The distribution for day 190 reveals that the expiry of the personal database validity is driven by individuals who significantly altered routines, with 40% of partic- ipants spending only around 10% of time in locations they have visited in the first week. In contrast, when the inferred locations of routers are shared among people (the global database case, represented by a blue line) the information does not expire and shows no decreasing trend during the observation period. This implies that rather than moving to entirely new PLOS ONE \| DOI:10.1371/journal.pone.0130824 July 1, 2015 5 / 11 Tracking Human Mobility Using WiFi Signals locations, people begin to visit places that are new to them, but familiar to other participants. The histograms of time coverage distribution in both panels of Fig 1a reveal that the individuals are heterogeneous in their mobility. The coverage in most cases is highly affected in the non- personal case (where the person does not collect their own location information, but data from others is used, marked using green in the figures), but 20% of participants retain a coverage of above 80% throughout the observation period, see Fig 1a, left panel. People living and working close to each other (like students in a dormitory) share a major part of their mobility and thus location of the APs they encounter can be estimated using data collected by others. The demonstrated stability of human mobility patterns over long periods has real-life pri- vacy implications. Human mobility can be efficiently captured using infrequent location updates Sampling location randomly across time (Fig 1b), rather than through the initial period (Fig 1a) provides a higher time coverage, which is retained throughout the observation. With around one sample per day per person on average, the location can be inferred 80% of the time in case of global lookup base and 70% in personal case (see Fig 1c, at training fraction of 0.007). h h b f h d b f h l The histograms in Fig 1b confirm that distribution of coverage in the non-personal case is bimodal within our population: mobility of some individuals can effectively be modeled using data from people around them, while patterns of others are so distinct they require using self- collected information. The single-mode distribution of coverage in the personal case and the fact that the distribution is unchanged between day 7 and day 190 show the lack of temporal decline when sampling happens throughout the observation period. The GPS sensor on a mobile device constitutes a major battery drain when active [21], whereas the WiFi frequently scans for networks by default. Our results show that GPS-based location sampling rate can be significantly reduced in order to save battery, while retaining high resolution location information through WiFi scanning. Our analyses also point to another scenario where WiFi time series can result in leaks of personal information. Infrequent location data can be obtained from a person’s (often public) tweets, Facebook updates, or other social networking check-ins and then matched with their WiFi records to track their mobility. Stability of human mobility allows for efficient WiFi-based positioning Denying a mobile application access to location data, even after a short period, may not be enough to prevent it from tracking user’s mobility, as long as its access to WiFi scans is retained. PLOS ONE \| DOI:10.1371/journal.pone.0130824 July 1, 2015 Single-user analysis To illustrate the ubiquity of WiFi access points and how effectively they can be used to infer mobility patterns, we present a small example dataset containing measured and inferred loca- tion information of one of the authors, collected over two days. During the 48 hours of obser- vation, the researcher’s phone was scanning for WiFi with a median period of 44 seconds, measuring on average 19.8 unique devices per scan, recording 3 822 unique access points. Only one scan during the 48 hours was empty, and one scan yielded 113 unique results. Fig 3a shows the corresponding GPS trace collected with a median sampling period of 5 minutes. When dividing the 48 hours of the test period into 10 minute bins, a raw GPS trace provides location estimation in 89% of these bins. Four stop locations are marked with blue circles and include home, two offices, and a food market visited by the researcher. Fig 3b shows the estimation of this trace based on the inferred locations of WiFi routers, see S1 File for detailed information on the location inference. The four stop locations are clearly visible, but the transitions have lower temporal resolution and errors in location estimations. This method provides location information in 97% of temporal bins. Using WiFi increases overall coverage, but might intro- duce errors in location estimation of routers which were only observed shortly, for example during transition periods. Fig 3c shows the estimation of this trace based on the locations of top 8 (0.2%) WiFi routers. The four important locations have been correctly identified, but information on transitions is lost. Information in 95% of temporal bins is available. Finally, Fig 3d shows a graphical representation of how much time the researcher spends in any one of the top eight locations during the observation time. Note that the first four locations account for an overwhelming fraction of the 48 hours. Knowing the physical position of the top routers and having access to WiFi information reveals the location of the user for the majority of the timebins. The details of trajectories become lost as we decrease the number of routers we use to estimate locations. With too few routers might not be possible to determine which of possible routes the subject chose or how long she took to travel through each segment of the trip. Overall human mobility can be effectively captured by top WiFi access points As previously suggested [15], people’s mobility has low entropy and thus a few most prevalent routers can work effectively as proxies for their location. Fig 1d shows that inferring the loca- tion of just 20 top routers per person on average (which, given the median count of 22 000 routers observed per person, corresponds to 0.1% of all routers seen) translates to knowing the location of individuals 90% of the time. Since our population consists of students, who attend classes in different lecture halls in various buildings across the campus, we expect that the num- ber of access points necessary to describe mobility of persons with a fixed work location can be even lower. There are persons in our study, for whom just four access points correspond to 90% of time coverage (see Fig D in S1 File for details). That the mobility of individuals in our sample overlaps is apparent in Fig 1d as the time cov- erage of three top routers in the personal case is the same as in the global coverage using the total of 80 routers (instead of 189 disjoint routers). 6 / 11 PLOS ONE \| DOI:10.1371/journal.pone.0130824 July 1, 2015 Tracking Human Mobility Using WiFi Signals As a consequence, a third party with access to records of WiFi scans and no access to loca- tion data, can effectively determine the location of each individual 90% of time by sending less than 20 queries to commercial services such as Google Geolocation API or Skyhook. Single-user analysis On the other hand, the high temporal resolution of the scans allows for very precise discovery of arrival and departure times and of time spent in transit. Such information has important implications for security and privacy, as it can be used to discover night-watch schedules, find times when the occupants are not home, or efficiently check work time of the employees. PLOS ONE \| DOI:10.1371/journal.pone.0130824 July 1, 2015 Discussion Our world is becoming progressively more enclosed in infrastructures supporting communica- tion, mobility, payments, or advertising. Logs from mobile phone networks have originally been considered only for billing purposes and internal network optimization; today they con- stitute a global database of human mobility and communication networks [13]. Credit card records form high-resolution traces of our spending behaviors [22]. The omnipresent WiFi networks, intended primarily for communication, has now became a location tracking infra- structure, as described here. The pattern is clear: every new cell tower, merchant with credit card terminal, every new private or municipal WiFi network offer benefits to the connected society, but, at the same time, create opportunities and perils of unexpected tracking. Cities entirely covered by WiFi signal provide unprecedented connectivity to citizens and visitors alike; at the same time multiple parties have to incorporate this fact in their policies to limit PLOS ONE \| DOI:10.1371/journal.pone.0130824 July 1, 2015 7 / 11 Tracking Human Mobility Using WiFi Signals Fig 3. 48 hours of location data of one of the authors, with the four visited locations visited marked in blue: home, two offices, and a food market. Even though the author’s phone has sensed 3 822 unique routers in this period, only a few are enough to describe the location more than 90% of time. a. traces recorded with GPS; b. traces reconstructed using all available data on WiFi routers locations—the transition traces are distorted, but all stop locations are visible and the location is known 97% of the time. c. with 8 top routers it is still possible to discover stop locations in which the author spent 95% of the time. In this scenario transitions are lost. d. timeseries showing when during 48 hours each of the top routers were seen. It can be assumed that AP 1 is home, as it’s seen every night, while AP 2 and AP 3 are offices, as they are seen during working hours. The last row shows the combined 95% of time coverage provided by the top 8 routers. doi:10.1371/journal.pone.0130824.g003 Fig 3. 48 hours of location data of one of the authors, with the four visited locations visited marked in blu Fig 3. 48 hours of location data of one of the authors, with the four visited locations visited marked in blue: home, two offices, and a food market. doi:10.1371/journal.pone.0130824.g003 Discussion PLOS ONE \| DOI:10.1371/journal.pone.0130824 July 1, 2015 8 / 11 Tracking Human Mobility Using WiFi Signals In the results, we focused on outdoor positioning with spatial resolution corresponding to WiFi AP coverage: we assume that if at least one AP is discovered in a scan, we can assign the location of this AP to the user. This is a deliberately simple model, described in detail in S1 File, but we consider the resulting spatial resolution sufficient for many aspects of research, such as studying human mobility patterns. The spatial resolution of dozens of meters is higher than for example CDR data [13], which describes the location with the accuracy of hundreds of meters to a few kilometers. Incorporating WiFi routers as location beacons can aid research by drasti- cally increasing temporal resolution without additional cost in battery drain. Students live in multiple dormitories on and outside of campus, take multiple routes com- muting to the university, frequent different places in the city, travel across the country and beyond. While the students spend most of their time within a few dozens of kilometers from their homes, they also make international and intercontinental trips (see Figs B and C in S1 File for details). Such long distance trips are not normally captured in studies based on telecom operator data. Our population is densely-connected and in this respect it is biased, in the same sense as any population of people working in the same location. We do simulate a scenario in which the individuals do not form a connected group by analyzing the results for personal- only database. We expect the obtained results to generalize outside of our study. Our findings connect to an ongoing debate about the privacy of personal data [23]. Location data has been shown to be among the most sensitive categories of personal information [19]. Still, a record of WiFi scans is, in most contexts, not considered a location channel. In the Android ecosystem, which constitutes 85% of global smartphone market in Q2 2014 [24], the permission for applications to passively collect the results of WiFi scans is separate from the location permission; moreover, the Wi-Fi connection information (ACCESS_WIFI_STATE) permission is not considered ‘dangerous’ in the Android framework, whereas both high-accu- racy and coarse location permissions are tagged as such [25]. Discussion Even though the author’s phone has sensed 3 822 unique routers in this period, only a few are enough to describe the location more than 90% of time. a. traces recorded with GPS; b. traces reconstructed using all available data on WiFi routers locations—the transition traces are distorted, but all stop locations are visible and the location is known 97% of the time. c. with 8 top routers it is still possible to discover stop locations in which the author spent 95% of the time. In this scenario transitions are lost. d. timeseries showing when during 48 hours each of the top routers were seen. It can be assumed that AP 1 is home, as it’s seen every night, while AP 2 and AP 3 are offices, as they are seen during working hours. The last row shows the combined 95% of time coverage provided by the top 8 routers. doi:10.1371/journal.pone.0130824.g003 privacy abuse of such infrastructure. Understanding and quantifying the dynamics of privacy and utility of infrastructures is crucial for building connected and free society. Since the creation of comprehensive databases containing geolocation for APs is primarily carried out by large companies [4], one might assume that WiFi based location tracking by ‘small players’, such as research studies or mobile applications, is not feasible. As we have shown above, however, APs can be very efficiently geolocated in a way that covers a large majority of individuals’ mobility patterns. privacy abuse of such infrastructure. Understanding and quantifying the dynamics of privacy and utility of infrastructures is crucial for building connected and free society. Since the creation of comprehensive databases containing geolocation for APs is primarily carried out by large companies [4], one might assume that WiFi based location tracking by ‘small players’, such as research studies or mobile applications, is not feasible. As we have shown above, however, APs can be very efficiently geolocated in a way that covers a large majority of individuals’ mobility patterns. Since the creation of comprehensive databases containing geolocation for APs is primarily carried out by large companies [4], one might assume that WiFi based location tracking by ‘small players’, such as research studies or mobile applications, is not feasible. As we have shown above, however, APs can be very efficiently geolocated in a way that covers a large majority of individuals’ mobility patterns. PLOS ONE \| DOI:10.1371/journal.pone.0130824 July 1, 2015 Discussion While it has been pointed out that Android WiFi permissions may allow for inference of sensitive personal information [26], the effect has not been quantified through real-world data. Here we have shown that inferring location with high temporal resolution can be efficiently achieved using only a small percentage of the WiFi APs seen by a device. This makes it possible for any application to collect scanned access points, report them back, and inexpensively convert these access points into users’ loca- tions. The impact is amplified by the fact that apps may passively obtain results of scans rou- tinely performed by Android system every 15–60 seconds. Such routine scans are even run when the user disables WiFi. See S1 File for additional analysis on data privacy in the Android ecosystem. Developers whose applications declare both location and WiFi permissions are able to use WiFi information to boost the temporal resolution of any collected location information. We have shown that even if the location permission is revoked by the user, or removed by the app developers, an initial collection of both GPS and WiFi data is sufficient to continue high-reso- lution tracking of the user mobility for subsequent months. Many top applications in the Play Store request Wi-Fi connection information but not explicit location permission. Examples from the top charts include prominent apps with more than 100 million users each, such as Candy Crush Saga, Pandora, and Angry Birds, among others. We are not suggesting that these or other applications collect WiFi data for location tracking. These apps, however, do have a de facto capability to track location, effectively circumventing Android permission model and general public understanding. Due to uniqueness of location traces, users can be easily identified across multiple datasets [17]. Our results indicate that any application can use WiFi permission to link users to other public and private identities, using data from Twitter or Facebook (based on geo-tagged tweets and posts), CDR data, geo-tagged payment transactions; in fact any geo-tagged data set. Such PLOS ONE \| DOI:10.1371/journal.pone.0130824 July 1, 2015 9 / 11 Tracking Human Mobility Using WiFi Signals cross-linking is another argument why WiFi scans should be considered a highly sensitive type of data. In our dataset, 92% of WiFi scans have at least one visible AP. Supporting Information S1 File. Additional details on the properties of the data and the employed analysis methods. In this Supporting File we present an example method of inferring the locations of WiFi rout- ers, explain the interplay between the long term stability and low entropy of human mobility, provide a detailed description of the mobility properties of the participants (Figs B and C), show the distributions of time coverage of top routers (Fig D), and explain how Android per- mission model allows apps to access the WiFi information of the user. (PDF) Acknowledgments We thank Yves-Alexandre de Montjoye and Andrea Cuttone for useful discussions. Discussion Even in the most challenging scenario, when there are no globally shared locations and each individual frequents different places, top 20 WiFi access points per person can be efficiently converted into geolocations (using Google APIs or crowd-sourced data) and used as a stable location channel. These results should inform future thinking regarding the collection, use, and data security of WiFi scans. We recommend that WiFi records be treated as strictly as location data. Author Contributions Conceived and designed the experiments: SL AS PS. Performed the experiments: PS AS RG. Analyzed the data: PS RG. Wrote the paper: PS AS RG SL. Conceived and designed the experiments: SL AS PS. Performed the experiments: PS AS RG. A l d h d PS RG W h PS AS RG SL Conceived and designed the experiments: SL AS PS. Performed the experiments: PS AS RG. Analyzed the data: PS RG. Wrote the paper: PS AS RG SL. PLOS ONE \| DOI:10.1371/journal.pone.0130824 July 1, 2015 References 1. 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https://openalex.org/W2142703401	http://diposit.ub.edu/dspace/bitstream/2445/131437/1/558180.pdf	English	null	Search for<mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" display="inline"><mml:mi>b</mml:mi><mml:mo>→</mml:mo><mml:mi>u</mml:mi></mml:math>transitions in<mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" display="inline"><mml:msup><mml:mi>B</mml:mi><mml:mo>−</mml:mo></mml:msup><mml:mo>→</mml:mo><mml:mo stretchy="false">[</mml:mo><mml:msup><mml:mi>K</mml:mi><mml:mo>+</mml:mo></mml:msup><mml:msup><mml:mi>π</mml:mi><mml:mo>−</mml:mo></mml:msup><mml:msup><mml:mi>π</mml:mi><mml:mn>0…	Physical review. D. Particles, fields, gravitation, and cosmology/Physical review. D, Particles, fields, gravitation, and cosmology	2,007	public-domain	7,552	RAPID COMMUNICATIONS RAPID COMMUNICATIONS RAPID COMMUNICATIONS Search for b ! u transitions in B ! K0DK B. Aubert, M. Bona, D. Boutigny, Y. Karyotakis, J. P. Lees, V. Poireau, X. Prudent, V. Tisserand, A. Zghiche, J. Garra Tico,2 E. Grauges,2 L. Lopez,3 A. Palano,3 G. Eigen,4 B. Stugu,4 L. Sun,4 G. S. Abrams,5 M. Battaglia,5 D. N. Brown,5 J. Button-Shafer,5 R. N. Cahn,5 Y. Groysman,5 R. G. Jacobsen,5 J. A. Kadyk,5 L. T. Kerth,5 Yu. G. Kolomensky,5 G. Kukartsev,5 D. Lopes Pegna,5 G. Lynch,5 L. M. Mir,5 T. J. Orimoto,5 M. T. Ronan,5,* K. Tackmann,5 W. A. Wenzel,5 P. del Amo Sanchez,6 C. M. Hawkes,6 A. T. Watson,6 T. Held,7 H. Koch,7 B Lewandowski 7 M Pelizaeus 7 T Schroeder 7 M Steinke 7 D Walker 8 D J Asgeirsson 9 T Cuhadar Donszelmann , , g y, y , , , , , g , J. Garra Tico,2 E. Grauges,2 L. Lopez,3 A. Palano,3 G. Eigen,4 B. Stugu,4 L. Sun,4 G. S. Abrams,5 M. Battaglia,5 D. N. Brown,5 J. Button-Shafer,5 R. N. Cahn,5 Y. Groysman,5 R. G. Jacobsen,5 J. A. Kadyk,5 L. T. Kerth,5 Yu. G. Kolomensky,5 G. Kukartsev,5 D. Lopes Pegna,5 G. Lynch,5 L. M. Mir,5 T. J. Orimoto,5 M. T. Ronan,5,* K. Tackmann,5 W. A. Wenzel,5 P. del Amo Sanchez,6 C. M. Hawkes,6 A. T. Watson,6 T. Held,7 H. Koch,7 A. D. Bukin,11 V. P. Druzhinin,11 V. B. Golubev,11 A. P. Onuchin,11 S. I. Serednyakov,11 Yu. I. Skovpen,11 E. P. Solodov,11 E. C. Martin,12 D. P. Stoker,12 S. Abachi,13 C. Buchanan,13 S. D. Foulkes,14 J. W. Gary,14 F. Liu,14 O. Long,14 B. C. Shen,14 L. Zhang,14 H. P. Paar,15 S. Rahatlou,15 V. Sharma,15 J. W. Berryhill,16 C. Campagnari,16 A. Cunha,16 B. Dahmes,16 T. M. Hong,16 D. Kovalskyi,16 J. D. Richman,16 T. W. Beck,17 A. M. Eisner,17 C. J. Flacco,17 C. A. Heusch,17 1550-7998=2007=76(11)=111101(8) © 2007 The American Physical Society Search for b ! u transitions in B ! K0DK B. Aubert,1 M. Bona,1 D. Boutigny,1 Y. Karyotakis,1 J. P. Lees,1 V. Poireau,1 X. Prudent,1 V. Tisserand,1 A. Zghiche,1 J. Garra Tico,2 E. Grauges,2 L. Lopez,3 A. Palano,3 G. Eigen,4 B. Stugu,4 L. Sun,4 G. S. Abrams,5 M. Battaglia,5 D. N. Brown,5 J. Button-Shafer,5 R. N. Cahn,5 Y. Groysman,5 R. G. Jacobsen,5 J. A. Kadyk,5 L. T. Kerth,5 Yu. G. Kolomensky,5 G. Kukartsev,5 D. Lopes Pegna,5 G. Lynch,5 L. M. Mir,5 T. J. Orimoto,5 M. T. Ronan,5,* K. Tackmann,5 W. A. Wenzel,5 P. del Amo Sanchez,6 C. M. Hawkes,6 A. T. Watson,6 T. Held,7 H. Koch,7 B. Lewandowski,7 M. Pelizaeus,7 T. Schroeder,7 M. Steinke,7 D. Walker,8 D. J. Asgeirsson,9 T. Cuhadar-Donszelmann,9 B. G. Fulsom,9 C. Hearty,9 T. S. Mattison,9 J. A. McKenna,9 A. Khan,10 M. Saleem,10 L. Teodorescu,10 V. E. Blinov,11 A. D. Bukin,11 V. P. Druzhinin,11 V. B. Golubev,11 A. P. Onuchin,11 S. I. Serednyakov,11 Yu. I. Skovpen,11 E. P. Solodov,11 K. Yu. Todyshev,11 M. Bondioli,12 S. Curry,12 I. Eschrich,12 D. Kirkby,12 A. J. Lankford,12 P. Lund,12 M. Mandelkern,12 E. C. Martin,12 D. P. Stoker,12 S. Abachi,13 C. Buchanan,13 S. D. Foulkes,14 J. W. Gary,14 F. Liu,14 O. Long,14 B. C. Shen,14 L. Zhang,14 H. P. Paar,15 S. Rahatlou,15 V. Sharma,15 J. W. Berryhill,16 C. Campagnari,16 A. Cunha,16 B. Dahmes,16 T. M. Hong,16 D. Kovalskyi,16 J. D. Richman,16 T. W. Beck,17 A. M. Eisner,17 C. J. Flacco,17 C. A. Heusch,17 J. Kroseberg,17 W. S. Lockman,17 T. Schalk,17 B. A. Schumm,17 A. Seiden,17 M. G. Wilson,17 L. O. Winstrom,17 E. Chen,18 C. H. Cheng,18 F. Fang,18 D. G. Hitlin,18 I. Narsky,18 T. Piatenko,18 F. C. Porter,18 R. Andreassen,19 G. Mancinelli,19 B. T. Meadows,19 K. Mishra,19 M. D. Sokoloff,19 F. Blanc,20 P. C. Bloom,20 S. Chen,20 W. T. Ford,20 J. F. Hirschauer,20 A. Kreisel,20 M. Nagel,20 U. Nauenberg,20 A. Olivas,20 J. G. Smith,20 K. A. Ulmer,20 S. R. Wagner,20 J. Zhang,20 A. M. Gabareen,21 A. Soffer,21 W. H. Toki,21 R. J. Wilson,21 F. Winklmeier,21 D. D. Altenburg,22 E. Feltresi,22 A. Hauke,22 H. Jasper,22 J. Merkel,22 A. Petzold,22 B. Spaan,22 K. Wacker,22 V. Klose,23 M. J. Kobel,23 H. M. Lacker,23 W. F. Mader,23 R. Nogowski,23 J. Schubert,23 K. R. Schubert,23 R. Schwierz,23 J. E. Sundermann,23 A. Volk,23 D. Bernard,24 G. R. Bonneaud,24 E. Latour,24 V. Lombardo,24 Ch. Thiebaux,24 M. Verderi,24 P. J. Clark,25 W. Gradl,25 F. Muheim,25 S. Playfer,25 A. I. Robertson,25 Y. Xie,25 M. Andreotti,26 D. Bettoni,26 C. Bozzi,26 R. Calabrese,26 A. 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Del Buono,57 Ch. de la Vaissie`re,57 O. Hamon,57 Ph. Leruste,57 J. Malcle`s,57 J. Ocariz,57 A. Perez,57 L. Gladney,58 M. Biasini,59 R. Covarelli,59 E. Manoni,59 C. Angelini,60 G. Batignani,60 S. Bettarini,60 M. Carpinelli,60 R. Cenci,60 A. Cervelli,60 F. Forti,60 M. A. Giorgi,60 A. Lusiani,60 G. Marchiori,60 M. A. Mazur,60 M. Morganti,60 N. Neri,60 PHYSICAL REVIEW D 76, 111101(R) (2007) PHYSICAL REVIEW D 76, 111101(R) (2007) Hollar,79 P. E. Kutter,79 Y. Pan,79 M. Pierini,79 R. Prepost,79 S. L. Wu,79 and H. Neal80 B. A. Petersen,69 L. Wilden,69 S. Ahmed,70 M. S. Alam,70 R. Bula,70 J. A. Ernst,70 V. Jain,70 B. Pan,70 M. A. Saeed,70 F. R. Wappler,70 S. B. Zain,70 W. Bugg,71 M. Krishnamurthy,71 S. M. Spanier,71 R. Eckmann,72 J. L. Ritchie,72 A. M. Ruland,72 C. J. Schilling,72 R. F. Schwitters,72 J. M. Izen,73 X. C. Lou,73 S. Ye,73 F. Bianchi,74 F. Gallo,74 D. Gamba,74 M. Pelliccioni,74 M. Bomben,75 L. Bosisio,75 C. Cartaro,75 F. Cossutti,75 G. Della Ricca,75 L. Lanceri,75 L. Vitale,75 V. Azzolini,76 N. Lopez-March,76 F. Martinez-Vidal,76,x D. A. Milanes,76 A. Oyanguren,76 J. Albert,77 77 77 77 77 77 77 77 78 (The BABAR Collaboration) 1Laboratoire de Physique des Particules, IN2P3/CNRS et Universite´ de Savoie, F-74941 Annecy-Le-Vieux, France 2Universitat de Barcelona, Facultat de Fisica, Departament ECM, E-08028 Barcelona, Spain 3Dipartimento di Fisica and INFN, Universita` di Bari, I-70126 Bari, Italy 4University of Bergen, Institute of Physics, N-5007 Bergen, Norway 5Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA and University of California, Berkeley, California 94720, USA 6University of Birmingham, Birmingham, B15 2TT, United Kingdom 7Ruhr Universita¨t Bochum, Institut fu¨r Experimentalphysik 1, D-44780 Bochum, Germany 8University of Bristol, Bristol BS8 1TL, United Kingdom 9University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z1 10Brunel University, Uxbridge, Middlesex UB8 3PH, United Kingdom 11Budker Institute of Nuclear Physics, Novosibirsk 630090, Russia 12University of California at Irvine, Irvine, California 92697, USA 13University of California at Los Angeles, Los Angeles, California 90024, USA 14University of California at Riverside, Riverside, California 92521, USA 15University of California at San Diego, La Jolla, California 92093, USA 16University of California at Santa Barbara, Santa Barbara, California 93106, USA 17University of California at Santa Cruz, Institute for Particle Physics, Santa Cruz, California 95064, USA 18California Institute of Technology, Pasadena, California 91125, USA 19University of Cincinnati, Cincinnati, Ohio 45221, USA 20University of Colorado, Boulder, Colorado 80309, USA 21Colorado State University, Fort Collins, Colorado 80523, USA 22Universita¨t Dortmund, Institut fu¨r Physik, D-44221 Dortmund, Germany 23Technische Universita¨t Dresden, Institut fu¨r Kern- und Teilchenphysik, D-01062 Dresden, Germany 24Laboratoire Leprince-Ringuet, CNRS/IN2P3, Ecole Polytechnique, F-91128 Palaiseau, France 25University of Edinburgh, Edinburgh EH9 3JZ, United Kingdom 26Dipartimento di Fisica and INFN, Universita` di Ferrara, I-44100 Ferrara, Italy 27Laboratori Nazionali di Frascati dell’INFN, I-00044 Frascati, Italy 28Dipartimento di Fisica and INFN, Universita` di Genova, I-16146 Genova, Italy 29Harvard University, Cambridge, Massachusetts 02138, USA 3Dipartimento di Fisica and INFN, Universita` di Bari, I-70126 Bari, Italy 4 4University of Bergen, Institute of Physics, N-5007 Bergen, Norway 5 5Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA sity of California, Berkeley, California 94720, USA California Institute of Technology, Pasadena, California 91125, USA 19 Technology, Pasadena, California 91125, USA 19University of Cincinnati, Cincinnati, Ohio 45221, USA 20 20University of Colorado, Boulder, Colorado 80309, USA 21Colorado State University, Fort Collins, Colorado 80523, USA 2 22Universita¨t Dortmund, Institut fu¨r Physik, D-44221 Dortmund, Germany titut fu¨r Kern- und Teilchenphysik, D-01062 Dresden, German che Universita¨t Dresden, Institut fu¨r Kern- und Teilchenphysik, D-01062 Dresden, Germany f p y y oire Leprince-Ringuet, CNRS/IN2P3, Ecole Polytechnique, F-91128 Palaiseau, France 25University of Edinburgh, Edinburgh EH9 3JZ, United Kingdom 26Dipartimento di Fisica and INFN, Universita` di Ferrara, I-44100 Ferrara, Italy 27 27Laboratori Nazionali di Frascati dell’INFN, I-00044 Frascati, Italy imento di Fisica and INFN, Universita` di Genova, I-16146 Genova, Italy 29 28Dipartimento di Fisica and INFN, Universita` di Genova, I-16146 Genova, Italy 29Harvard University Cambridge Massachusetts 02138 USA 111101-1 111101-1 PHYSICAL REVIEW D 76, 111101(R) (2007) A. J. S. Smith,62 A. V. Telnov,62 E. Baracchini,63 F. Bellini,63 G. Cavoto,63 A. D’Orazio,63 D. del Re,63 E. Di Marco,63 A. J. S. Smith,62 A. V. Telnov,62 E. Baracchini,63 F. Bellini,63 G. Cavoto,63 A. D’Orazio,63 D. del Re,63 E. Di Marco,63 R. Faccini,63 F. Ferrarotto,63 F. Ferroni,63 M. Gaspero,63 P. D. Jackson,63 L. Li Gioi,63 M. A. Mazzoni,63 S. Morganti,63 G. Piredda,63 F. Polci,63 F. Renga,63 C. Voena,63 M. Ebert,64 T. Hartmann,64 H. Schro¨der,64 R. Waldi,64 T. Adye,65 G. Castelli,65 B. Franek,65 E. O. Olaiya,65 S. Ricciardi,65 W. Roethel,65 F. F. Wilson,65 R. Aleksan,66 S. Emery,66 M. Escalier,66 A. Gaidot,66 S. F. Ganzhur,66 G. Hamel de Monchenault,66 W. Kozanecki,66 G. Vasseur,66 Ch. Ye`che,66 R. Faccini,63 F. Ferrarotto,63 F. Ferroni,63 M. Gaspero,63 P. D. Jackson,63 L. Li Gioi,63 M. A. Mazzoni,63 S. Morganti,63 G. Piredda,63 F. Polci,63 F. Renga,63 C. Voena,63 M. Ebert,64 T. Hartmann,64 H. Schro¨der,64 R. Waldi,64 T. Adye,65 G. Castelli,65 B. Franek,65 E. O. Olaiya,65 S. Ricciardi,65 W. Roethel,65 F. F. Wilson,65 R. Aleksan,66 S. Emery,66 R. Faccini,63 F. Ferrarotto,63 F. Ferroni,63 M. Gaspero,63 P. D. Jackson,63 L. Li Gioi,63 M. A. Mazzoni,63 S. Morganti,63 G. Piredda,63 F. Polci,63 F. Renga,63 C. Voena,63 M. Ebert,64 T. Hartmann,64 H. Schro¨der,64 R. Waldi,64 T. Adye,65 G. Castelli,65 B. Franek,65 E. O. Olaiya,65 S. Ricciardi,65 W. Roethel,65 F. F. Wilson,65 R. Aleksan,66 S. Emery,66 M. Escalier,66 A. Gaidot,66 S. F. Ganzhur,66 G. Hamel de Monchenault,66 W. Kozanecki,66 G. Vasseur,66 Ch. Ye`che,66 M. Zito,66 X. R. Chen,67 H. Liu,67 W. Park,67 M. V. Purohit,67 J. R. Wilson,67 M. T. Allen,68 D. Aston,68 R. Bartoldus,68 P. Bechtle,68 N. Berger,68 R. Claus,68 J. P. Coleman,68 M. R. Convery,68 J. C. Dingfelder,68 J. Dorfan,68 G. P. Dubois-Felsmann,68 W. Dunwoodie,68 R. C. Field,68 T. Glanzman,68 S. J. Gowdy,68 M. T. Graham,68 P. Grenier,68 C. Hast,68 T. Hryn’ova,68 W. R. Innes,68 J. Kaminski,68 M. H. Kelsey,68 H. Kim,68 P. Kim,68 M. L. Kocian,68 D. W. G. S. Leith,68 S. Li,68 S. Luitz,68 V. Luth,68 H. L. Lynch,68 D. B. MacFarlane,68 H. Marsiske,68 R. Messner,68 D. R. Muller,68 C. P. O’Grady,68 I. Ofte,68 A. Perazzo,68 M. Perl,68 T. Pulliam,68 B. N. Ratcliff,68 A. Roodman,68 A. A. Salnikov,68 R. H. Schindler,68 J. Schwiening,68 A. Snyder,68 J. Stelzer,68 D. Su,68 M. K. Sullivan,68 K. Suzuki,68 S. K. Swain,68 J. M. Thompson,68 J. Va’vra,68 N. van Bakel,68 A. P. Wagner,68 M. Weaver,68 W. J. Wisniewski,68 M. Wittgen,68 D. H. RAPID COMMUNICATIONS B. AUBERT et al. B. AUBERT et al. PHYSICAL REVIEW D 76, 111101(R) (2007) Wright,68 A. K. Yarritu,68 K. Yi,68 C. C. Young,68 P. R. Burchat,69 A. J. Edwards,69 S. A. Majewski,69 B. A. Petersen,69 L. Wilden,69 S. Ahmed,70 M. S. Alam,70 R. Bula,70 J. A. Ernst,70 V. Jain,70 B. Pan,70 M. A. Saeed,70 F. R. Wappler,70 S. B. Zain,70 W. Bugg,71 M. Krishnamurthy,71 S. M. Spanier,71 R. Eckmann,72 J. L. Ritchie,72 A. M. Ruland,72 C. J. Schilling,72 R. F. Schwitters,72 J. M. Izen,73 X. C. Lou,73 S. Ye,73 F. Bianchi,74 F. Gallo,74 D. Gamba,74 M. Pelliccioni,74 M. Bomben,75 L. Bosisio,75 C. Cartaro,75 F. Cossutti,75 G. Della Ricca,75 L. Lanceri,75 L. Vitale,75 V. Azzolini,76 N. Lopez-March,76 F. Martinez-Vidal,76,x D. A. Milanes,76 A. Oyanguren,76 J. Albert,77 Sw. Banerjee,77 B. Bhuyan,77 K. Hamano,77 R. Kowalewski,77 I. M. Nugent,77 J. M. Roney,77 R. J. Sobie,77 P. F. Harrison,78 J. Ilic,78 T. E. Latham,78 G. B. Mohanty,78 M. Pappagallo,78,k H. R. Band,79 X. Chen,79 S. Dasu,79 K. T. Flood,79 J. J. Hollar,79 P. E. Kutter,79 Y. Pan,79 M. Pierini,79 R. Prepost,79 S. L. Wu,79 and H. Neal80 y y M. Escalier,66 A. Gaidot,66 S. F. Ganzhur,66 G. Hamel de Monchenault,66 W. Kozanecki,66 G. Vasseur,66 Ch. Ye`che,66 M. Zito,66 X. R. Chen,67 H. Liu,67 W. Park,67 M. V. Purohit,67 J. R. Wilson,67 M. T. Allen,68 D. Aston,68 R. Bartoldus,68 P. Bechtle,68 N. Berger,68 R. Claus,68 J. P. Coleman,68 M. R. Convery,68 J. C. Dingfelder,68 J. Dorfan,68 G. P. Dubois-Felsmann,68 W. Dunwoodie,68 R. C. Field,68 T. Glanzman,68 S. J. Gowdy,68 M. T. Graham,68 P. Grenier,68 C. Hast,68 T. Hryn’ova,68 W. R. Innes,68 J. Kaminski,68 M. H. Kelsey,68 H. Kim,68 P. Kim,68 M. L. Kocian,68 B. A. Petersen,69 L. Wilden,69 S. Ahmed,70 M. S. Alam,70 R. Bula,70 J. A. Ernst,70 V. Jain,70 B. Pan,70 M. A. Saeed,70 F. R. Wappler,70 S. B. Zain,70 W. Bugg,71 M. Krishnamurthy,71 S. M. Spanier,71 R. Eckmann,72 J. L. Ritchie,72 A. M. Ruland,72 C. J. Schilling,72 R. F. Schwitters,72 J. M. Izen,73 X. C. Lou,73 S. Ye,73 F. Bianchi,74 F. Gallo,74 D. Gamba,74 M. Pelliccioni,74 M. Bomben,75 L. Bosisio,75 C. Cartaro,75 F. Cossutti,75 G. Della Ricca,75 L. Lanceri,75 L. Vitale,75 V. Azzolini,76 N. Lopez-March,76 F. Martinez-Vidal,76,x D. A. Milanes,76 A. Oyanguren,76 J. Albert,77 Sw. Banerjee,77 B. Bhuyan,77 K. Hamano,77 R. Kowalewski,77 I. M. Nugent,77 J. M. Roney,77 R. J. Sobie,77 P. F. Harrison,78 J. Ilic,78 T. E. Latham,78 G. B. Mohanty,78 M. Pappagallo,78,k H. R. Band,79 X. Chen,79 S. Dasu,79 K. T. Flood,79 J. J. x ‡Also with Universita` della Basilicata, Potenza, Italy. F-91898 ORSAY Cedex, France Deceased. x ‡Also with Universita` della Basilicata, Potenza, Italy. g p k xAlso with Universitat de Barcelona, Facultat de Fisica, Departament ECM, E-08028 Barcelona, Spain , , y F-91898 ORSAY Cedex, France 37Lawrence Livermore National Laboratory, Livermore, California 94550, USA f Liverpool, Liverpool L69 7ZE, United Kingdom 38University of Liverpool, Liverpool L69 7ZE, United Kingdom y f p p g 39Queen Mary, University of London, E1 4NS, United Kingdom y y f g 40University of London, Royal Holloway, Surrey TW20 0EX, United Kingdom 41University of Louisville, Louisville, Kentucky 40292, USA 42University of Manchester, Manchester M13 9PL, United Kingdom 43University of Maryland, College Park, Maryland 20742, USA 44University of Massachusetts, Amherst, Massachusetts 01003, USA ts Institute of Technology, Laboratory for Nuclear Science, Cambridge, Massachusetts 02139, USA 46McGill University, Montre´al, Que´bec, Canada H3A 2T8 i Fisica and INFN, Universita` di Milano, I-20133 M 48University of Mississippi, University, Mississippi 38677, USA 49Universite´ de Montre´al, Physique des Particules, Montre´al, Que´bec, Canada H3C 3J7 50 50Mount Holyoke College, South Hadley, Massachusetts 01075, USA College, South Hadley, Massachusetts 01075, USA 51Dipartimento di Scienze Fisiche and INFN, Universita` di Napoli Federico II, I-80126, Napoli, Italy 53University of Notre Dame, Notre Dame, Indiana 46556, USA 54 of Notre Dame, Notre Dame, Indiana 46556, USA 54Ohio State University, Columbus, Ohio 43210, USA 55 55University of Oregon, Eugene, Oregon 97403, USA 56Dipartimento di Fisica and INFN, Universita` di Padova, I-35131 Padova, Italy di Fisica and INFN, Universita` di Padova, I-35131 e Physique Nucle´aire et de Hautes Energies, IN2P3/CNRS, Universite´ Pierre et Marie Curie-Paris6, site´ Denis Diderot-Paris7, F-75252 Paris, France Universite´ Denis Diderot-Paris7, F-75252 Paris, France ty of Pennsylvania, Philadelphia, Pennsylvania 191 59Dipartimento di Fisica and INFN, Universita` di Perugia, I-06100 Perugia, Italy nto di Fisica, Universita` di Pisa, Scuola Normale Superiore and INFN, I-56127 Pisa, Italy 61 61Prairie View A&M University, Prairie View, Texas 77446, USA 6 62Princeton University, Princeton, New Jersey 08544, USA 64Universita¨t Rostock, D-18051 Rostock, Germany y 65Rutherford Appleton Laboratory, Chilton, Didcot, Oxon, OX11 0QX, United Kingdom 66DSM/Dapnia, CEA/Saclay, F-91191 Gif-sur-Yvette, France 6 67University of South Carolina, Columbia, South Carolina 29208, USA 68 68Stanford Linear Accelerator Center, Stanford, California 94309, USA 69 69Stanford University, Stanford, California 94305-4060, USA 70State University of New York, Albany, New York 12222, USA 1 71University of Tennessee, Knoxville, Tennessee 37996, USA 72University of Texas at Austin, Austin, Texas 78712, USA 3 73University of Texas at Dallas, Richardson, Texas 75083, USA 4 74Dipartimento di Fisica Sperimentale and INFN, Universita` di Torino, I-10125 Torino, Ita 75 76IFIC, Universitat de Valencia-CSIC, E-46071 Valencia, Spain 77 77University of Victoria, Victoria, British Columbia, Canada V8W 3P6 78Department of Physics, University of Warwick, Coventry CV4 7AL, United Kingdom 79 79University of Wisconsin, Madison, Wisconsin 53706, USA 80 80Yale University, New Haven, Connecticut 06511, USA ‡ †Also with Universita` di Perugia, Dipartimento di Fisica, Perugia, Italy. PHYSICAL REVIEW D 76, 111101(R) (2007) 30Universita¨t Heidelberg, Physikalisches Institut, Philosophenweg 12, D-69120 Heidelberg, Germany 31Imperial College London, London, SW7 2AZ, United Kingdom 32University of Iowa, Iowa City, Iowa 52242, USA 33Iowa State University, Ames, Iowa 50011-3160, USA 34Johns Hopkins University, Baltimore, Maryland 21218, USA 35Universita¨t Karlsruhe, Institut fu¨r Experimentelle Kernphysik, D-76021 Karlsruhe, Germany 36Laboratoire de l’Acce´le´rateur Line´aire, IN2P3/CNRS et Universite´ Paris-Sud 11, Centre Scientiﬁque d’Orsay, B. P. PHYSICAL REVIEW D 76, 111101(R) (2007) 34, F-91898 ORSAY Cedex, France 37Lawrence Livermore National Laboratory, Livermore, California 94550, USA 38University of Liverpool, Liverpool L69 7ZE, United Kingdom 39Queen Mary, University of London, E1 4NS, United Kingdom 40University of London, Royal Holloway, Surrey TW20 0EX, United Kingdom and Bedford New College, Egham, Surrey TW20 0EX, United Kingdom 41University of Louisville, Louisville, Kentucky 40292, USA 42University of Manchester, Manchester M13 9PL, United Kingdom 43University of Maryland, College Park, Maryland 20742, USA 44University of Massachusetts, Amherst, Massachusetts 01003, USA 45Massachusetts Institute of Technology, Laboratory for Nuclear Science, Cambridge, Massachusetts 02139, USA 46McGill University, Montre´al, Que´bec, Canada H3A 2T8 47Dipartimento di Fisica and INFN, Universita` di Milano, I-20133 Milano, Italy 48University of Mississippi, University, Mississippi 38677, USA 49Universite´ de Montre´al, Physique des Particules, Montre´al, Que´bec, Canada H3C 3J7 50Mount Holyoke College, South Hadley, Massachusetts 01075, USA 51Dipartimento di Scienze Fisiche and INFN, Universita` di Napoli Federico II, I-80126, Napoli, Italy 52NIKHEF, National Institute for Nuclear Physics and High Energy Physics, NL-1009 DB Amsterdam, The Netherlands 53University of Notre Dame, Notre Dame, Indiana 46556, USA 54Ohio State University, Columbus, Ohio 43210, USA 55University of Oregon, Eugene, Oregon 97403, USA 56Dipartimento di Fisica and INFN, Universita` di Padova, I-35131 Padova, Italy 57Laboratoire de Physique Nucle´aire et de Hautes Energies, IN2P3/CNRS, Universite´ Pierre et Marie Curie-Paris6, Universite´ Denis Diderot-Paris7, F-75252 Paris, France 58University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA 59Dipartimento di Fisica and INFN, Universita` di Perugia, I-06100 Perugia, Italy 60Dipartimento di Fisica, Universita` di Pisa, Scuola Normale Superiore and INFN, I-56127 Pisa, Italy 61Prairie View A&M University, Prairie View, Texas 77446, USA 62Princeton University, Princeton, New Jersey 08544, USA 63Dipartimento di Fisica and INFN, Universita` di Roma La Sapienza, I-00185 Roma, Italy 64Universita¨t Rostock, D-18051 Rostock, Germany 65Rutherford Appleton Laboratory, Chilton, Didcot, Oxon, OX11 0QX, United Kingdom 66DSM/Dapnia, CEA/Saclay, F-91191 Gif-sur-Yvette, France 67University of South Carolina, Columbia, South Carolina 29208, USA 68Stanford Linear Accelerator Center, Stanford, California 94309, USA 69Stanford University, Stanford, California 94305-4060, USA 70State University of New York, Albany, New York 12222, USA 71University of Tennessee, Knoxville, Tennessee 37996, USA 72University of Texas at Austin, Austin, Texas 78712, USA 73University of Texas at Dallas, Richardson, Texas 75083, USA 74Dipartimento di Fisica Sperimentale and INFN, Universita` di Torino, I-10125 Torino, Italy 75Dipartimento di Fisica and INFN, Universita` di Trieste, I-34127 Trieste, Italy 76IFIC, Universitat de Valencia-CSIC, E-46071 Valencia, Spain 77University of Victoria, Victoria, British Columbia, Canada V8W 3P6 78Department of Physics, University of Warwick, Coventry CV4 7AL, United Kingdom 79University of Wisconsin, Madison, Wisconsin 53706, USA 80Yale University, New Haven, Connecticut 06511, USA Also with IPPP, Physics Department, Durham University, Durham DH1 3LE, United Kingdom. ‡ †Also with Universita` di Perugia, Dipartimento di Fisica, Perugia, Italy. Deceased. kAlso with IPPP, Physics Department, Durham University, Durham DH1 3LE, United Kingdom. PHYSICAL REVIEW D 76, 111101(R) (2007) Also with Universitat de Barcelona, Facultat de Fisica, Departament ECM, E-08028 Barcelona, Spain. Also with Universita` della Basilicata, Potenza, Italy. Also with Universita` di Perugia, Dipartimento di Fisica, Perugia, Italy. Deceased. ARCH FOR b ! u TRANSITIONS IN . . . PHYSICAL REVIEW D 76, 111101(R) (2 30Universita¨t Heidelberg, Physikalisches Institut, Philosophenweg 12, D-69120 Heidelberg, Germany 31Imperial College London, London, SW7 2AZ, United Kingdom 32University of Iowa, Iowa City, Iowa 52242, USA 33 33Iowa State University, Ames, Iowa 50011-3160, USA 4 34Johns Hopkins University, Baltimore, Maryland 21218, USA 35Universita¨t Karlsruhe, Institut fu¨r Experimentelle Kernphysik, D-76021 Karlsruhe, Germany 36Laboratoire de l’Acce´le´rateur Line´aire, IN2P3/CNRS et Universite´ Paris-Sud 11, Centre Scientiﬁque d’Orsay, B. P. 34, F 91898 ORSAY C d F k xAlso with Universitat de Barcelona, Facultat de Fisica, Departament ECM, E-08028 Barcelona, Spain. y I. INTRODUCTION where ~m indicates a point in the Dalitz plane m2 K; m2 K0, AD ~m; ~m ADm; m the absolute value, and the strong phase of the D0 ( D0) decay amplitude, and B the strong phase difference between the two interfering B decay amplitudes. Equations (1) and (2) hold when ne- glecting D-mixing effects, which in the standard model (SM) give negligible corrections to [5] and do not affect the rB measurement. Following the discovery of CP violation in B meson decays and the measurement of the angle of the unitarity triangle [1] associated with the Cabibbo-Kobayashi- Maskawa (CKM) quark mixing matrix, the focus has turned toward the measurements of the other angles and . Following Ref. [2], several methods have been proposed to measure the relative weak phase between the B ! D0K amplitude, proportional to the CKM matrix element Vcb (Fig. 1), and the B ! D0K amplitude, proportional to Vub. This weak phase, which by deﬁnition is argV ubVud=V cbVcd, can be measured from the interference that occurs when the D0 and the D0 decay to common ﬁnal states. Determining the angle from the measurements of RADS and AADS requires extracting the strong phases by means of a Dalitz analysis of the three-body decay of the neutral D meson, for which the available statistics are insufﬁcient. However, with the current statistics we can measure RADS and constrain rB by exploiting the fact that in Eq. (1) jCj 1. Since the value of rB is related to the level of interference between the diagrams of Fig. 1, high values of rB lead to a better sensitivity to in any mea- surement involving B ! D0K decays. Thus, rB is a key ingredient for the extraction of from other measurements [6]. As an extension of the method proposed in Ref. [3], we search for B ! K0DK [4], where the CKM- favored B ! D0K decay, followed by the doubly Cabibbo-suppressed D0 ! K0 decay, interferes with the CKM-suppressed B ! D0K decay, followed by the Cabibbo-favored D0 ! K0 decay. Both the Belle and BABAR collaborations have pub- lished similar measurements but in a different decay chain, B ! DK with D ! K [7]. Unlike those measurements, we can take advantage of the smaller value of rD, given by r2 D 0:214 0:008 0:008% [8] in D ! I. INTRODUCTION K0 de- cays as opposed to r2 D 0:362 0:020 0:027% [9] in D ! K decays. This implies that for a given error on RADS, the sensitivity to rB is better. In order to reduce the systematic uncertainties, we mea- sure ratios of decay rates: RADS K0DK K0DK K0DK K0DK r2 B r2 D 2rBrDC cos; (1) AADS K0DK K0DK K0DK K0DK RADS K0DK K0DK K0DK K0DK r2 B r2 D 2rBrDC cos; (1) (1) AADS K0DK K0DK K0DK K0DK PHYSICAL REVIEW D 76, 111101(R) (2007) PHYSICAL REVIEW D 76, 111101(R) (2007) (Received 2 August 2007; published 21 December 2007) We search for decays of a B meson into a neutral D meson and a charged kaon, with the D meson decaying into a charged kaon, a charged pion, and a neutral pion. This ﬁnal state can be reached through the b ! c transition B ! D0K followed by the doubly Cabibbo-suppressed D0 ! K0, or the b ! u transition B ! D0K followed by the Cabibbo-favored D0 ! K0. The interference of these two amplitudes is sensitive to the angle of the unitarity triangle. We present results based on 226 106 ee ! 4S ! B B events collected with the BABAR detector at SLAC. We ﬁnd no signiﬁcant evidence for these decays and we set a limit RADS K0DKK0DK K0DKK0DK < 0:039 at 95% conﬁdence level, which we translate with a Bayesian approach into rB jAB ! D0K=AB ! D0Kj < 0:19 at 95% conﬁdence level. PACS numbers: 13.25.Hw, 14.40.Nd DOI: 10.1103/PhysRevD.76.111101 DOI: 10.1103/PhysRevD.76.111101 B. AUBERT et al. B. AUBERT et al. 111101-3 RAPID COMMUNICATIONS PHYSICAL REVIEW D 76, 111101(R) (2007) not used to reconstruct the B candidate; the angle in the CM frame between the thrust axes of the B candidate and of the detected remainder of the event; the polar angle of the B candidate in the CM frame; the distance of closest approach between the track of the kaon candidate from the B and the trajectory of the reconstructed D meson (this is consistent with zero for signal events, but can be larger in cc events); the distance along the beams between the reconstructed vertex of the B candidate and the vertex of the other tracks in the event, this is consistent with zero for continuum events, but is sensitive to the B lifetime for signal events. The NNet is trained with simulated continuum and signal events. We ﬁnd agreement between the distributions of all six variables in simulation, off-resonance data, and B ! D events. We apply a loose preselection on the NNet (0:4 < NNet < 1:0) with a 90% efﬁciency for signal and a 68% rejection power for continuum, and then use the NNet itself in the likelihood ﬁt described below to fully exploit its discriminating power. The event selection was developed from studies of off- resonance data and B B and continuum events simulated with Monte Carlo (MC) techniques. A large on-resonance data sample of B ! D0, D0 ! K0 events was used to validate several aspects of the simulation and analysis procedure. We refer to this mode as B ! D. A B-meson candidate is characterized by the energy- substituted mass mES s 2 ~p0 ~pB2=E2 0 p2 B q and en- ergy difference E E B s p 2 , where E and p are energy and momentum, the asterisk denotes the CM frame, the subscripts 0 and B refer to the initial ee state and B candidate, respectively, and s is the square of the CM energy. For signal events, mES is centered around the B mass with a resolution of about 2:5 MeV=c2, and E is centered at zero with a resolution of 17 MeV. y p Both kaon candidates are required to satisfy kaon iden- tiﬁcation criteria, which are based on the speciﬁc ioniza- tion loss measured in the tracking devices and on the Cherenkov angles measured in the DIRC and are typically 85% efﬁcient, depending on momentum and polar angle. PHYSICAL REVIEW D 76, 111101(R) (2007) Misidentiﬁcation rates are at the 2% level. The 0 candi- dates are reconstructed as pairs of photon candidates in the EMC, each with energy larger than 70 MeV and a lateral shower proﬁle consistent with an electromagnetic deposit. These pairs must have a total energy greater than 200 MeV and 118 < m < 145 MeV=c2. To account for the corre- lation between the tails in the distribution of the K0 invariant mass and the 0 candidate mass, we require the difference between the two measured masses to be within 32:5 MeV=c2 of the expected value of mD0 m0 1729:5 MeV=c2 [12], retaining 90% of the signal. The remaining background from other B ! h1h20Dh 3 [4] modes is reduced by removing events where the invari- ant mass of any h1h20 candidate, with any particle-type assignment other than the signal hypothesis, is consistent with the D0 meson mass, retaining 92% of the signal. Considering both the case where the two kaons have the same and the opposite charge (referred to as ‘‘same-sign’’ and ‘‘opposite-sign’’ samples, respectively), 28 621 events survive the selection described above and the loose re- quirements jEj < 100 MeV and mES > 5:2 GeV=c2. While the dominant background comes from continuum events, there is still a nonnegligible contribution from 4S ! B B events (denoted ‘‘B B’’ in the following). We consider separately the B ! D background, since it differs from the signal only in the E distribution. For this decay mode the opposite-sign B ! D0 amplitude is suppressed by a factor rB2, where 0:22 is the sine of the Cabibbo angle. Therefore we expect to ﬁnd a non- negligible B ! D background only in the same-sign sample. PHYSICAL REVIEW D 76, 111101(R) (2007) PHYSICAL REVIEW D 76, 111101(R) (2007) with the BABAR detector at the PEP-II B factory at SLAC [10]. Approximately 7% of the collected data (15:8 fb1) have a center-of-mass (CM) energy 40 MeV below the 4S resonance. These ‘‘off-resonance’’ data are used to study backgrounds from continuum events, ee ! q q (q u; d; s, or c). The BABAR detector is described else- where [11]. Charged-particle tracking is provided by a ﬁve- layer silicon vertex tracker (SVT) and a 40-layer drift chamber (DCH). In addition to providing precise position information for tracking, the SVT and DCH also measure the speciﬁc ionization (dE=dx), which is used for particle identiﬁcation of low-momentum charged particles. At higher momenta (p > 0:7 GeV=c) pions and kaons are identiﬁed by Cherenkov radiation detected in a ring- imaging device (DIRC). The position and energy of pho- tons are measured with an electromagnetic calorimeter (EMC) consisting of 6580 thallium-doped CsI crystals. These systems are mounted inside a 1.5T solenoidal super- conducting magnet. not used to reconstruct the B candidate; the angle in the CM frame between the thrust axes of the B candidate and of the detected remainder of the event; the polar angle of the B candidate in the CM frame; the distance of closest approach between the track of the kaon candidate from the B and the trajectory of the reconstructed D meson (this is consistent with zero for signal events, but can be larger in cc events); the distance along the beams between the reconstructed vertex of the B candidate and the vertex of the other tracks in the event, this is consistent with zero for continuum events, but is sensitive to the B lifetime for signal events. II. EVENT RECONSTRUCTION AND SELECTION The results presented in this paper are based on 226 1064S ! B B decays collected between 1999 and 2004 2rBrDS sin=RADS; (2) where rB j AB! D0K AB!D0K j, r2 D BD0!K0 BD0!K0 . The C and S parameters are deﬁned as 2rBrDS sin=RADS; (2) FIG. 1. Feynman diagrams for the CKM-favored B ! D0K and the CKM- and color-suppressed B ! D0K decays. C R AD ~m AD ~m cos ~m ~m Bd ~m R j AD ~mj2d ~m R jAD ~mj2d ~m q ; (3) (3) S R AD ~m AD ~m sin ~m ~m Bd ~m R j AD ~mj2d ~m R jAD ~mj2d ~m q ; (4) (4) FIG. 1. Feynman diagrams for the CKM-favored B ! D0K and the CKM- and color-suppressed B ! D0K decays. 111101-4 RAPID COMMUNICATIONS RAPID COMMUNICATIONS SEARCH FOR b ! u TRANSITIONS IN . . III. LIKELIHOOD FIT AND RESULTS After these requirements, the background is mostly due to D0- D0 pair production in ee ! cc events, with D0 ! K0 and D ! K. To discriminate between the sig- nal and this dominant background we use a neural network (NNet) with six quantities that distinguish continuum and B B events: L0 P ipi and L2 P ipicos2i, both calcu- lated in the CM frame, where pi is the momentum of particle i, i is its angle relative to the thrust axis of the B candidate, and the sum runs over all tracks and clusters The signal and background yields are extracted by maximizing the extended likelihood L eN0 QN i 1 Li~xi=N!. Here N0 NDK Ncont NBB ND is the sum of the yields of the signal and the three background contributions (including both the same- sign and the opposite-sign components), ~x fNNet; E; mESg, N is the number of events in the selected sample, and the likelihood of the individual events (Li) is 111101-5 RAPID COMMUNICATIONS IG. 2 (color online). Likelihood ﬁt projections of the NNet, E, and mES distributions separately for the same (top) and opposit bottom) sign samples. To visually enhance the signal, the distributions for the latter sample are shown after cuts, with a 67% signa fﬁciency, on the ratios between the signal and the total likelihood of all the variables other than the one shown. The points with erro ars represent the data, while the dashed, dashed-dotted, and solid lines represent the contributions from continuum, B B, and D ackgrounds, respectively. The dotted line represents the signal contribution, visible only in the same-sign sample. . AUBERT et al. PHYSICAL REVIEW D 76, 111101(R) (2007 B. AUBERT et al. PHYSICAL REVIEW D 76, 111101(R) PHYSICAL REVIEW D 76, 111101(R) (2007) B. AUBERT et al. PHYSICAL REVIEW D 76, 111101(R) (2007) PHYSICAL REVIEW D 76, 111101(R) (2007) FIG. 2 (color online). Likelihood ﬁt projections of the NNet, E, and mES distributions separately for the same (top) and opposite (bottom) sign samples. To visually enhance the signal, the distributions for the latter sample are shown after cuts, with a 67% signal efﬁciency, on the ratios between the signal and the total likelihood of all the variables other than the one shown. PHYSICAL REVIEW D 76, 111101(R) (2007) region, for each variable r RADS or rB, as the interval where Lr > Lmin and 68% R Lr>Lmin Lrdr. Figure 3 shows LRADS and LrB. We set a 95% conﬁdence level (C.L.) limit by integrating the likelihood, starting from RADS 0 (rB 0), thus excluding unphys- ical values, and we deﬁne the 68% C.L. region, for each variable r RADS or rB, as the interval where Lr > Lmin and 68% R Lr>Lmin Lrdr. Equation (5) assumes equal efﬁciencies for the same- and opposite-sign signal samples, regardless of the differ- ence in the Dalitz structure. This has been demonstrated to be true in MC within a relative statistical error of 4%. We then consider this as a systematic error on RADS. We also repeat the ﬁt by varying the PDF parameters obtained from MC within their statistical errors and by estimating fRS=WS cont on off-resonance data and fRS=WS DK on exclusively recon- structed D events. To account for the observed variations, we assign a 0.0076 systematic error on RADS. The uncer- tainty due to B decays with distributions similar to the signal, in particular B ! D , D K, D K , and KK0, is estimated by varying their branching fractions within their known errors and found to be 6 105 on RADS, and therefore negligible. The quality of the simula- tion of B decays to ﬁnal states with charm mesons that might mimic the signal has been checked by comparing data and MC samples in the sidebands of the E distribu- tion where these decays dominate. Similarly, we searched the sidebands of the mD0 m0 distribution for back- ground from charmless B decays and found no evidence of it. IV. CONCLUSIONS In summary, we measure the ratio of the rate for the B ! K 0DK decay to the favored decay B ! K 0DK to be RADS 0:0130:012 0:010. While this re- sult is consistent with and similar in sensitivity to the completely independent previously published results [7], it is obtained using a different D decay mode. Because the measurement is not statistically signiﬁcant, we set a 95% C.L. limit RADS < 0:039. We use this information to infer the ratio between the rates of the B ! D0K and B ! D0K decays to be rB 0:091 0:059 and con- sequently set a limit rB < 0:19 at 95% C.L. III. LIKELIHOOD FIT AND RESULTS The points with error bars represent the data, while the dashed, dashed-dotted, and solid lines represent the contributions from continuum, B B, and D backgrounds, respectively. The dotted line represents the signal contribution, visible only in the same-sign sample. deﬁned as for opposite-sign events. In these equations we have de- ﬁned R parameters for the backgrounds analogous to those for the signal, deﬁned in Eq. (1). The individual probability densitity functions (PDFs) f are derived from MC and are built as the product of one-dimensional distributions of the three variables. The only exception is the mES and E PDF for the D background, where we use a two-dimensional nonparametric distribution [13] due to a nonnegligible correlation between these two variables. The NNet distri- butions are all modeled with a histogram with eight bins between 0.4 and 1. The mES distributions are modeled with a Gaussian in the case of the signal, a threshold function L i~xi NDK 1 RADS fRS DK~xi Ncont 1 Rcont fRS cont~xi NBB 1 RBB fRS BB~xi NDfD~xi (5) for same-sign events and L i~xi NDKRADS 1 RADS fWS DK~xi NcontRcont 1 Rcont fWS cont~xi NBBRBB 1 RBB fWS BB ~xi (6) L i~xi NDKRADS 1 RADS fWS DK~xi NcontRcont 1 Rcont fWS cont~xi NBBRBB 1 RBB fWS BB ~xi (6) (6) 111101-6 PHYSICAL REVIEW D 76, 111101(R) (2007) PHYSICAL REVIEW D 76, 111101(R) (2007) SEARCH FOR b ! u TRANSITIONS IN . . FIG. 3 (color online). Likelihood function for RADS (left) and rB (right). The latter is obtained in a Bayesian approach, assum- ing ﬂat prior distributions for rB and C cos. The 68% and 95% regions are shown in dark and light shading, respectively. [14] in the case of the continuum background, and the sum of a threshold function and a Gaussian function with an exponential tail in the case of the B B background. Finally, the E distributions are parametrized with the sum of two Gaussians in the case of the signal, an exponential in the case of the continuum background, and a sum of two exponentials in the case of the B B background. For mES and E of the B B and continuum background, we use different parameters for same-sign and opposite-sign sample. p We perform the ﬁt by ﬂoating the four total yields (NDK, Ncont, NBB, and ND), the three R variables and the shape parameters of the threshold function used to parametrize the mES distribution for the same- and opposite-sign con- tinuum background separately. Figure 2 shows the distri- butions of the three variables in the selected sample, with the likelihood projections overlaid. The ﬁt yields RADS 0:0130:010 0:004, NDK 14:7 0:6 102, Ncont 239:3 2:1 102, NBB 25:5 1:6 102, ND 6:7 0:4 102, Rcont 3:05 0:07, RBB 0:42 0:07. FIG. 3 (color online). Likelihood function for RADS (left) and rB (right). The latter is obtained in a Bayesian approach, assum- ing ﬂat prior distributions for rB and C cos. The 68% and 95% regions are shown in dark and light shading, respectively. consistent with r2 D 0:214 0:008 0:008% [8]. The likelihood LRADS is obtained by convolving the like- lihood returned by the ﬁt with a Gaussian of width 0.0076, equivalent to the systematic uncertainty. consistent with r2 D 0:214 0:008 0:008% [8]. The likelihood LRADS is obtained by convolving the like- lihood returned by the ﬁt with a Gaussian of width 0.0076, equivalent to the systematic uncertainty. Figure 3 shows LRADS and LrB. We set a 95% conﬁdence level (C.L.) limit by integrating the likelihood, starting from RADS 0 (rB 0), thus excluding unphys- ical values, and we deﬁne the 68% C.L. RAPID COMMUNICATIONS B. AUBERT et al. ACKNOWLEDGMENTS Following a Bayesian approach, we extract rB by deﬁn- ing the posterior distribution We are grateful for the excellent luminosity and machine conditions provided by our PEP-II colleagues, and for the substantial dedicated effort from the computing organiza- tions that support BABAR. The collaborating institutions wish to thank SLAC for its support and kind hospitality. This work is supported by DOE and NSF (USA), NSERC (Canada), IHEP (China), CEA and CNRS-IN2P3 (France), BMBF and DFG (Germany), INFN (Italy), FOM (The Netherlands), NFR (Norway), MIST (Russia), MEC (Spain), and PPARC (United Kingdom). Individuals have received support from the Marie Curie EIF (European Union) and the A. P. Sloan Foundation. L rB R prB; rD; LRADSrB; rD; drDd R prB; rD; LRADSrB; rD; drDddrB ; (7) (7) where C cos, RADSrB; rD; is given in Eq. (1), and prB; rD; is the prior distribution for these three quanti- ties. They are considered uncorrelated, with and rB uniformly distributed in the range of [ 1, 1] and [0, 1], respectively. The prior distribution for rD is a Gaussian 111101-7 PHYSICAL REVIEW D 76, 111101(R) (2007) [1] B. Aubert et al. (BABAR Collaboration), Phys. Rev. Lett. 89, 201802 (2002); K. Abe et al. (Belle Collaboration), Phys. Rev. D 66, 071102 (2002). [7] B. Aubert et al. (BABAR Collaboration), Phys. Rev. D 72, 032004 (2005); M. Saigo et al. (Belle Collaboration), Phys. Rev. Lett. 94, 091601 (2005). [2] M. Gronau and D. Wyler, Phys. Lett. B 265, 172 (1991); M. Gronau and D. London, Phys. Lett. B 253, 483 (1991). [8] B. Aubert et al. (BABAR Collaboration), Phys. Rev. Lett. 97, 221803 (2006). [9] B. Aubert et al. (BABAR Collaboration), Phys. Rev. Lett. 91, 171801 (2003). [3] D. Atwood, I. Dunietz, and A. Soni, Phys. Rev. Lett. 78, 3257 (1997); Phys. Rev. D 63, 036005 (2001). PEP-II Conceptual Design Report, SLAC-0418, 19 [4] Charge conjugation is implied throughout the paper. Also, we use the notation B ! h 1 h 2 0Dh 3 (with hi or K) for the decay chains B ! ~D0h 3 , where ~D0 is either a D0 or a D0 and ~D0 ! h 1 h 2 0. We also refer to h3 as the or K from the B. [11] B. Aubert et al. (BABAR Collaboration), Nucl. Instrum. Methods Phys. Res., Sect. A 479, 1 (2002). [12] S. Eidelman et al. (Particle Data Group), Phys. Lett. B 1 592, 031501 (2004) and 2005 partial update for the 2006 edition. [5] Y. Grossman, A. Soffer, and J. Zupan, Phys. Rev. D 72, 031501 (2005). [13] K. Cranmer, Comput. Phys. Commun. 136, 198 (2001). [6] B. Aubert et al. (BABAR Collaboration), Phys. Rev. Lett. 95, 121802 (2005). [14] H. Albrecht et al. (ARGUS Collaboration), Z. Phys. C 48, 543 (1990). 111101-8 111101-8
https://openalex.org/W4206486127	https://revistas.ucm.es/index.php/PSIC/article/download/PSIC1111120001A/35042	es		Datos de la revista	Psicooncología	2,011	cc-by	177	La editorial Asociación de Psicooncología a los efectos previstos en el artículo 32.1 párrafo segundo de la vigente TRLPI, se opone expresamente a que cualquiera de las páginas de (nombre de la revista o periódico), o partes de ellas, sean utilizadas para la realización de revistas de prensa. Cualquier forma de reproducción, distribución, comunicación pública o transformación de esta obra solo puede ser realizada con la autorización de sus titulares, salvo excepción prevista porla ley. Diríjase a CEDRO (Centro Español de Derechos Reprográficos) si necesita fotocopiar, escanear, reproducir, distribuir, transformar o poner a disposición en Internet algún fragmento de esta obra (www.conlicencia.com ; 91 702 19 70 / 93 272 04 47). PSICO ONCOLOGÍA INVESTIGACIÓN Y CLÍNICA BIOPSICOSOCIAL EN ONCOLOGÍA Volumen 8, Número 1, 2011 Edita: Asociación de Psicooncología de Madrid Facultad de Psicología. Buzón 24. Universidad Complutense de Madrid Campus de Somosaguas. 28223 Madrid. España Tfno: 00 34 913 943 126 Dirección de Internet: http://www.ucm.es/info/apsom/revistapsicooncologia http://revistas.ucm.es/portal/modulos.php?name=Revistas2_Editorial&id=PSIC E-mail: psicooncologia@psi.ucm.es © Asociación de Psicooncología de Madrid. ISSN: 1696-7240 S.V.: 46/03-R-CM Depósito Legal: M. 35130-2003 Imprime: COMPOBELL, S.L. Murcia
https://openalex.org/W3014532031	https://discovery.ucl.ac.uk/id/eprint/10107935/1/1362.full.pdf	English	null	Rapid implementation of mobile technology for real-time epidemiology of COVID-19	medRxiv (Cold Spring Harbor Laboratory)	2,020	cc-by	6,110	on August 14, 2020 http://science.sciencemag.org/ d from An evolving body of literature suggests that COVID-19 incidence and outcomes vary accord- ing to age, sex, race, ethnicity, and underlying health status, with inconsistent evidence sug- gesting that commonly used medications— such as angiotensin-converting enzyme inhib- itors, thiazolidinediones, and ibuprofen—may alter the natural disease course (3–9). Further, symptoms of COVID-19 vary widely, with fever and dry cough reportedly the most prevalent, though numerous investigations have dem- onstrated that asymptomatic carriage is a significant determinant of community spread (5–7, 10–13). In addition, the full spectrum of clinical presentation, which is still being char- acterized, may differ markedly across patient subgroups. RecentadvisoriesfromtheAmerican Gastroenterological Association, the American Academy of Otolaryngology–Head and Neck Surgery, and the British Geriatrics Society have emphasized the potential association between COVID-19 infection and previously underappreciated gastrointestinal symptoms (e.g., nausea, anorexia, and diarrhea), loss of taste and/or smell, and common geriatric syndromes (e.g., falls and delirium). The pan- demic has considerably outpaced our collec- tive efforts to fully characterize who is most at risk or may suffer the most serious sequelae of infection. To meet this challenge, we established a multinational collaboration, the COronavirus Pandemic Epidemiology (COPE) Consortium, composed of leading investigators from sev- eral large clinical and epidemiological cohort studies. COPE brings together a multidiscipli- nary team of scientists with expertise in big data research and translational epidemiology to investigate the COVID-19 pandemic in a large and diverse patient population. Several large cohorts have already agreed to join these efforts, including the Nurses’ Health Study series, the Growing Up Today Study (GUTS), the Health Professionals Follow-Up Study (HPFS), TwinsUK, American Cancer Society Cancer Prevention Study 3 (CPS-3), the Multi- ethnic Cohort Study, the California Teachers Study (CTS), the Black Women’s Health Study (BWHS), the Sister Study, Aspirin in Reducing Events in the Elderly (ASPREE), the Stanford Nutrition Studies, the Gulf Long-term Follow- up (GuLF) Study, the Agricultural Health Study, the National Institute of Environmen- tal Health Sciences Environmental Polymor- phisms Registry, the Predicting Progression of Developing Myeloma in a High-Risk Screened Population (PROMISE) study, and the Pre- cursor Crowdsourcing (PCROWD) study. To aid in our data harmonization efforts in the United States, we developed the COVID Symp- tom Study (previously known as the COVID Mobile phone applications and web-based tools facilitate self-guided collection of population- level data at scale (14), the results of which can be rapidly redeployed to inform partic- ipants of urgent health information (14, 15). 1Clinical and Translational Epidemiology Unit, Massachusetts General Hospital and Harvard Medical School, 55 Fruit St., Boston, MA 02114, USA. 2Department of Twin Research and Genetic Epidemiology, King’s College London, Westminster Bridge Road, London SE1 7EH, UK. 3Department of Ageing and Health, Guys and St. Thomas’ NHS Foundation Trust, Lambeth Palace Road, London SE1 7EH, UK. 4School of Biomedical Engineering & Imaging Sciences, King’s College London, 1 Lambeth Palace Road, London SE1 7EU, UK. 5Zoe Global Limited, 164 Westminster Bridge Road, London SE1 7RW, UK. 6Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, 665 Huntington Ave., Boston, MA 02114, USA. These authors contributed equally to this work. †These authors contributed equally to this work. ‡Corresponding author. Email: achan@mgh.harvard.edu §COPE Consortium members and affiliations are listed in the supplementary materials. RESEARCH RESEARCH CORONAVIRUS Rapid implementation of mobile technology for real-time epidemiology of COVID-19 David A. Drew1, Long H. Nguyen1, Claire J. Steves2,3, Cristina Menni2, Maxim Freydin2, Thomas Varsavsky4, Carole H. Sudre4, M. Jorge Cardoso4, Sebastien Ourselin4, Jonathan Wolf5, Tim D. Spector2,5†, Andrew T. Chan1,6†‡, COPE Consortium§ in more controlled research settings, and these studies benefit from greater lead time for field testing, question curation, and recruitment. Although many digital collection tools for COVID-19 are being developed and launched in the Unites States and abroad (see http:// mhealth-hub.org/mhealth-solutions-against- covid-19 for a continuously updated resource list from the European Union and the World Health Organization), including some in part- nership with government health agencies such as the Centers for Disease Control and Prevention, most applications have largely been configured to offer a single assessment of symptoms to tailor semipersonalized rec- ommendations for further evaluation. Infec- tious disease surveillance web-based tools (e.g., http://flunearyou.org) have been rap- idly adapted for COVID-19–specific collection (e.g., http://covidnearyou.org). Alternatively, web portals have been developed for research- ers to report patient-level information on behalf of participants already enrolled in clinical registries (e.g., ccc19.org). Integra- tion with approaches that use remote data capture (e.g., wearable technology or symptom checkers such as real-time reporting ther- mometers) is also being considered. Although these approaches offer critical public health insights, they are often not tailored for the type of scalable longitudinal data capture that epidemiologists need to perform comprehen- sive, well-powered investigations. CORONAVIRUS Rapid implementation of mobile technology for real-time epidemiology of COVID-19 David A. Drew1, Long H. Nguyen1, Claire J. Steves2,3, Cristina Menni2, Maxim Freydin2, Thomas Varsavsky4, Carole H. Sudre4, M. Jorge Cardoso4, Sebastien Ourselin4, Jonathan Wolf5, Tim D. Spector2,5†, Andrew T. Chan1,6†‡, COPE Consortium§ David A. Drew1, Long H. Nguyen1*, Claire J. Steves2,3, Cristina Menni2, Maxim Freydin2, Thomas Varsavsky4, Carole H. Sudre4, M. Jorge Cardoso4, Sebastien Ourselin4, Jonathan Wolf5, Tim D. Spector2,5†, Andrew T. Chan1,6†‡, COPE Consortium§ The rapid pace of the coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) presents challenges to the robust collection of population-scale data to address this global health crisis. We established the COronavirus Pandemic Epidemiology (COPE) Consortium to unite scientists with expertise in big data research and epidemiology to develop the COVID Symptom Study, previously known as the COVID Symptom Tracker, mobile application. This application—which offers data on risk factors, predictive symptoms, clinical outcomes, and geographical hotspots—was launched in the United Kingdom on 24 March 2020 and the United States on 29 March 2020 and has garnered more than 2.8 million users as of 2 May 2020. Our initiative offers a proof of concept for the repurposing of existing approaches to enable rapidly scalable epidemiologic data collection and analysis, which is critical for a data-driven response to this public health challenge. T T he exponentially increasing number of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections has led to “an urgent need to expand public health activities in order to eluci- date the epidemiology of the novel virus and characterize its potential impact” (1). Under- standing risk factors for infection and pre- dictors of subsequent outcomes is crucial to gain control of the coronavirus disease 2019 (COVID-19) pandemic (2). However, the speed at which the pandemic is unfolding poses an unprecedented challenge for the collection of exposure data to characterize the full breadth of disease severity, hampering efforts for time- ly dissemination of accurate information to affect public health planning and clinical man- agement. Thus, there is an urgent need for an adaptable real-time data-capture platform to rapidly and prospectively collect actionable high-quality data that encompass the spectrum of subclinical and acute presentations and identify disparities in diagnosis, treatment, and clinical outcomes. Addressing this priority will allow for more accurate estimates of dis- ease incidence, inform risk mitigation strat- egies, facilitate allocation of scarce testing resources, and encourage appropriate quar- antine and treatment of those afflicted. on August 14, 2020 http://science.sciencemag.org/ d from Between 24 and 29 March 2020, more than 1.6 million unique individuals downloaded the application and shared clinical and demographic information, as well as daily symptoms and high-intensity occupational exposures (blue map). Population density of those presenting with any symptoms (left purple map) varied according to region, with widespread reports of fatigue, cough, and diarrhea, followed by anosmia and, relatively infrequently, fever (inset). Examination of individuals who reported complex symptoms (right purple map), defined as having cough or fever and at least Fig. 2. COVID Symptom Study use, reported symptoms, and testing results according to geographic location in the United Kingdom. Between 24 and 29 March 2020, more than 1.6 million unique individuals downloaded the application and shared clinical and demographic information, as well as daily symptoms and high-intensity occupational exposures (blue map). Population density of those presenting with any symptoms (left purple map) varied according to region, with widespread reports of fatigue, cough, and diarrhea, followed by anosmia and, relatively infrequently, fever (inset). Examination of individuals who reported complex symptoms (right purple map), defined as having cough or fever and at least Fig. 2. COVID Symptom Study use, reported symptoms, and testing results according to geographic location in the United Kingdom. Between 24 and one other symptom (diarrhea, anosmia, or fever), reveals areas that potentially need more testing. For the subset of the population that received a COVID-19 test (black map), areas with larger proportions of positive tests (orange map) appear to coincide with areas in which high proportions of the population reported complex symptoms. By contrast, some areas with low prevalence of complex symptoms have received higher rates of testing and, consequently, more negative tests (green map). This example of real-time visualization of data captured by the COVID Symptom Study may help public health and government officials reallocate resources, identify areas with unmet testing needs, and detect emerging hotspots. Fig. 2. COVID Symptom Study use, reported symptoms, and testing results according to geographic location in the United Kingdom. Between 24 and 29 March 2020, more than 1.6 million unique individuals downloaded the application and shared clinical and demographic information, as well as daily symptoms and high-intensity occupational exposures (blue map). Population density of those presenting with any symptoms (left purple map) varied according to region, with widespread reports of fatigue, cough, and diarrhea, followed by anosmia and, relatively infrequently, fever (inset). on August 14, 2020 http://science.sciencemag.org/ d from Both technologies are particularly advanta- geous when many individuals are advised to maintain physical distance from others (16). Such digital tools have already been applied 1 of 6 Drew et al., Science 368, 1362–1367 (2020) 19 June 2020 RESEARCH \| REPORT Fig. 1. Schematic of the participant workflow. After downloading the COVID interactions, potential exposure to infected patients, and use of PPE. Fig. 1. Schematic of the participant workflow. After downloading the COVID Symptom Study app and providing consent, users are prompted to enter baseline demographic and clinical information and are serially queried about new or ongoing symptoms, testing results, and extent of isolation. Health care workers offer additional information about the intensity of their patient interactions, potential exposure to infected patients, and use of PPE. With informed consent, users also participating in a variety of ongoing cohorts or clinical trials (Nurses’ Health Study, TwinsUK, and others) have the option of linking COVID Symptom Study information to their extant research data. interactions, potential exposure to infected patients, and use of PPE. With informed consent, users also participating in a variety of ongoing cohorts or clinical trials (Nurses’ Health Study, TwinsUK, and others) have the option of linking COVID Symptom Study information to their extant research data. Users Any Symptoms Complex Symptoms Received Testing COVID-19 Positive COVID-19 Negative Fatigue Cough Diarrhea Anosmia Fever on August 14, 2020 http://science.sciencemag.org/ Received Testing Received Testing Users Any Symptoms Complex Symptoms COVID-19 Positive Users Any Symptoms Complex Symptoms Received Testing Fatigue Cough Diarrhea Anosmia Fever Fatigue Cough Diarrhea Anosmia Fever one other symptom (diarrhea, anosmia, or fever), reveals areas that potentially need more testing. For the subset of the population that received a COVID-19 test (black map), areas with larger proportions of positive tests (orange map) appear to coincide with areas in which high proportions of the population reported complex symptoms. By contrast, some areas with low prevalence of complex symptoms have received higher rates of testing and, consequently, more negative tests (green map). This example of real-time visualization of data captured by the COVID Symptom Study may help public health and government officials reallocate resources, identify areas with unmet testing needs, and detect emerging hotspots. Fig. 2. COVID Symptom Study use, reported symptoms, and testing results according to geographic location in the United Kingdom. Drew et al., Science 368, 1362–1367 (2020) 19 June 2020 on August 14, 2020 http://science.sciencemag.org/ d from 39066 38791 17767 13758 11027981495348652746962183071306821041674 1667 512 110 0 25000 Sympto Fatigue Cough Diarrhea Fever Anosmia Shortness of Breath 0 50000 100000 150000 200000 Total (n) Participants who tested COVID positive 53 53 37 29 22 21 17 16 14 13 12 12 9 9 5 5 4 4 3 2 0 20 40 60 Symptom(s) Frequency Fatigue Cough Anosmia Fever Diarrhea Shortness of Breath 0 100 200 300 Total (n) The COVID Symptom Study enables self- reporting of data related to COVID-19 expo- sure and infections (Fig. 1). On first use, the app queries location, age, and core health risk fac- tors. Dailypromptsqueryforupdates oninterim symptoms, health care visits, and COVID-19 testing results. For those self-quarantining or seeking health care, the level of intervention and related outcomes are collected. Individu- als without obvious symptoms are also encour- aged to use the app. Through pushed software updates, we can add or modify questions in real time to test emerging hypotheses about COVID-19 symptoms and treatments. Notably, participants enrolled in ongoing epidemio- logic studies, clinical cohorts, or clinical trials can provide informed consent to link survey data collected through the app to their preexist- ing study cohort data and any relevant bio- specimens in a Health Insurance Portability and Accountability Act (HIPAA)–compliant and General Data Protection Regulation (GDPR)– compliant manner. A specific module is also provided for health care workers to determine the intensity and type of their direct patient care experiences, the availability and use of personal protective equipment (PPE), and work- related stress and anxiety. Participants who tested COVID positive 60 Participants who tested COVID positive 53 53 37 29 22 21 17 16 14 13 12 12 9 9 5 5 4 4 3 2 0 20 40 60 Symptom(s) Frequency Fatigue Cough Anosmia Fever Diarrhea Shortness of Breath 0 100 200 300 Total (n) 216 183 151 45 34 31 27 24 22 22 16 15 15 15 14 8 4 4 2 2 1 0 50 100 150 200 Symptom(s) Frequency Fatigue Cough Diarrhea Fever Anosmia Shortness of breath 0 200 400 600 Total (n) Participants who tested COVID negative 216 Participants who tested COVID negative Through rapid deployment of this tool, we can gain key insights into population dynamics of the disease (Fig. 2). on August 14, 2020 http://science.sciencemag.org/ d from Examination of individuals who reported complex symptoms (right purple map), defined as having cough or fever and at least 2 of 6 Drew et al., Science 368, 1362–1367 (2020) 19 June 2020 RESEARCH \| REPORT 91549 39066 38791 17767 13758 11027981495348652746962183071306821041674 1667 512 110 0 25000 50000 75000 100000 Symptom(s) Frequency Fatigue Cough Diarrhea Fever Anosmia Shortness of Breath 0 50000 100000 150000 200000 Total (n) All participants who have reported any COVID-related symptoms Symptom Tracker) mobile app with in-kind contributions from Zoe Global Ltd., a digital health care company, and academic scientists from Massachusetts General Hospital and King’s College London. By leveraging the established digital backbone of an applica- tion used for personal nutrition studies, the COVID Symptom Study app was launched in the United Kingdom on 24 March 2020 and became available in the United States on 29 March 2020 (https://covid.joinzoe.com/us). The COPE Consortium is committed to the shared international pursuit of combating COVID-19 and has worked with scientific collaborators and thought leaders in real-time epidemiology to prioritize data harmonization and sharing as part of the Coronavirus Census Collective (17). Drew et al., Science 368, 1362–1367 (2020) 19 June 2020 39066 38791 17767 13758 11027981495348652746962183071306821041674 1667 512 110 0 25000 50000 75000 Symptom(s) Frequenc Fatigue Cough Diarrhea Fever Anosmia Shortness of Breath 0 50000 100000 150000 200000 Total (n) 216 183 151 45 34 31 27 24 22 22 16 15 15 15 14 8 4 4 2 2 1 0 50 100 150 200 Symptom(s) Frequency Fatigue Cough Diarrhea Fever Anosmia Shortness of breath 0 200 400 600 Total (n) Participants who tested COVID positive Participants who tested COVID negative 53 53 37 29 22 21 17 16 14 13 12 12 9 9 5 5 4 4 3 2 0 20 40 60 Symptom(s) Frequency Fatigue Cough Anosmia Fever Diarrhea Shortness of Breath 0 100 200 300 Total (n) Fig. 3. Symptoms reported through the COVID Symptom Study app. By 27 March 2020, 265,851 individuals in the United Kingdom reported any symptom potentially associated with COVID-19 (top). Participants provided data on whether they were tested for COVID-19, as well as the test result. 1176 individuals reported having received a COVID-19 test (0.4% of those with symptoms). Symptom frequencies among those who tested positive (middle; n = 340) versus negative (bottom; n = 836) are shown. on August 14, 2020 http://science.sciencemag.org/ d from The prediction mapping included data from 1,339,670 users of the COVID Symptom Study on 1 April; 998,244 users on 10 April; and 1,234,918 users on 20 April. Public Health Wales NHS Trust data were current as of 21 April 2020 at 13:00 local time, taken from the “Rapid COVID-19 Virology - Public” dashboard (accessed via https://phw.nhs.wales/), and downloaded on 22 April 2020 at 12:30 p.m. Eastern Standard Time. Fig. 4. Predicting COVID-19 cases on the basis of real-time symptom reporting in Wales, United Kingdom. This time series (bar graph) displays the number of new confirmed cases (gray bars) reported by the Public Health Wales NHS Trust between 31 March 2020 and 20 April 2020. After 2 April, case numbers appear to have declined through 5 April. However, our symptom-based prediction model (18), developed from symptom reports from untested users of the COVID Symptom Study app, showed a high proportion of predicted COVID-19 cases in southern Wales on 1 April [dark red areas in (A)]. Six days later, Welsh health authorities reported a subsequent peak in cases over a 4-day period (6 to 9 April), driven primarily by these southern regions (colored bars). By 10 April, new confirmed cases across Wales declined. However, on the basis of reported symptoms (B), regions in South Wales still had high predicted levels of COVID-19, which became apparent as a viduals who tested positive. Indeed, in subse- quent analyses with a larger sample set, we have shown that anosmia appears to be a strong predictor for COVID-19 (18). By con- trast, fever alone was not particularly dis- criminatory. However, when fever was present in combination with less appreciated symp- toms, a greater frequency of positive tests was observed. These findings suggest that perhaps individuals with complex or multiple- symptom (three or more) presentations should be prioritized for testing. Concerningly, 20% of individuals reported complex symptoms bination commonly led to testing but was not a particularly accurate predictor of a positive test. Similarly, no individuals who reported diarrhea in the absence of other symptoms tested positive. Notably, more complex pre- sentations with cough and/or fatigue and at least one additional symptom, including less commonly appreciated complaints such as diarrhea and anosmia, appeared to be enriched among those with positive test results relative to those with negative results. on August 14, 2020 http://science.sciencemag.org/ d from By collecting participant- reported geospatial data, highlighted as a crit- ical need for pandemic epidemiologic research (15), we can rapidly identify populations with highly prevalent symptoms in regions that may emerge as outbreak hotspots. An early snapshot of the first 1.6 million users in the United Kingdom over the first 5 days of use confirms the variability in symptoms reported across suspected COVID-19 cases and is useful for generating and testing broader hypothe- ses. At the time, users had a mean age of 41, ranged from 18 to 90 years old, and were 75% female. Graphic visualization of our initial results (Fig. 3) demonstrates that among those reporting symptoms by 27 March 2020 (n = Fig. 3. Symptoms reported through the COVID Symptom Study app. By 27 March 2020, 265,851 individuals Fig. 3. Symptoms reported through the COVID Symptom Study app. By 27 March 2020, 265,851 individuals in the United Kingdom reported any symptom potentially associated with COVID-19 (top). Participants provided data on whether they were tested for COVID-19, as well as the test result. 1176 individuals reported having received a COVID-19 test (0.4% of those with symptoms). Symptom frequencies among those who tested positive (middle; n = 340) versus negative (bottom; n = 836) are shown. Fig. 3. Symptoms reported through the COVID Symptom Study app. By 27 March 2020, 265,851 individuals in the United Kingdom reported any symptom potentially associated with COVID-19 (top). Participants provided data on whether they were tested for COVID-19, as well as the test result. 1176 individuals reported having received a COVID-19 test (0.4% of those with symptoms). Symptom frequencies among those who tested positive (middle; n = 340) versus negative (bottom; n = 836) are shown. Drew et al., Science 368, 1362–1367 (2020) 19 June 2020 3 of 6 RESEARCH \| REPORT http://science.sciencem Downloaded from second spike in confirmed cases between 15 and 16 April. As of 20 April (C), predicted COVID-19 prevalence across Wales according to symptom reporting appears to be low, which corresponds to a flattening of the cumulative incidence curve. However, several regions in southern Wales still have relatively high reports of symptoms and appear at risk for subsequent cases of COVID-19. Black dots on the maps represent the relative proportion of positive tests reported by health authorities across Wales that day by region. Drew et al., Science 368, 1362–1367 (2020) 19 June 2020 on August 14, 2020 http://science.sciencemag.org/ d from In particular, anosmia may be a more predictive symptom, as it was more common than fever in indi- 265,851 individuals), the most common symp- toms were fatigue and cough, followed by diarrhea, fever, and anosmia. Shortness of breath was reported relatively rarely. Only 0.4% (n = 1176) of individuals reporting pos- sible COVID-19 symptoms reported receiv- ing a quantitative polymerase chain reaction test for COVID-19. A comparison of symptomatic users who reported receiving a test within the initial launch period generated several hypotheses for future study with the growing dataset. The frequency of cough or fatigue alone or in com- Drew et al., Science 368, 1362–1367 (2020) 19 June 2020 4 of 6 RESEARCH \| REPORT enrolled in clinical trials. At the Massachusetts General Hospital and Brigham and Women’s Hospital, we are deploying the tool within several clinical studies, centralized biobank- ing efforts, and health care worker surveil- lance programs. Health care workers are particularly vulnerable to COVID-19’s effects beyond infection, including work hazards from PPE shortages, emotional stress, and absenteeism. Real-time data generation fo- cused within these populations will be criti- cal to optimally allocate resources to protect our health care workforce and assess its efficacy. on engaging local public health leaders. For example, we have partnered with the Uni- versity of Texas School of Public Health to conduct statewide surveillance to support pub- lic health decision-making, especially as the Texas state government begins softening miti- gation strategies. (cough and/or fatigue plus at least one of anosmia, diarrhea, or fever) but had not yet been tested, representing a substantial pop- ulation that appears to be at elevated risk for the disease. Additional work is warranted to confirm whether complex or multiple- symptom cases can accurately predict COVID-19 incidence. Our approach demonstrates a proof of con- cept for rapid repurposing of existing data collection methods to implement scalable real-time collection of population-level data during a fast-moving global health crisis. We call on our colleagues to work with us so that we may deploy all of the tools at our disposal to address this unprecedented public health challenge. Building on these initial findings, our team subsequently developed a weighted prediction model based on the symptoms of more than 2 million individual app users (18). on August 14, 2020 http://science.sciencemag.org/ d from By using this prediction model, we demonstrate the po- tential utility of the COVID Symptom Study app to collect data for long-term studies as well as for immediate public health planning. In southern Wales in the United Kingdom, users reported symptoms that predicted, 5 to 7 days in advance, two spikes in the number of con- firmed positive COVID-19 cases reported by public health authorities (Fig. 4). Conversely, a decline in reports of symptoms preceded a drop in confirmed cases by several days. These results demonstrate that this app pro- spectively captures the dynamics of COVID-19 incidence days in advance of traditional mea- sures, such as positive tests, hospitalizations, or mortality. We are currently planning ad- ditional studies using a broadly representa- tive sample of individuals who will undergo uniform COVID-19 testing to further vali- date our approach to symptom-based mod- eling of incidence. These data demonstrate compelling evidence for the potential pre- dictive power of our approach, which will improve as more data are collected to inform the model. Further, our data highlight the potential utility of real-time symptom track- ing to help guide allocation of resources for testing and treatment as well as recommen- dations for lockdown or easement in spe- cific areas. Even so, our approach has limitations. We recognize that a smartphone application does not represent a random sampling of the pop- ulation. However, this is an inherent limita- tion of any epidemiologic study that relies on voluntary participation. Our approach has the benefit of allowing rapid deployment across a large cross section of the population during a major public health crisis. With time and continued use, the large number of participants will include a sufficient quantity of users within key subgroups such that we can adjust our methodology for potential sources of confound- ing. By engaging cohorts with underrepre- sented populations, such as the BWHS in the United States, we also hope to leverage exist- ing investigator-participant relationships to encourage enrollment of individuals from populations that have traditionally been chal- lenging to recruit. Moreover, by encouraging longitudinal, prospective data collection, we can capture associations based on within- person variation over time, a notable advan- tage over repeated cross-sectional surveys that introduce considerable between-person varia- tion. In the near future, we hope to release our app as fair-use open source software to facili- tate translation and development in other re- gions. 360, 2153–2157 (2009). 15. B. Xu, M. U. G. Kraemer; Open COVID-19 Data Curation Group Lancet Infect. Dis. 20, 534 (2020). 15. B. Xu, M. U. G. Kraemer; Open COVID-19 Data Curation Group, Lancet Infect. Dis. 20, 534 (2020). 16. P. J. Lyons, “Coronavirus Briefing: What Happened Today,” The New York Times (30 March 2020). 16. P. J. Lyons, “Coronavirus Briefing: What Happened Today,” The New York Times (30 March 2020). 16. P. J. Lyons, “Coronavirus Briefing: What Happened Today,” The New York Times (30 March 2020). 17. E. Segal et al., Nat. Med. 10.1038/s41591-020-0929-x (2020). 17. E. Segal et al., Nat. Med. 10.1038/s41591-020-0929-x (2020). 18. C. Menni et al., Nat. Med. 10.1038/s41591-020-0916-2 (2020). 19. D. A. Drew et al., medRxiv [Preprint]. 6 April 2020; https://doi.org/10.1101/2020.04.02.20051334. 19. D. A. Drew et al., medRxiv [Preprint]. 6 April 2020; https://doi.org/10.1101/2020.04.02.20051334. With additional data collection, we will also apply big data approaches (e.g., machine learn- ing) to identify emerging patterns in dynam- ic settings of exposure, onset of symptoms, disease trajectory, and clinical outcomes. Our launch of the app within several large epidemiology cohorts that have previously gathered longitudinal data on lifestyle, diet and health factors, and genetic information will allow investigation of a much broader range of putative risk factors for COVID-19 outcomes. With additional follow-up, we will also be positioned to investigate long-term effects of COVID-19, including mental health, disability, mortality, and financial outcomes. Mobile technology can also supplement re- cently launched clinical trials or biobanking protocols already embedded within clinical settings. In collaboration with the Stand Up to Cancer foundation, we have also devel- oped a strategy to track information among individuals living with cancer, including those 20. MGHcteu, MGHcteu/ScienceCOPEMethodsCode: Science v1.0.1, Version 1.0.1, Zenodo (2020); https://doi.org/10.5281/ zenodo.3765955. 20. MGHcteu, MGHcteu/ScienceCOPEMethodsCode: Science v1.0.1, Version 1.0.1, Zenodo (2020); https://doi.org/10.5281/ zenodo.3765955. on August 14, 2020 http://science.sciencemag.org/ d from We have begun working with colleagues in Canada, Australia, and Sweden to implement this tool within their countries. We have also developed a practical toolkit to assist clinical researchers with local institutional review board and regulatory approval to facilitate deployment within research studies (www. monganinstitute.org/cope-consortium). This toolkit includes full details of the mobile app’s questions, consent documents, privacy policies, and terms of use. With broader implementa- tion, data generated from the COVID Symptom Study app are increasingly being linked to the public health response within the National Health Service (NHS) in the United Kingdom. The app is endorsed by the Welsh government, NHS Wales, the Scottish government, and NHS Scotland, and our scientific team updates the U.K. chief scientific officer daily. We are work- ing to develop a similar approach in the United States. However, the lack of a national health care system has required a strategy focused Drew et al., Science 368, 1362–1367 (2020) 19 June 2020 REFERENCES AND NOTES 1. M. Lipsitch, D. L. Swerdlow, L. Finelli, N. Engl. J. Med. 382, 1. M. Lipsitch, D. L. Swerdlow, L. Finelli, N. Engl. J. Med. 382, 1194–1196 (2020). 1. M. Lipsitch, D. L. Swerdlow, L. Finelli, N. Engl. J. Med. 382, 1194–1196 (2020). 2. G. A. FitzGerald, Science 367, 1434 (2020). 3. M. Day, BMJ 368, m1086 (2020). 4. L. Fang, G. Karakiulakis, M. Roth, Lancet Respir. Med. 8, e21 (2020). 4. L. Fang, G. Karakiulakis, M. Roth, Lancet Respir. Med. 8, e21 (2020). 5. W. J. Guan et al., N. Engl. J. Med. 382, 1708–1720 (2020). 5. W. J. Guan et al., N. Engl. J. Med. 382, 1708–1720 (2020). 6. D. 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Code used to generate maps is published and available in Zenodo (20). Data collected in the app are being shared with other health researchers through the NHS-funded Health Data Research UK (HDRUK)/SAIL Consortium, housed in the UK Secure e-Research Platform (UKSeRP) in Swansea, Wales. Anonymized data are available to be shared with bona fide researchers through HDRUK according to their protocols in the public interest. See https://healthdatagateway.org/detail/9b604483-9cdc-41b2-b82c- 14ee3dd705f6. U.S. investigators are encouraged to coordinate data Drew et al., Science 368, 1362–1367 (2020) 19 June 2020 ACKNOWLEDGMENTS Use of the COVID-19 app in cohorts and informed consent as described was approved by the Partners Human Research Committee (Protocol 2020P000909) and is registered on ClinicalTrials.gov as NCT04331509. We acknowledge J. Capdevila Pujol of Zoe Global Ltd. for his contributions to this work. Funding: Zoe Global Ltd. provided in kind support for all aspects of building, running, and supporting the tracking app and service to all users worldwide. C.J.S., C.M., M.F., T.V., C.H.S., M.J.C., S.O., and T.D.S. were supported by the Wellcome Trust and EPSRC (WT212904/Z/18/Z, WT203148/Z/16/Z, and WT213038/Z/18/Z), the NIHR GSTT/KCL Biomedical Research Centre, MRC/BHF (MR/M016560/1), the NIHR, and the Alzheimer’s Society (AS-JF-17-011). A.T.C. is the Stuart and Suzanne Steele MGH Research Scholar and Stand Up to Cancer scientist. L.H.N., D.A.D., and A.T.C. were supported by the Massachusetts Consortium on Pathogen Readiness (MassCPR), M. Schwartz, and L. Schwartz. Author contributions: D.A.D. and L.H.N. drafted the manuscript. All authors contributed to the conceptualization, methodology, formal analysis, investigation, resources, data curation, and review and editing of the manuscript. A.T.C. and T.D.S. supervised the study and acquired funding.Competing interests:T.D.S. is a consultant for Zoe Global Ltd. J.W. is an employee of Zoe Global Ltd. D.A.D. and A.T.C. previously served as investigators in a clinical trial of diet and lifestyle using a Drew et al., Science 368, 1362–1367 (2020) 19 June 2020 5 of 6 RESEARCH \| REPORT article that is credited to a third party; obtain authorization from the rights holder before using such material. requests through the COPE Consortium (www.monganinstitute.org/ cope-consortium). Data updates can be found on https://covid.joinzoe. com. For aggregate deidentified “snapshot” datasets used for analyses provided here, please request time-stamped datasets by email to predict@mgh.harvard.edu for data files,“COVIDSymptomTrackerData_ 03292020” (Figs. 2 and 3), or “COVIDSymptomTrackerData_04/21/ 20” (Fig. 4). This work is licensed under a Creative Commons Attribution 4.0 International (CC BY 4.0) license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. To view a copy of this license, visit https://creativecommons.org/licenses/by/4.0/. This license does not apply to figures/photos/artwork or other content included in the article that is credited to a third party; obtain authorization from the rights holder before using such material. distinct mobile application that was supported by Zoe Global Ltd. The other authors have no conflicts of interest to declare. Data and materials availability: This manuscript was previously made available as a preprint on medRxiv.org (19). ACKNOWLEDGMENTS Code used to generate maps is published and available in Zenodo (20). Data collected in the app are being shared with other health researchers through the NHS-funded Health Data Research UK (HDRUK)/SAIL Consortium, housed in the UK Secure e-Research Platform (UKSeRP) in Swansea, Wales. Anonymized data are available to be shared with bona fide researchers through HDRUK according to their protocols in the public interest. See https://healthdatagateway.org/detail/9b604483-9cdc-41b2-b82c- 14ee3dd705f6. U.S. investigators are encouraged to coordinate data originally published online May 5, 2020 DOI: 10.1126/science.abc0473 (6497), 1362-1367. 368 Science originally published online May 5, 2020 DOI: 10.1126/science.abc0473 (6497), 1362-1367. 368 Science SUPPLEMENTARY MATERIALS SUPPLEMENTARY MATERIALS science.sciencemag.org/content/368/6497/1362/suppl/DC1 Figs. S1 to S9 COPE Consortium Members MDAR Reproducibility Checklist 2 April 2020; accepted 30 April 2020 Published online 5 May 2020 10.1126/science.abc0473 SUPPLEMENTARY MATERIALS science.sciencemag.org/content/368/6497/1362/suppl/DC1 Figs. S1 to S9 COPE Consortium Members MDAR Reproducibility Checklist 6 of 6 Science. No claim to original U.S. Government Works Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Rapid implementation of mobile technology for real-time epidemiology of COVID-19 Jorge Cardoso, Sebastien Ourselin, Jonathan Wolf, Tim D. Spector, Andrew T. Chan and COPE Consortium David A. Drew, Long H. Nguyen, Claire J. Steves, Cristina Menni, Maxim Freydin, Thomas Varsavsky, Carole H. Sudre, M. Rapid implementation of mobile technology for real-time epidemiology of COVID-19 Jorge Cardoso, Sebastien Ourselin, Jonathan Wolf, Tim D. Spector, Andrew T. Chan and COPE Consortium David A. Drew, Long H. Nguyen, Claire J. Steves, Cristina Menni, Maxim Freydin, Thomas Varsavsky, Carole H. Sudre, M. (6497), 1362-1367. 368 Science is a registered trademark of AAAS. Science Science, 1200 New York Avenue NW, Washington, DC 20005. The title (print ISSN 0036-8075; online ISSN 1095-9203) is published by the American Association for the Advancement of Science Science. No claim to original U.S. Government Works Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Mobile symptom tracking health reports. Kingdom, mathematical modeling predicted geographical hotspots of incidence 5 to 7 days in advance of official public anosmia, was predictive of a positive test verification for SARS-CoV-2. As exemplified by data from Wales, United and/or prevalence of combinations of symptoms (three or more), including fatigue and cough, followed by diarrhea, fever, (previously known as the COVID Symptom Tracker) from across the United Kingdom and the United States. The symptoms. The authors recruited about 2 million users (including health care workers) to the COVID Symptom Study pushed software updates to participants to encourage reporting of potential coronavirus disease 2019 (COVID-19) al. et for real-time epidemiology. Taking advantage of existing longitudinal health care and research patient cohorts, Drew population is defying attempts at tracking it, and quantitative polymerase chain reaction testing so far has been too slow The rapidity with which severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spreads through a Mobile symptom tracking , this issue p. 1362 Science p ARTICLE TOOLS http://science.sciencemag.org/content/368/6497/1362 MATERIALS SUPPLEMENTARY http://science.sciencemag.org/content/suppl/2020/05/04/science.abc0473.DC1 CONTENT RELATED http://stm.sciencemag.org/content/scitransmed/12/549/eabb9401.full http://stm.sciencemag.org/content/scitransmed/12/546/eabc1931.full http://stm.sciencemag.org/content/scitransmed/11/499/eaat0360.full http://stm.sciencemag.org/content/scitransmed/12/541/eabb5883.full http://stm.sciencemag.org/content/scitransmed/9/396/eaal3653.full http://stm.sciencemag.org/content/scitransmed/12/534/eabb1469.full REFERENCES http://science.sciencemag.org/content/368/6497/1362#BIBL This article cites 18 articles, 4 of which you can access for free PERMISSIONS http://www.sciencemag.org/help/reprints-and-permissions http://science.sciencemag.org/content/368/6497/1362 http://science.sciencemag.org/content/suppl/2020/05/04/science.abc0473.DC1 http://stm.sciencemag.org/content/scitransmed/12/549/eabb9401.full http://stm.sciencemag.org/content/scitransmed/12/546/eabc1931.full http://stm.sciencemag.org/content/scitransmed/11/499/eaat0360.full http://stm.sciencemag.org/content/scitransmed/12/541/eabb5883.full http://stm.sciencemag.org/content/scitransmed/9/396/eaal3653.full http://stm.sciencemag.org/content/scitransmed/12/534/eabb1469.full http://science.sciencemag.org/content/368/6497/1362#BIBL This article cites 18 articles, 4 of which you can access for free PERMISSIONS http://www.sciencemag.org/help/reprints-and-permissions http://www.sciencemag.org/help/reprints-and-permissions Terms of Service Use of this article is subject to the
https://openalex.org/W3103445696	https://lifescienceglobal.com/pms/index.php/jrge/article/download/8147/4387	English	null	The Effects of Determinants of Government Expenditure on Education and Health: Evidence From SADC Economies	Journal of reviews on global economics	2,020	cc-by	6,535	1. INTRODUCTION12 Furthermore, SADC member states spend large amount of money on education to affect growth and development in their economies. According to SADC (2017), SADC member countries spent an average of 23% on education expenditure, which makes it the largest component of government expenditure. There are a few policies in place to address the education problems in SADC economies. In 1997 SADC countries signed education protocol which address the quality and cohesion of education within the members states. The protocol acknowledged the need to develop the human resource capacity of the community, and the purpose of the Protocol is to overcome the difficulties faced by individual economy in their attempts to build successful education systems. However, there are still challenges that prevent total access of education in the region. A healthy population is an essential catalyst for economic and social development (SADC3, 2019a). Health care is the major element of human capital investment, thus rising national expenditure on health will increase life quality, labour productivity, and general being of the people (Culyer and Newhouse, 2000). Communicable diseases are a serious concern for social and human development in the region (WHO, 2019). SADC accounts for 33% of all people living with HIV and AIDS in the world; eight SADC member countries are among those economies with the highest percentages of tuberculosis; and 75% of the SADC population is in danger of contracting malaria (SADC,2019b). In 1999, SADC signed health protocol aimed among other, to create a joint strategy to address the health problems for children, women and another defenceless populace. SADC economies spend an average of 10% of their national budget on health, which is the second highest in the budget following education (SADC, 1999). However, health and social indicators are the worst in the region (WHO, 2019). According to the SADC (2018) majority of the people residing in the region suffer mainly from preventable illness which could be prevented at little cost. Thus, it is important to understand the impacts of the determinants of government expenditure on health in SADC. Population of SADC economies is youthful, where 76.4% of the population is below the age of 35 (SADC, 2011). Literature shows that in developed economies as population grow, the aging will increase political pressure to move the composition of social expenditure in favour of the elderly but sacrificing other government expenditure such as education (Dormont, Grignon and Huber, 2006. Journal of Reviews on Global Economics, 2020, 9, 378-386 Journal of Reviews on Global Economics, 2020, 9, 378-386 378 Horisani Mhlari and Teboho Jeremiah Mosikari* chool of Economic Sciences, Department of Economics, North West University, South Africa of Economic Sciences, Department of Economics, North West University, South Africa Abstract: Health and education expenditure play a major role in determining the well-being and development of the people. However, SADC economies have poor health and education indicators, despite high expenditure by governments. The purpose of this study is to assess the determinants of government expenditure on health and education in SADC countries. This study used the annual data covering the period 1997 to 2017. FMOLS 1 and DOLS 2 are applied to estimate the parameters of each variable understudy. The techniques were developed to estimate and test hypothesis about cointegrating vector to panel data. Overall the results revealed that gross domestic product, population growth and corruption are crucial determinants of health and education expenditure in SADC countries. Therefore, governments in SADC can implement policy that are pro economic growth, and measures that discourages corruption. This can be achieved by allocating high budget on education expenditure, and implementing independent corruption agencies. Keywords: Government Expenditure, Government Expenditure on Education, Government Expenditure on Health Corruption, Economic Growth, Population, Panel data. The Effects of Determinants of Government Expenditure on Education and Health: Evidence From SADC Economies Horisani Mhlari and Teboho Jeremiah Mosikari* Address correspondence to this author at the School of Economic Sciences, Department of Economics, North West University, South Africa; E-mail: tebohomosikari@gmail.com JEL classification: H51, H52, D57, 04, P23, C23. 1. INTRODUCTION12 Therefore, this gaps warrants an investigation in SADC economies. Therefore, the purpose of this study is to identify the determinants of government expenditure on health and education in SADC. In which it can be established how this factors affect government expenditure on health and education in SADC countries. This will be done by explaining the determinants and their behavior on government expenditure on Education and Health. Positively, this study will assist policy makers to design strong socioeconomic policies designated to SADC economies. In addition, this study will serve as a recent source of information for researchers as the findings will inform debates on the subject. This study is structured as follows: Section 2 is the literature survey studies; Section 3 presents research methodology; Section 4 presents empirical results. Section 5 concludes the study. Adolph Wagner (1835 – 1917), a German economist, in 1880 theorised a well-known association between the growth of the economy and relative growth in public expenditure activities. The law state that as per capita gross national product raise, public expenditure will increase. That is, as income rises, the demand for goods and services provided by the state increases, mainly because of the technological requirements of industrialization and urbanization that supplement the income growth. Furthermore, Wagner thinks of government expenditure as the endogenous factor that is initiated by economic growth rather than exogenous factor. Wagner provides two explanations for the growth of the public sector expenditure as income escalates. Firstly, when these services provided are basic needs, such as administration, education, protection, law and order, health, redistribution of income, and capital expenditure that complement the process of industrialization. Secondly, a significant expansion of cultural and welfare expenditures, with respect to education and income redistribution, takes place as income increases. Literature reveals that there is a number of determinants for health and education expenditure such as corruption, federalism, economic growth, population and so forth (Kotera and Okada, 2017; Loto, 2011; Masenyetse and Motelle, 2012; Musaba, Chilonda and Matchaya, 2013; Paul and Furahisha, 2017; Thamae, 2013). All these determinants have been backed by a strong theoretical and empirical review. Nonetheless there is inconsistency on empirical findings on the determinants of education and health expenditure, see the work of (Angko, 2013; Chaabouni and Abednnadher, 2014; Guisan and Exposito, 2010; Corsetti, Meier and Müller, 2012; Kotera and Okada, 2017; Loto, 2011; Paul and Furahisha, 2017; Thamae, 2013). 1. INTRODUCTION12 Corruption is growing at a geometric rate in the SADC region, most government in the region blame past racial segregation for the presence of corruption in their respective economies. According to Gumede (2010) corruption comes in two forms: Firstly, what he calls big time corruption, when public official changes the rules to propel patronage to relatives. Secondly, is what he called pretty corruption, when government workers intentionally neglect their responsibilities to deliver public service. SADC has enjoyed positive economic growth recently. According to the African Development Bank Group (2019) SADC economies recorded 2.1% growth in 2018 and rose to E-ISSN: 1929-7092/20 Address correspondence to this author at the School of Economic Sciences, Department of Economics, North West University, South Africa; E-mail: tebohomosikari@gmail.com JEL classification: H51, H52, D57, 04, P23, C23. 1Fully modified Ordinary Least Squares 2Dynamic Ordinary Least Squares 3Southern African Development Community (SADC) 1Fully modified Ordinary Least Squares 2Dynamic Ordinary Least Squares 3Southern African Development Community (SADC) E-ISSN: 1929-7092/20 © 2020 Lifescience Global Journal of Reviews on Global Economics, 2020, Vol. 9 379 The Effects of Determinants of Government Expenditure on Education and Health 2.4% in 2019. Service sector has been widely cited as the main contributor of increase in gross domestic product in the region. However, growth rate is below macroeconomic convergence rate of 7% per year. consumption service which can be divided equal between individuals. He added, that education is the only instrument in which the have-not can build up their capital. Furthermore, the theory claimed that government expenditure on education especial on high education serve the interest of government better, because it makes the youth or student to be more compliant citizens. This study is motivated by the following factors: firstly, there is no existing empirical literature on government expenditure on health and education in SADC countries. Except for the work of Mussagy and Babatunde (2015) which whoever focused on education and economic growth in Mozambique exclusively than considering SADC economies as an economic unit. Lastly, the literature survey shows that no attention was given to explore the econometric method of panel Fully Modified Ordinary Least Squared (FMOLS) and Dynamic Ordinary Least Square (DOLS). The interest of such use of techniques is that in a case of pre-existence of cointegration among the system it is best to use this parameter estimates since they do not require post-estimation diagnostics. 1. INTRODUCTION12 This section on literature survey is divided into twofold, the first part discusses the empirical review that was conducted based on general government expenditure on health and, second part conclude with literature on government expenditure on education. Colombier (2012) assessed the drivers of public health expenditure with special attention on Baumol’s cost disease theory. The author used the Gourieroux-Holly-Monfort (GHM) test and assessed the consistency of the panel by fixed effect ad random effect carried out by Hausman specification test. The 2. LITERATURE SURVEY This study follows two theoretical studies, the theory of pure public expenditure and Wagner’s law. The theory of public expenditure was brought to existence by Paul Samuelsson in 1954, in his study the pure theory of public expenditure. The study divides the national budget into two sections; (i) algebraic taxes and transfers, known as income redistribution which can be changed until the society is moved to optimal condition; (ii) the provision of the collective goods. Samuelsson (1954) defines collective goods as goods that are consumed by everyone in common. Therefore, the consumption of these goods and services by one person, does not reduce the consumption of the same goods to the next person. Samuelson (1954) further extended the theory specifically to cover for government expenditure on education. He acknowledges the fact that education is private Journal of Reviews on Global Economics, 2020, Vol. 9 Mhlari and Mosikari 380 determinants to health expenditure in these economies while positive correlation between health expenditure and income was found to be significant. This suggest that health expenditure is responsive to income in the region, with elasticity value of 0.78. Furthermore, the authors suggested that the government of this economies should provide more of health expenditure, since health care is responsive to income change. On education studies, Mussagy and Babatunde (2015) studies the relationship between public expenditure on education and economic growth in Mozambique using quarterly data from 1996 to 2012. The researchers used Johansen Cointegration approach to study the long run relationship between the variables and the error correction was used to evaluate the short run adjustment dynamics. The results revealed that government expenditure on education in Mozambique is low and have an adverse effect on economic growth. The researchers further suggested that the government of Mozambique should increase government expenditure on education in order to accelerate economic growth in the economy. Johansen cointegration techniques was applied by Loto (2011) in studying government expenditure of Nigeria at a sectoral level for the period 1980 to 2008. In particular, five sectors of government which are security, health, education, transportation, and communication. In testing stationarity and cointegration amongst the variables the author applied Augmented Dickey Fuller (ADF) and Johansen cointegration techniques. The result revealed that education was negatively and insignificantly connected to gross domestic product. results depict that income is a key factor of public health expenditure. 2. LITERATURE SURVEY This suggested that Baumol’s cost disease exert positive significant impact on health expenditure. In particular, the findings reveal that a 1% increase in the excess of wage increase over productivity growth in health care will increase the growth rate of public health expenditure by almost 0.2%. The author further suggested that Baumol’s hypothesis is more applicable in public health sector. Bates and Santerre (2013) examined the determinants of public expenditure on health in 50 United States of America. The main focus was to determine whether the Baumol cost disease applies in America. The authors used fixed effect model and two stage least squared estimation to estimate the parameters. The results provided that if health care expenditure increase more rapidly when the states wage increases exceed productivity gains. In line with the above mention study, this study concluded that the United States health care sector supports the Baumol’s cost disease. Public expenditure on health, government effectiveness, education and the quality of life in Asia and Africa was assessed by Guisan and Exposito (2010). The results revealed that the only way to improve health spending is to raise spending on education. Moreover, the effects of education on health have both defensive measures to address malnutrition, water impurity and other negative circumstances. Grigoli and Kapsoli (2013) examined public expenditure on health with a special focus on its efficiency in emerging economies and developing economies. The authors used the Stochastic Frontier Model that control the socioeconomic determinants of public expenditure on health and provide economies specific estimates. The results indicated the at a current expenditure level, life expectancy can be push up by five years, when the economies are efficient in public expenditure on health. Novignon, Olakojo and Nonvignon (2012) examined the effect of both public and private expenditure on health and health status in Sub-Saharan Africa (SSA). Panel data from 1995 to 2010 for 44 Sub-Saharan Africa economies was applied. The authors used both fixed and random effect models to analysis the effect of health expenditure on health outcomes. The results revealed that health care expenditure has significantly and positive influence on health status by improving life expectancy at birth, reducing mortality and death rate. The work by Nyamongo and Schoeman (2010) studied the effect of quality of governance and education expenditure in Africa countries. Their study paid attention to corruption, political stability or instability and democracy. 3.1. Model Description Equation (1) is the adopted and modified model of Yildirim and Sezgin (2002), which the study will apply to examine the determinants of government expenditure on health. !!" = !!" + !!" + !!!!" + !!" (3) !!" = !!!!"!! + !!" (4) (3) (4) !"!" = !! + !!!"#!" + !!!"#!" + !!!"!!" + !!!"#! + !"#$%!" + !!" (1) !"!" = !! + !!!"#!" + !!!"#!" + !!!"!!" + !!!"#! + !"#$%!" + !!" (1) Where for !=1, 2 .., ! for each unit in the panel, !=1, 2…, !. Where γ denote the fixed effect and ! represent the slope coefficient permitted to change cross individuals’ unit in equation 3. In model 4, ϕ is the autoregressive coefficient of the residual εit in equation 3. This study applies non-stationary estimation techniques, Fully-Modified OLS and Dynamic OLS models. Phillips and Hansen (1990) modified OLS to produce FM-OLS, to provide optimal estimates of cointegrating regression. The reason behind modifying OLS was to account for serial correlation effects and for the endogeneity in the regressors that results from the existence of cointegrating relationship between variables. The results from FM-OLS estimators are asymptotically unbiased and has fully effective mixture normal asymptotic permitting for standard Wald test, using asymptotic Chi-squares statistical inference. The panel Fully Modified OLS estimator for long run parameters can be defined geometrical as follows: (1) Where, HE denotes health expenditure, COR represent corruption, EDU for education expenditure, POP for population growth rate, GDP denotes gross domestic product, and LIFE for life expectancy at birth. On the education, this study assesses the determinants government expenditure on education by adapting and modifying the model of Busemeyer (2007). Thus, the following equation is used in this study. !"#!" = !! + !!!!"!" + !!!"#!"+ !!!"!!" + !!!"!" + !!!"!" + !!" (2) (2) Where, EDU represents education expenditure, COR for corruption, POP for population growth rate, HE for health expenditure as percentage of GDP, GDP for gross domestic product, TE for tertiary enrolment and ε for error term. The subscripts i and t indicates the cross section and times series respectively. !!!"#$% = !!! ! !!! !!" −Ẍ!" ! ! !!! !!" − ! !!! Ẍ!" !!" ∗−!!! −1 (5) (5) 2. LITERATURE SURVEY The authors used both bivariate and multivariate estimation analysis to achieve the objectives set. Their results indicated that corruption has an adverse effect on education expenditure, where most corrupt developing economies devote a small share of government expenditure on education. Moreover, political instability has also a negative impact on education, and democracy will have no impact on education plans. Urhie, Ewetan and Okodua (2016) conducted a comparative study on national income, government spending on education and education attainment for 91 economies. The objectives of the study were to determine the relationship between education expenditure and national income. The study deployed the correlation analysis to determine how public expenditure on education differs with national income groups over time. The results revealed that there is positive association among government expenditure on education and national income at a global level over Phi (2017) assessed the determinants of health expenditure in OECD economies with focus on economic growth, demographics, medical progress and health care systems. The author uses both fixed effect and random effect models to examine the key drives of health expenditure in these economies. The results revealed the economic growth is the key The Effects of Determinants of Government Expenditure on Education and Health Journal of Reviews on Global Economics, 2020, Vol. 9 381 from 1997 to 2017. The education expenditure collected from African Development Indicators and UNESCO data, it worth noting that it had missing values in all the sources, thus the education expenditure data was extrapolated to fill in the miss values. The unit of measure of all the variables are in percentages except of corruption which is an index. time. In conclusion, more generally several factors have been identified in this literature section above. Firstly; there is no existing empirical literature on government expenditure on health and education in SADC countries. Except for the work of Mussagy and Babatunde (2015) which whoever focused on education and economic growth in Mozambique exclusively than considering SADC economies as an economic unit. Finally, the literature survey shows that no attention was given to study the phenomena within panel FMOLS and DOLS techniques. The interest of such use of techniques is that in a case of pre-existence of cointegration among the system it is best to use this parameter estimates since they do not require post-estimation diagnostics. Therefore, this gap warrants an investigation in SADC economies. 3. RESEARCH METHODOLOGY The purpose of the section is to outline the methods used to study the determinants of government expenditure on education and health. 3.3. Estimation Methods The difficulty with time series data is that independent variables can appear to be significant than they actually are, if it has the same trend as the dependent variables. Thus, non-stationary variables to appear to be related even if they are not related. Therefore, the current study needs to test for stationarity conditions of variables in order to avoid spurious regression. The stationarity of the variables is examined using both the unit root tests developed by Levin, Lin and Chu (LLC) (2002) and Im, Pesaran and Shin (2003). The next step is to test for cointegration among those variables using Pedroni (1999) and Pedroni (2001). Pedroni proposed the following model in test for cointegration among the variables which is residual based: 4. EMPIRICAL RESULTS Where !!! , is the long-run scalar variance of the residual !!" and !!! is the M × M long-run covariance among. !!". is ! ×1 vector that provides the long-run covariance among the residuals !!" and each of the !!" . The study assesses panel unit root for all the variables included in the model. The study deploys the LLC and Im, Pesaran and Shin for panel unit root. Table 1, shows Levin, Lin and Chu and Im, Pesaran and Shin panel unit root for SADC economies. The first column shows the order of integration, followed the variable interest. The second column is the unit root test Levin, Lin and Chu, which can be tested under Constant, Constant and Trend, and None. The third column reveal Im, Pesaran, and shin panel unit root test, which can be test under Constant, Constant and Trend only. Levin, Lin, and Chu test reveal that corruption, population and GDP are stationary at level, whereas the other are stationary at first difference. Im, Pesaran and Shin test reveal that education, population, corruption and GDP are stationary at levels, whereas the others are stationary at first difference. The dynamic ordinary least squares were established by Saikkonen (1991) and Stock and Watson (1993) to estimate and test hypothesis about cointegrating vector to panel data. Panel dynamic ordinary least squares is wholly parametric and gives a calculation convenient. The panel dynamic ordinary least Squares estimator for long run parameters can be defined geometrical as follows: !!" = !! + !"#!" + ! !!! !!"∆!!"!!+!!" (8) (8) Where !! Where: !!" ∗= !!" − Ĺ!!" Ĺ!!! ∆!!"ỹ!" ≡ Ѓ!!" + !!!" ! − Ĺ!!" Ĺ!!" Ѓ!!! + !!!! ! (6) All the data is obtained from the African Development Indicators published by the World Bank. The data applied is the annul data covering the period (6) Journal of Reviews on Global Economics, 2020, Vol. 9 382 Mhlari and Mosikari The former equation describes the transformed variables of !!" to attain endogeneity correction. The latter equation also defines the serial correlation term and !! is a lower triangular decomposition of !! which is the covariance metrics and can be defined as follows: differenced exogenous variables. While !!" is the error term which is assumed to be integrated for order zero. The parameter estimates of DOLS can be defined as follows: latter equation also defines the serial correlation term and !! is a lower triangular decomposition of !! which is the covariance metrics and can be defined as follows: !"#$%&!" = !!! ! !!! ! !!! ᴢ!!Ẑ!" ! !!! ᴢ!"!!" ∗ !! (9) Where ᴢ!"= !!" −Ẍ!"∆!!"!! … . . ∆!!"!! !"#$%&!" = !!! ! !!! ! !!! ᴢ!!Ẑ!" ! !!! ᴢ!"!!" ∗ !! (9) (9) Where ᴢ!"= !!" −Ẍ!"∆!!"!! … . . ∆!!"!! !! = !!! !!" !!" !!! (7) (7) Notes: (), (), (*) represent 10%, 5% 1% level of significant respectively. Notes: (), (), () represent 10%, 5% 1% level of significant respectively. esent 10%, 5% 1% level of significant respectively. 4. EMPIRICAL RESULTS represent cross section specific effects and !!" is the coefficient of a lead or lags of first Table 1: Panel Unit Root Test Results Table 1: Panel Unit Root Test Results Levin, Lin and Chu (LLC) Im, Pesaran and Shin Order of integration variable constant Trend & constant None Constant trend & constant Level EDU 0.119 (1.087) 0.154 (-1.985) 0.864 (0.370) 0.000 (-6.6320 0.012 (-6.672) First difference DEDU 0.000* (-20.949) 0.000* (-18.221) 0.000* (-23.848) 0.000* (-19.753) 0.000* (-17.581) Level HE 0.218 (-1.043) 0.5931 (-1.435) 0.8592 (1.097) 0.181 (-1.666) 0.383 (-1.225) First difference DHE 0.000* (-13.656) 0.000* (-12.238) 0.000* (-16.160) 0.000* (-11.919) 0.000* (-9.619) Level GDP 0.000* (-7.946) 0.000* (-7.493) 0.002* (-4.553) 0.000* (-7.761) 0.007* (-5.262) Level POP 0.000* (-4.845) 0.000*** (5.345) 0.053* (-6.564) 0.000* (-10.676) 0.000* (-12.765) Level LIFE 0.996 (0.496) 0.657 (-0.448) 1.000 (8.626) 0.999 (6.949) 0.522 (0.322) First difference DLIFE 0.014* (-7.167) 0.925 (8.505) 0.000* (-11.398) 0.000* (-11.735) 0.000* (-7.537) Level TE 0.996 (2.697) 0.313 (-0.548) 1.000 (7.626) 1.000 (5.949) 0.467 (0.486) First difference DTE 0.018 (-7.167) 0.017 (-8.505) 0.000* (-11.398) 0.000* (-11.735 0.000* (-10.327) Level COR 0.000* (-2.435) 0.02* (-4.456) 0.421 (0.168) 0.011** (-5.787) 0.056* (-3.987) Notes: () () () represent 10% 5% 1% level of significant respectively Table 1: Panel Unit Root Test Results The Effects of Determinants of Government Expenditure on Education and Health Journal of Reviews on Global Economics, 2020, Vol. 9 383 Table 2: Pedroni Panel Cointegration Test on Education and Health Expenditure Health expenditure Education expenditure Methods (t-statistic) Probability value (t-statistic) Probability value Within dimension/ panel statistic Panel v-statistics (-2.632), 0.995 (-2.086), 0.981 Panel rho-statistics (1.234), 0.891 (-0.171), 0.432 Panel PP-statistic (-11.007) 0.000* (-23.785) 0.000* Panel ADF-statistics (-4.368) 0.000* (-4.692) 0.000* Between dimension / group mean statistic Group rho-statistics (2.910) 0.998 (0.905) 0.817 Group PP-statistics (-15.777) 0.000* (-35.392) 0.000* Group ADF-statistics (-3.839) 0.000* (-4.675) 0.000*** Notes: () () (*) represent 10% 5% 1% level of significant respectively Pedroni Panel Cointegration Test on Education and Health Expenditure Notes: (), (), () represent 10%, 5% 1% level of significant respectively. Notes: (), (), () represent 10%, 5% 1% level of significant respectively. Thus, this study will apply non-stationary models, since the variables are integrated for different orders. On education model, within test shows that PP-statistic and ADF statistic are equally below 1% level of significant. Therefore, under this test we can reject the null hypothesis and conclude that there is cointegration between the variables. 4. EMPIRICAL RESULTS Panel V-statistics and Panel rho statistics are both above any level of significance. This indicates that under panel rho and panel v-statistics we failed to reject the null hypothesis and conclude that there is no cointegration between the variables. Therefore, we cannot conclude about cointegration relationship without considering the between dimension results. Since there is a draw between the four tests, two claiming no cointegration and the other two claiming cointegration between the variables Table 2 report the Pedroni cointegration test on health and education, which is divided into two groups, within dimension and Between Dimension statistics. The value on the brackets shows the estimation statistics. The null hypothesis of Pedroni cointegration test is that there is no cointegration between the variables. Therefore, if the probability values of the within and between dimension are below level of significant, we reject the null hypothesis and concluded that there is cointegration between the variables. On health model, within test shows that PP-statistic and ADF statistic are both below any indicated significance level. Therefore, under this test we can reject the null hypothesis and conclude that there is cointegration between the variables. Panel rho statistics and panel V-statistics are both above level of significance indicated. This implies that under panel rho and panel v-statistics we failed to reject the null hypothesis and conclude that there is no cointegration between the variables. Therefore, we cannot conclude about cointegration relationship without considering the between dimension results. Since there is a stalemate between the four tests, two claiming no cointegration and the other two claiming cointegration between the variables. The between dimension statistics reveals 0.986 p-value of group rho statistics, which is above any significance level. However, the table shows 0.000 and 0.000 p-values of PP-statistics and ADF statistics which are below 1% level of significant respectively. Therefore, two out of three between dimension group statistics rejects the null hypothesis that there is no cointegration. Majority of between dimension statistics reveals that the variables are cointegrated. The aggregate results of Pedroni cointegration test contend that there is cointegration between education expenditure and its potential determinants. Table 3 present the fully modified OLS and Dynamic OLS results with health expenditure as the dependent variable. The first column is the FMOLS results and last column is the DOLS results. FMOLS indicates that all the independent variables are significant except life expectancy variable. 4. EMPIRICAL RESULTS However, under DOLS only two variables that are significant which is education expenditure and corruption. In both FMOLS and DOLS shows that there is a positive insignificant relationship between health expenditure and life expectancy in SADC. FMOLS results show that there is positive and significant relationship between education expenditure and health expenditure in SADC. Therefore, 1% The between dimension statistics reveals 0.891 p-value of group rho statistics, which is above 10% level of significant. However, the table shows 0.000 and 0.000 p-values of PP-statistics and ADF statistics which are below 1% level of significant respectively. Therefore, two out of three between dimension group statistics rejects the null hypothesis that there is no cointegration. Majority of between dimension statistics reveals that the variables are cointegrated. The aggregate results of Pedroni cointegration test contend that there is cointegration between health expenditure and their potential determinants. Journal of Reviews on Global Economics, 2020, Vol. 9 Mhlari and Mosikari 384 Table 3: Health Expenditure in SADC FMOLS and DOLS Variables Fully modified OLS Dynamic OLS Coefficient Probability value Coefficient Probability value LIFE 0.075 0.197 0.007 0.9642 EDU 0.264 0.000 0.785 0.006* GDP -0.206 0.001* 0.015 0.586 POP 0.080 0.000* 0.012 0.192 COR 0.743 0.000*** -0.253 0.060* Adjusted R-squared 0.63213 0.7958 Notes: (), (), (*) represent 10%, 5% 1% level of significant respectively. Table 3: Health Expenditure in SADC FMOLS and DOLS increase in education expenditure will lead to 0.264% increase in health expenditure. Under DOLS the results show that there is positive significant relationship between health expenditure and education expenditure in SADC. One percent increase in education expenditure will results in 0.785% increase in health expenditure in SADC. Economic growth was found to be negative and significantly related to health expenditure in SADC under FMOLS. A 1% increase in economic growth will lead to 0.264% drop in health expenditure. DOLS revealed that positive and insignificant relationship between economic growth and health expenditure. A 1% increase in economic growth leading to 0.015% increase in health expenditure in SADC. Population has a positive significant relationship with health expenditure in SADC. One per cent increase in population will lead to 0.080% increase in health expenditure. The dynamic OLS model shows that there is positive and insignificant relationship between health expenditure and population, and 1% increase in population growth will lead to 0.012% increase in health expenditure. Notes: (), (), () represent 10%, 5% 1% level of significant respectively. Notes: (), (), () represent 10%, 5% 1% level of significant respectively. 4. EMPIRICAL RESULTS Corruption has a positive and significant relationship with health expenditure according to FMOLS, and 1% increase in corruption will lead to 0.743% increase in health expenditure. On the other hand, DOLS shows that there is negative and significant relationship between health expenditure and corruption, and 1% increase in corruption will lead to 0.253% drop in health expenditure. Table 4, under FMOLS it shows that education expenditure is positively and significantly related to higher education enrolments. A 1% increase in health expenditure will lead to 0.599% increase in education expenditure. Additionally, the dynamic OLS test also indicates that there is positive relationship between education and health expenditure in SADC. A 1% increase in health expenditure will lead to 0.925% increase in education expenditure. This results also indicates that education expenditure and health expenditure are complement rather than rivals. FMOLS reveals that gross domestic product has a positive and significant relationship with education expenditure in SADC. A 1% increase in gross domestic product will results in 0.0156% increase in education expenditure. In addition, the results show that population growth has a positive significant impact on education expenditure in SADC under FMOLS model. Thus 1% in population growth will lead into 0.038% increase in education expenditure in the region. Dynamic OLS also show that population growth has a positive impact on education expenditure, where 1% increase in population growth will lead to 0.056% increase in education expenditure in SADC. Lastly, the results under FMOLS shows that corruption has a negative and significant relationship with education expenditure. Thus, 1% increase in corruption in SADC will lead to 0.337% increase in education expenditure. Whereas dynamic OLS model reveal a negative significant relationship between Table 4: Education Expenditure in SADC FMOLS and DOLS Variable Fully modified OLS Dynamic OLS Coefficient Probability value Coefficient Probability value TE -0.646 0.000 -0.018 0.588 HE 0.599 0.000* 0.925 0.000* GDP 0.156 0.028 -0.017 0.016 POP 0.381 0.000* 0.056 0.000* COR 0.337 0.000* -0.106 0.001*** Adjusted R-squared 0.565963 0.96 Table 4: Education Expenditure in SADC FMOLS and DOLS Journal of Reviews on Global Economics, 2020, Vol. 9 385 The Effects of Determinants of Government Expenditure on Education and Heal Culyer, A.J. & Newhouse, J.P. 2000. Handbook of health economics. Elsevier. Culyer, A.J. & Newhouse, J.P. 2000. Handbook of health economics. Elsevier. education expenditure and corruption in SADC economies. A 1% increase in corruption will lead to 0.106 drop in education expenditure. 4. EMPIRICAL RESULTS Grigoli, F. & Kapsoli, J. 2013. Waste not, want not: the efficiency of health expenditure in emerging and developing economies. https://doi.org/10.5089/9781484364260.001 https://doi.org/10.1016/S0304-4076(03)00092-7 Kotera, G. & Okada, K. 2017. How does democratization affect the composition of government expenditure? Journal of Economic Behavior & Organization. https://doi.org/10.1016/j.jebo.2017.03.004 Levin, A., Lin, C.-F. & Chu, C.-S.J. 2002. Unit root tests in panel data: asymptotic and finite-sample properties. Journal of econometrics, 108 (1):1-24. https://doi.org/10.1016/S0304-4076(01)00098-7 Loto, M. 2011. Impact of government sectoral expenditure on economic growth. Journal of Economics and International Finance, 3 (11):646-652 Masenyetse, R.F. and Motelle, S.I., 2012. Government revenue-expenditure nexus in Lesotho: the decline in SACU revenue. American Journal of Economics, 2(1), pp.8-14. https://doi.org/10.5923/j.economics.20120201.02 Musaba, E.C., Chilonda, P. and Matchaya, G., 2013. Impact of government sectoral expenditure on economic growth in Malawi, 1980-2007. Journal of Economics and Sustainable Development, 4(2), pp.71-78. Mussagy, I.H. & Babatunde, M.A. 2015. Government Spending on Education and Economic Growth in Mozambique: A Cointegration Approach. Revista Electrónica de Investigação e Desenvolvimento, (5). 5. CONCLUSION OF THE STUDY Devkota, S.P., Chaulagain, R. & Bagale, S. 2016. Public expenditure in education sector of Nepal. The Online Journal of New Horizons in Education-April, 6 (2). The aim of the study was to examine the effects of determinants of government expenditure on education and health in SADC countries. Thereafter, assess how these determinants affect the latter variables. This study employed panel data approach by applying data unit root tests such as Im, Pesaran and Shin (IPS) and Levin, Lin and Chu (LLC). In determining the long run relationship between the variables understudy the technique of Pedroni cointegration tests was applied. Furthermore, DOLS and FMOLS were applied to estimate the parameters for the variables. The study highlights that corruption and education expenditure are the significant determinants of health expenditure, whereas these results differ from those of Novignon, et al. (2012). Other results show that health expenditure, gross domestic product, population growth and corruption are the significant determinants of education expenditure in SADC countries. The findings are supported by both FMOLS and DOLS estimators which shows significant impact of health expenditure, corruption and higher education enrolments. Thus, there is a need for SADC governments to embark on implementing policies that increase education expenditure and corruption control policies to affect government expenditure. This can be achieved by allocating high budget on education expenditure and implementing independent agencies to combat corruption. Dormont, B., Grignon, M. and Huber, H., 2006. Health expenditure growth: reassessing the threat of ageing. Health economics, 15(9), pp.947-963. https://doi org/10 1002/hec 1165 ( ) pp https://doi.org/10.1002/hec.1165 Guisan, M.-C. & Exposito, P. 2010. Health Expenditure, Education, Government Effectiveness and Quality of Life in Africa and Asia. Regional and Sectoral Economic Studies, 10 (1):115-126. Gumede, W. 2010. Tackling Corruption Jacana., 02:15-23. https://doi.org/10.1088/2058-7058/23/02/25 Im, K.S., Pesaran, M.H. & Shin, Y. 2003. Testing for unit roots in heterogeneous panels. 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Paper presented at the SADC Health protocol. Retrieved 27 july 2017. from, www.sadc.int/ documents-publication/show/804 UN. 2015. The Millennium Development Goals Report 2015 UNITED. New York. Retrieved, 27/july/2018.From,/www.google.com/ search?q=The+Millennium+Development+Goals+Report+20 15+UNITED&rlz=1C1GCEA_enZA775ZA775&oq=The+Mille nnium+Development+Goals+Report+2015+UNITED&aqs=c hrome..69i57j0.2696j0j8&sourceid=chrome&ie=UTF-8 SADC. 2011 population review in SADC. Retrieved, http://www.hst. org.za/publications/NonHST%20Publications/SADC_Region al_Assessment_2011-2012.pdf SADC. 2017. Action Plan for SADC Industrialization Strategy and Roadmap. Paper presented at the ROADMAP. Retrieved 27 july 2017 https://www.google.com/search?rlz. Urhie, E., Ewetan, O.O. & Okodua, H. 2016. National Income, Public Expenditure on Education and Educational Attainment: A Comparative Analysis. Covenant Journal of Business and Social Sciences, 6 (2). SADC. 2018 health review in SADC. 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https://openalex.org/W2125726508	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0082533&type=printable	English	null	Cost Analysis of Integrating the PrePex Medical Device into a Voluntary Medical Male Circumcision Program in Zimbabwe	PloS one	2,014	cc-by	6,700	Abstract doi:10.1371/journal.pone.0082533 Received July 28, 2013; Accepted October 23, 2013; Published May 6, 2014 Received July 28, 2013; Accepted October 23, 2013; Published May 6, 2014 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or ot any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. icle, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for work is made available under the Creative Commons CC0 public domain dedication. Funding: Funding support for this work was provided by PEPFAR (President’s Emergency Plan for AIDS Relief) through the USAID \| Health Policy Initiative Costing Task Order, contract number GPO-I-00-05-00040-00, beginning July 1, 2010. The Costing Task Order is implemented by Futures Group, in collaboration with the Futures Institute and the Centre for Development and Population Activities. The funder played a significant technical role in study design, analysis, decision to publish, and preparation of the manuscript. The views expressed in this manuscript do not represent the opinion of USAID or the U.S. Government. Competing Interests: The authors have declared that no competing interest exist. * E-mail: kkripke@futuresinstitute.org . These authors contributed equally to this work. adult VMMC devices are currently being assessed for safety, effectiveness, cost, and client and provider acceptability [8,9]. Devices could potentially simplify the procedure, enabling non- physician providers to conduct the surgery in a wider array of settings. The availability of devices might also offer an alternative mode of VMMC for men who have fears related to conventional surgery. Cost Analysis of Integrating the PrePex Medical Device into a Voluntary Medical Male Circumcision Program in Zimbabwe Emmanuel Njeuhmeli1., Katharine Kripke2., Karin Hatzold3, Jason Reed4, Dianna Edgil1, Juan Jaramillo5, Delivette Castor1, Steven Forsythe2, Sinokuthemba Xaba6, Owen Mugurungi6 1 United States Agency for International Development, Washington, DC, United States of America, 2 Health Policy Initiative, Futures Institute, Washington, DC, United States of America, 3 Population Services International, Harare, Zimbabwe, 4 Office of the U.S. Global AIDS Coordinator, Washington, DC, United States of America, 5 The Partnership for Supply Chain Management System, Arlington, Virginia, United States of America, 6 Zimbabwe Ministry of Health and Child Welfare, Harare, Zimbabwe Abstract Background: Fourteen African countries are scaling up voluntary medical male circumcision (VMMC) for HIV prevention. Several devices that might offer alternatives to the three WHO-approved surgical VMMC procedures have been evaluated for use in adults. One such device is PrePex, which was prequalified by the WHO in May 2013. We utilized data from one of the PrePex field studies undertaken in Zimbabwe to identify cost considerations for introducing PrePex into the existing surgical circumcision program. Methods and Findings: We evaluated the cost drivers and overall unit cost of VMMC at a site providing surgical VMMC as a routine service (‘‘routine surgery site’’) and at a site that had added PrePex VMMC procedures to routine surgical VMMC as part of a research study (‘‘mixed study site’’). We examined the main cost drivers and modeled hypothetical scenarios with varying ratios of surgical to PrePex circumcisions, different levels of site utilization, and a range of device prices. The unit costs per VMMC for the routine surgery and mixed study sites were $56 and $61, respectively. The two greatest contributors to unit price at both sites were consumables and staff. In the hypothetical scenarios, the unit cost increased as site utilization decreased, as the ratio of PrePex to surgical VMMC increased, and as device price increased. Conclusions: VMMC unit costs for routine surgery and mixed study sites were similar. Low service utilization was projected to result in the greatest increases in unit price. Countries that wish to incorporate PrePex into their circumcision programs should plan to maximize staff utilization and ensure that sites function at maximum capacity to achieve the lowest unit cost. Further costing studies will be necessary once routine implementation of PrePex-based circumcision is established. Citation: Njeuhmeli E, Kripke K, Hatzold K, Reed J, Edgil D, et al. (2014) Cost Analysis of Integrating the PrePex Medical Device into a Voluntary Medical Male Circumcision Program in Zimbabwe. PLoS ONE 9(5): e82533. doi:10.1371/journal.pone.0082533 Citation: Njeuhmeli E, Kripke K, Hatzold K, Reed J, Edgil D, et al. (2014) Cost Analysis of Integrating the PrePex Medical Device into a Voluntary Medical Male Circumcision Program in Zimbabwe. PLoS ONE 9(5): e82533. Citation: Njeuhmeli E, Kripke K, Hatzold K, Reed J, Edgil D, et al. (2014) Cost Analysis of Integrating the PrePex Medical Device into a Voluntary Medical Male Circumcision Program in Zimbabwe. PLoS ONE 9(5): e82533. doi:10.1371/journal.pone.0082533 Introduction In 2005–2007, three randomized controlled clinical trials demonstrated that voluntary medical male circumcision (VMMC) reduced male acquisition of HIV through heterosexual intercourse by approximately 60% [1–3]. Since then mathematical modeling studies have suggested that scaling up VMMC in 13 Eastern and Southern African countries to 80% coverage over five years and maintaining that coverage through 2025 could avert 3.4 million HIV infections over that time period and save approximately $16.5 billion in the cost of HIV treatment and care [4]. One adult VMMC device, PrePex, developed by Circ MedTech (Herzelia, Israel), works by compressing the foreskin between a fitted ring and elastic band, leading to distal tissue necrosis. In most cases, PrePex does not require the use of injected anesthesia and does not require suturing. Clients who undergo VMMC using the PrePex device are required to wear the device for seven days and then return to the clinic for removal on the seventh day. In a Despite the promise of VMMC to substantially impact the HIV epidemic in these settings, scale-up of VMMC programs has been challenged by issues related to demand creation and service availability in remote and resource-constrained areas [5–7]. New May 2014 \| Volume 9 \| Issue 5 \| e82533 May 2014 \| Volume 9 \| Issue 5 \| e82533 1 PLOS ONE \| www.plosone.org Cost of Integrating PrePex into VMMC Figure 1. Key characteristics of the different implementation models. The routine surgery site and the hypothetical mixed site employed 1 medical doctor, 6 nurses, 3 theatre assistants, and 1 receptionist in a 4-bed facility, and the mixed study site employed 1 medical doctor, 8 nurses, 3 theatre assistants, and 1 receptionist in a 6-bed facility. doi:10.1371/journal.pone.0082533.g001 Figure 1. Key characteristics of the different implementation models. The routine surgery site and the hypothetical mixed site employed 1 medical doctor, 6 nurses, 3 theatre assistants, and 1 receptionist in a 4-bed facility, and the mixed study site employed 1 medical doctor, 8 nurses, 3 theatre assistants, and 1 receptionist in a 6-bed facility. doi:10.1371/journal.pone.0082533.g001 Figure 1. Key characteristics of the different implementation models. The routine surgery site and the hypothetical mixed site employed 1 medical doctor, 6 nurses, 3 theatre assistants, and 1 receptionist in a 4-bed facility, and the mixed study site employed 1 medical doctor, 8 nurses, 3 theatre assistants, and 1 receptionist in a 6-bed facility. doi:10.1371/journal.pone.0082533.g001 available. Introduction Therefore, we sought to examine potential cost implications of integrating the PrePex device into an existing VMMC program. Instead of estimating a unit cost specifically for PrePex-based VMMC vs. surgical VMMC, as others have [16,17], we examined the site-level unit cost of VMMC at a high- throughput public facility providing only routine surgical VMMC (‘‘routine surgery site’’) and at a similar facility in which staff and equipment were added to also conduct PrePex-based circumci- sions for a research study (‘‘mixed study site’’). We used data collected during routine surgical implementation in Zimbabwe and as part of the Zimbabwe PrePex field study, respectively. Because of the differing staffing pattern and equipment used at each site, the unit costs of the two sites are not comparable. Therefore, to enable comparison of the unit costs, we also created a hypothetical mixed site scenario that retained the same staffing pattern and number of beds as the routine surgery site. Rwanda field study, the PrePex procedure took 4.3 minutes for device application (including preparation) and 3.8 minutes for device removal seven days later [10], compared with 23– 30 minutes for conventional surgery (from scrubbing the patient in preparation for the operation to cleaning the wound after suturing) in a multi-country study [11]. A series of three studies (safety case study, comparative study, and field study) of the PrePex device was first completed in Rwanda [10,12,13]. This same series of studies was subsequently completed independently in Zimbabwe. The information gener- ated from these six studies informed the WHO decision in May 2013 to add PrePex to its prequalification list [14,15]. Claims have been advanced that the PrePex procedure would result in significantly decreased unit costs per VMMC compared with conventional surgery; this has not been borne out in analyses published to date. Using hypothetical costing information from Kenya, Obiero and colleagues derived a unit cost of $44.54– $49.02 for PrePex, not including the device cost, and $54.52– $55.29 for forceps-guided surgical VMMC [16]. Duffy and colleagues collected costs of PrePex-based and surgical VMMC at a high-volume urban hospital in Uganda and found a unit cost of $30.55 for PrePex-based and $22.65 for surgical VMMC, using a PrePex device price of $20 and excluding demand creation costs [17]. This study poses several questions: (1) What are the major drivers of unit costs in each type of site (routine surgery and the two different mixed sites)? Introduction (2) How would the unit cost at a mixed site change with varying ratios of surgical to PrePex-based circumcisions? (3) How would unit costs change with varying levels of site utilization? (4) What impact would different device prices have on the unit cost? Methods Countries in Eastern and Southern Africa have already begun scaling up VMMC using conventional surgical approaches. Because of age and other exclusions (PrePex is only qualified for use with men over the age of 18), programs that wish to introduce PrePex will need to continue to make conventional surgery PLOS ONE \| www.plosone.org May 2014 \| Volume 9 \| Issue 5 \| e82533 Cost Model All analyses were performed using Microsoft Excel 2010 (Microsoft Corporation, Redmond, WA). All costs are presented in USD. All analyses were performed using Microsoft Excel 2010 (Microsoft Corporation, Redmond, WA). All costs are presented in USD. The routine surgery site had a four-bed capacity and employed one medical doctor, six nurses, three theatre assistants, and one receptionist. The doctors had overall responsibility for the surgical procedure, provided local anesthesia, removed the foreskin, stopped bleeding either using sutures or electrocauterization, and attended to post-operative complications and treatment of adverse events involving wound revisions or severe infections with abscesses. The nurses conducted group education sessions, conducted pre-operative examination and counseling, conducted HIV testing and counseling, assisted with circumcisions, conduct- ed post-operative examination and counseling, and conducted post-operative review of the client on day 2,day 7 and day 42 post- surgery. The theatre assistant was responsible for cleaning the operation room, cleaning instruments after the operation, prepar- ing instruments and operation room in case of adverse events, and cleaning the waiting area, counseling and examination rooms. The receptionist was responsible for taking clients’ personal details, explaining the procedure, and booking the client for operation and follow-up review. Unit costs per circumcision (regardless of site type) comprised seven cost categories: consumables, device, supply chain manage- ment, staff, durable equipment, training, and waste management. Indirect costs, costs for demand creation, and costs for complica- tions were not included in this analysis. Unit costs represent costs to the service provider and do not include costs to clients, such as transport to and from the intervention sites. The VMMC unit cost for the mixed sites is the average unit cost of all circumcisions provided, including both conventional surgical VMMCs and PrePex-based VMMCs; we did not disaggregate unit costs by modality. Site-level unit costs were derived by calculating the per circumcision cost for each of the seven cost categories and then adding the costs of all the categories: Unit cost~ cspszcppp \|ﬄﬄﬄﬄﬄﬄﬄ{zﬄﬄﬄﬄﬄﬄﬄ} consumable cost zdppz 31:4%cspsz31:4%(cpzd)pp \|ﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄ{zﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄ} supply chain management cost z(szezt)=mzw Unit cost~ cspszcppp \|ﬄﬄﬄﬄﬄﬄﬄ{zﬄﬄﬄﬄﬄﬄﬄ} consumable cost zdppz 31:4%cspsz31:4%(cpzd)pp \|ﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄ{zﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄﬄ} supply chain management cost z(szezt)=mzw The mixed study site had a six-bed capacity and employed one medical doctor, eight nurses (an additional two nurses and two beds to conduct PrePex circumcisions), three theatre assistants, and one receptionist. Cost Model In addition to the responsibilities outlined above for the surgery site, the doctor supervised the nurses conducting PrePex circumcisions, handled device-related compli- cations, and conducted surgery in case of displacement of the device. The nurses conducted the PrePex procedure: fitting the PrePex device size, placing the device, removing the dried foreskin, inner ring, and elastic band on day 7, dressing the wound, and conducting post-operative review of the client after removal of the device on day 7 and day 42. The theatre assistant sterilized instruments used for removal of the foreskin and device. All staff at the mixed site were qualified to conduct both types of circumcisions based on client demand. where cs is the per circumcision cost of consumables for conventional surgical circumcisions ps is the percentage of conventional surgical circumcisions at the site (for the routine surgery site, ps = 100%) cp is the per circumcision cost of consumables for PrePex-based circumcisions cp is the per circumcision cost of consumables for PrePex-based circumcisions pp is the percentage of PrePex-based circumcisions at the site (for the routine surgery site, pp = zero) pp is the percentage of PrePex-based circumcisions at the site (for the routine surgery site, pp = zero) d is the device price 31.4% of the cost of consumables (including device) constitutes the supply chain cost [18] 31.4% of the cost of consumables (including device) constitutes the supply chain cost [18] s is the annual staff cost at that type of site s is the annual staff cost at that type of site e is the annual durable equipment cost at that type of site e is the annual durable equipment cost at that type of site t is the annual training cost at that type of site t is the annual training cost at that type of site m is the median number of circumcisions performed per day at that type of site, multiplied by 220 working days per year The hypothetical mixed site had a four-bed capacity and employed the same staff as the routine surgery site. w is the cost of waste management per circumcision Data on service delivery were collected between May 8 and July 9, 2012, at the Harare site for the mixed study site model and at the Bulawayo site for the routine surgery site model. For PrePex- based circumcisions, clients who presented for placements were counted as PrePex VMMCs conducted. Implementation models Three implementation models were evaluated (Figure 1): a routine surgery site based on the configuration (staffing, equip- ment) of the Bulawayo VMMC Centre, a mixed study site based PLOS ONE \| www.plosone.org May 2014 \| Volume 9 \| Issue 5 \| e82533 May 2014 \| Volume 9 \| Issue 5 \| e82533 2 Cost of Integrating PrePex into VMMC described below when calculating the unit cost at the mixed study site. on the configuration of the Harare PrePex Field Study Site, and a hypothetical mixed site with a configuration comparable to that of the routine surgery site. The Bulawayo and Harare sites are dedicated VMMC sites, where efficiencies such as task sharing, use of VMMC kits with disposable surgical instruments for the conventional surgery, and use of multiple surgical beds for each surgeon are being implemented. Cost Model All devices were removed at day 7. The median number of daily circumcisions per site during this period was used in all of the unit cost calculations except the analysis that examined different levels of site capacity utilization, which used the observed minimum, first quartile, median, third quartile, maximum, and theoretical maximum (explained in the Results section) numbers of clients per day. The population to be circumcised included males ages 10 to 49 years; those under 18 years of age, those declining to participate in the study, and those with exclusions such as HIV infection, cracked foreskin, phimosis, short or tight frenulum, or preputial adhesions were not eligible to be circumcised using PrePex and were offered conventional surgery. Those with active genital infections were treated and asked to come back for circumcision at a later date. In the mixed study sites during the period of data collection, 84% of the circumcisions were conducted using conventional surgery and 16% were conducted using PrePex. This ratio was applied as Details about each cost category follow. Details about each cost category follow. Consumables. Lists of required consumables for both surgical VMMC using the forceps-guided technique and PrePex- based circumcisions were provided by the Zimbabwe PrePex Field Study team, as defined in the study protocol. Consumables prices were provided by the PEPFAR funded through USAID Partner- ship for Supply Chain Management System (SCMS) project and are available in Table S1 and Table S2. For conventional surgical VMMC, the study utilized pre-sterilized, disposable commodities bundled into kits (market price sourced by SCMS as of February 15, 2013), while a combination of disposable and reusable commodities was used for the PrePex procedure. The commodity costs for the PrePex procedure were derived from average SCMS pricing from procurements in 2009–2012, with data compiled on January 10, 2013, or from a quotation obtained from Circ MedTech on December 19, 2012, as indicated in Table S2. The actual PrePex device is disposable/single-use. Each reusable commodity cost was divided across 150 procedures to derive the unit cost, based upon experience in the field study. Device. The baseline PrePex device cost applied was USD $20 per unit based on a quotation from Circ MedTech to SCMS Device. Utilization of site capacity Generating, maintaining, and predicting fluctuations in demand for VMMC over time can be challenging. In some countries scaling up VMMC, sites have been fully staffed and equipped, only to experience suboptimal utilization during some periods. We examined the impact of different levels of site utilization on the unit cost. In these analyses the daily costs for staffing, durable equipment, and training were held constant, while consumables, device, supply chain management, and waste management costs varied by number of circumcisions. The distribution of numbers of circumcisions per day was based on site utilization data from the routine surgery and mixed study sites between May 8 and July 9, 2012. Because the sites were not operating at full capacity, a theoretical maximum number of circumcisions per day was estimated based on the complement of staff and equipment at each type of site in an eight-hour day if demand for circumcisions met or exceeded supply. The median number of VMMC procedures per day was 26 at both the routine surgery site and the mixed study site. The theoretical maximum number of daily VMMCs was 80 at the routine surgery site and 120 at the mixed study site. VMMC unit cost was highly sensitive to the level of site utilization in both types of sites, ranging at the mixed study site from $45 at the theoretical maximum utilization to $98 when the site performed only nine VMMCs per day (the minimum observed during the two-month period at this site). Similarly, at the routine surgery site, the unit cost ranged from $45 at theoretical maximum site utilization to $122 when only five VMMCs (the minimum observed during the two-month period at this site) were performed per day (Table 3). Training. All nurses and doctors in the routine surgery and mixed sites received the same initial competency-based team training. Staff turnover was approximately 50% per year, so on average an entirely new cohort of staff would need to be retrained every 3.6 years. Therefore, training costs were allocated uniformly over 3.6 years. At the routine surgery site, the training cost in 2012 was USD $884 per trainee for a 6-day course that included 2.5 days of theory and 3.5 days of applied work. For the mixed sites, the training cost was USD $1,252 per trainee for an 8.5-day course that included an additional 2.5 days for applied training with PrePex. Impact of device price The price of the PrePex device outside of small procurements for research studies has not been negotiated. Because only 16% of circumcisions used PrePex at the mixed sites, we modeled the variations in unit cost as a function of a variety of PrePex device prices using a fictional scenario in which 68% of the circumcisions were performed using PrePex (the maximum possible in this population given age and physiological exclusions), in order to see the maximum possible impact of variations in device price. Under these assumptions, the unit cost ranged from $50 at a device price of $2.00 (device 3% of the unit cost) to $63 at a device price of $20 (device 22% of unit cost) (Table 4). Therefore, the unit cost is sensitive to variations in the device price. Key cost drivers The unit cost per VMMC for the routine surgery site was $55.83, for the mixed study site it was $60.58, and for the hypothetical mixed site it was $57.45 (Table 1). The two largest contributors to the unit cost were consumables and staff. At the routine surgery site, consumables ($30.36) and staff ($14.90) contributed a combined 81% to the unit cost; in the mixed study site, consumables ($30.87), including device, and staff ($17.83) contributed a combined 80% to the unit cost; in the hypothetical mixed site, consumables ($30.87), including device, and staff ($14.90) contributed a combined 80% to the unit cost. Training, durable equipment, and waste management contributed negligibly to the unit costs at all three sites. Utilization of site capacity All nurses and doctors at the mixed site were trained in both surgical and PrePex-based circumcision. Waste management. At the routine surgery site, according to 2012 site records, the cost of waste management of USD $1.00 per kg of waste was based on current program generation of 25 kg of waste per week, resulting from 130 circumcisions per week. The same unit cost for waste management per VMMC was applied to the mixed sites. Cost of Integrating PrePex into VMMC Cost of Integrating PrePex into VMMC years old, and 5.6% were ineligible for physiological reasons such as phimosis or tight foreskin. Since acceptability of the PrePex device in Zimbabwe outside the study environment is currently unknown, we looked at the effect on the unit cost of varying the ratio of conventional surgical circumcisions to PrePex-based circumcisions. The maximum percentage of PrePex-based cir- cumcisions with 100% acceptability would be 68%, given age and physiological exclusions. We kept the staffing and the total number of circumcisions per day constant in this analysis: although in theory both the staffing and the total number of circumcisions per day might change with different ratios of the different types of circumcisions, we did not have robust data upon which to base changes in these variables for this analysis. The unit cost in this analysis ranged from $60 per circumcision when 100% of circumcisions were performed using conventional surgery to $63 per circumcision when 68% of circumcisions were performed using PrePex (Table 2). on December 19, 2012. This is not the price that was used in the series of clinical studies in Zimbabwe, but it was the price quoted by the manufacturer for pilot implementation studies in several countries. Supply chain management. Based on a study of the existing supply chain management system in Zimbabwe conducted by the USAID DELIVER Project in 2010, supply chain costs per circumcision were calculated by multiplying the costs of consum- ables (including device) by 31.4%, comprising 11.4% for procurement costs plus 20% for logistics expenses [18]. Staff salaries. Current (2012) salaries of public-sector staff involved in the VMMC program in Zimbabwe were used: medical doctor, $2,200 per month; nurse, $700 per month; theatre assistant, $150 per month; receptionist, $250 per month. These salary costs are uniform for all sites where public-sector cadres are used. Monthly salaries were multiplied by twelve to produce an annual salary and then divided by the number of circumcisions per year to produce per circumcision staff costs. Durable equipment. The list of durable equipment utilized by each type of site was provided by the Zimbabwe PrePex Field Study team. Costs for each item were derived from average SCMS pricing from procurements in 2009–2012, with data compiled on January 10, 2013, and listed in Table S3. Equipment costs were allocated uniformly over three years based on estimated life span. Cost Model The baseline PrePex device cost applied was USD $20 per unit based on a quotation from Circ MedTech to SCMS May 2014 \| Volume 9 \| Issue 5 \| e82533 PLOS ONE \| www.plosone.org 3 Discussion of underutilization would be even greater if overhead costs had been included in the analysis. This result highlights the importance of optimal demand creation and a balanced relationship between service supply (availability) and demand for VMMC services, regardless of the circumcision method used. In this analysis we sought to examine the impact on VMMC unit costs of introducing PrePex into an existing routine surgical VMMC program in Zimbabwe. Introduction of PrePex at the study site did not have a large impact on the overall unit cost. The key cost drivers for both the routine surgery and the mixed sites were consumables and staff salary costs, suggesting areas of focus for lowering the price per VMMC. At the mixed study site, the unit cost only increased by $3 when the ratio of surgical and PrePex-based circumcisions was hypothetically varied from 100% surgery to 32% surgery and 68% PrePex. Unit costs were highly sensitive to potential variations in the device price when 68% of the circumcisions were performed using the device; therefore, a responsibly low public-sector price could result in cost savings and avail VMMC services to more people in need, if acceptability of PrePex turns out to be high. Further research examining alternative service delivery models in different settings and country contexts might find unit costs that are different from those presented in this study. For example, if implementation is carried out in a site dedicated entirely to VMMC, rather than co-located or integrated within a public health care facility, increased costs for waste transportation and disposal and supply chain management might be expected with either the routine surgery or mixed site model. If circumcisions are outsourced to the private sector, staff salaries, facility rental or construction, and profit may increase or decrease the unit costs. The use of temporary or locum staff to adjust capacity to accommodate fluctuations in demand could change the cost structure. Different implementation models—for example, those involving mobile teams and outreach campaigns—might include additional or higher costs for expenses such as transportation. If a new supply chain management system needs to be created or the existing one strengthened, supply chain management costs would The largest impact on the unit cost was underutilization of site capacity. At the mixed study site, the minimum observed daily service utilization resulted in unit costs twice those of maximum observed service utilization. Impact of ratio of conventional surgical to PrePex-based circumcisions at mixed site At the mixed study site between May 8 and July 9, 2012, 28% of the clients were ineligible for PrePex due to being less than 18 May 2014 \| Volume 9 \| Issue 5 \| e82533 PLOS ONE \| www.plosone.org 4 Cost of Integrating PrePex into VMMC Table 1. Cost drivers for unit cost of different implementation models. Routine surgery site Mixed study site Hypothetical mixed site* Cost category cost per circumcision % of unit cost Cost per circumcision % of unit cost Cost per circumcision % of unit cost Staff $14.90 27% $17.83 29% $14.90 26% Training $0.30 0.5% $0.58 1.0% $0.45 0.8% Consumables $30.36 54% $27.62 46% $27.62 48% Device $0.00 0% $3.25 5% $3.25 6% Durable equipment $0.55 1.0% $1.42 2.3% $1.35 2.4% Supply chain management $9.53 17% $9.69 16% $9.69 17% Waste management $0.19 0.3% $0.19 0.3% $0.19 0.3% Total unit cost/circumcision $55.83 $60.58 $57.45 84% surgery+16% PrePex. doi:10.1371/journal.pone.0082533.t001 Discussion Sensitivity analysis of device price on unit cost at mixed study site with 68% of circumcisions conducted using the PrePex device. Routine surgery site1 Mixed study site2 doi:10.1371/journal.pone.0082533.t004 demand creation, this question merits further research. At this time, little is known about the acceptability of the PrePex device outside of research settings, either in Zimbabwe or in other cultural contexts. Costs of supervision, community engagement, and program management were not included in this analysis. These costs should be collected as the introduction of PrePex is rolled out on a larger scale in a number of different settings. demand creation, this question merits further research. At this time, little is known about the acceptability of the PrePex device outside of research settings, either in Zimbabwe or in other cultural contexts. Costs of supervision, community engagement, and program management were not included in this analysis. These costs should be collected as the introduction of PrePex is rolled out on a larger scale in a number of different settings. likely be higher, at least temporarily. If commodity transportation requires air freight rather than ocean freight, procurement costs will be higher. The teams in Zimbabwe were highly experienced and efficient, so they could conduct many circumcisions per day. Newly trained teams usually need more time to conduct each circumcision, reducing productivity and driving up unit costs. likely be higher, at least temporarily. If commodity transportation requires air freight rather than ocean freight, procurement costs will be higher. The teams in Zimbabwe were highly experienced and efficient, so they could conduct many circumcisions per day. Newly trained teams usually need more time to conduct each circumcision, reducing productivity and driving up unit costs. This study provides new data on the cost of introducing the PrePex device into an existing surgery-based VMMC program and highlights the importance of controlling consumable and staff costs and ensuring high levels of site utilization through effective demand creation strategies. We found no evidence in these analyses that introducing the PrePex device would result in increased efficiency of the VMMC program in terms of reducing the unit cost. To assist countries that wish to incorporate PrePex into their VMMC scale-up plans, it will be necessary to collect a broader diversity of actual cost data from different implementation models, across different countries. It is also reasonable to speculate that unit costs could decrease as a result of PrePex availability. Acknowledgments The authors gratefully acknowledge the Zimbabwe Ministry of Health, including the PrePex Field Study team, for providing the data. We thank Renee Ridzon and Geoff Garnett from the Bill and Melinda Gates Foundation, Julia Samuelson from the World Health Organization, Tim Farley, Kevin Duffy, Moses Galukande, Tigistu Adamu, Kelly Curran, Hally Mahler, Robert Bailey, and Walter Obiero for their critical feedback, helpful discussion, and information sharing. Finally, the authors would like to thank the publications staff at MCHIP for their assistance with copy editing and formatting. Table S2 Itemized consumables costs for PrePex-based circumcisions. Table S3 Durable equipment costs for the routine surgery site, mixed study site, and hypothetical mixed site. This study has several limitations. The per-VMMC unit costs provided did not include all cost components, and they were based on limited data using a single service delivery model. PrePex procedures were performed as part of a study, and thus the costs of those procedures do not reflect routine service delivery conditions (for example, in Zimbabwe the mixed sites employed more staff than those sites offering routine circumcision, but did not observe an increase in the total number of circumcisions. In routine service delivery, it is doubtful that such additional staff would be hired without any increase in uptake). Conventional surgical circumci- sions, on the other hand, were provided as a routine service, in a model perfected over years. Actual unit costs should be determined by careful costing studies in a large number of sites once routine implementation of both circumcision methods is well under way. The unit costs did not include indirect costs or costs for demand creation, each of which might contribute significantly to the unit cost. Other groups have attempted to derive unit costs for demand creation for VMMC in other settings, but they have found too much variation in implementation and costs to be able to produce a standard unit cost [19]. Because of the importance of (DOCX) Discussion At the point that they are no longer experimental, PrePex circumcisions provided as a routine service could be reduced in cost due to potential reductions in consumables and supply chain costs and higher service utilization. A responsible public-sector price for the PrePex device when purchased in large volumes, which reflects the costs of materials and production plus a reasonable mark-up for profit, should reduce the overall consumables cost. Because supply chain costs are related to consumables costs, by extension they might also decrease. Supporting Information Table S1 Itemized consumables costs for forceps- guided routine surgical circumcisions. (DOCX) In Zimbabwe, nurses are not allowed to perform certain aspects of the surgical male circumcision procedure, but the PrePex Field Study demonstrated that nurses are able to conduct the entire PrePex VMMC procedure. Different staffing patterns can affect the overall unit cost. For countries without task shifting, instituting a task-shifting policy is one way to significantly decrease staff costs and therefore decrease unit costs of VMMC. Table S2 Itemized consumables costs for PrePex-based circumcisions. Discussion Similar cost increases were seen from underutilization at the routine surgery sites. The negative impact on of conventional surgical versus PrePex-based circumcisions on unit cost at the mixed study site. Table 2. Effect of proportion of conventional surgical versus PrePex-based circumcisions on unit cost at the mixed study site. % conventional surgical circumcisions % PrePex-based circumcisions Unit cost 100% 0% $60 95% 5% $60 90% 10% $60 80% 20% $61 70% 30% $61 60% 40% $62 50% 50% $62 40% 60% $62 32% 68% $63 * This number is different from the unit cost at the routine surgery site because additional equipment and staff were added at the mixed study site, but there was no increase in the number of circumcisions conducted per day. doi:10.1371/journal.pone.0082533.t002 Table 2. Effect of proportion of conventional surgical versus PrePex-based circumcisions on unit cost * This number is different from the unit cost at the routine surgery site because additional equipment and staff were added at the mixed study site, but there was no increase in the number of circumcisions conducted per day. doi:10.1371/journal.pone.0082533.t002 * This number is different from the unit cost at the routine surgery site because additional equipment and staff were added at the mixed study site, but there was no increase in the number of circumcisions conducted per day. doi:10.1371/journal.pone.0082533.t002 May 2014 \| Volume 9 \| Issue 5 \| e82533 PLOS ONE \| www.plosone.org Cost of Integrating PrePex into VMMC Table 3. Effect of site capacity utilization on unit cost at routine surgery site and mixed study site. Routine surgery site1 Mixed study site2 # circ/day Unit cost # circ/day Unit cost Minimum 5 $122 9 $98 First quartile 15 $67 17 $71 Median 26 $56 26 $61 Third quartile 31 $53 33 $56 Actual maximum 56 $47 58 $50 Theoretical maximum 80 $45 120 $45 14 beds, 7 medical staff. 26 beds, 9 medical staff. doi:10.1371/journal.pone.0082533.t003 Table 4. Sensitivity analysis of device price on unit cost at mixed study site with 68% of circumcisions conducted using the PrePex device. Device price Unit cost Device % of unit cost $2 $50 3% $5 $52 6% $10 $56 12% $15 $59 17% $20 $63 22% doi:10.1371/journal.pone.0082533.t004 Table 3. Effect of site capacity utilization on unit cost at routine surgery site and mixed study site. Table 4. References 1. Auvert B, Taljaard D, Lagarde E, Sobngwi-Tambekou J, Sitta R, et al. (2005) Randomized controlled intervention trial of male circumcision for reduction of HIV infection risk: the ANRS 1265 Trial. PLoS Med 2: e298. 11. Bertrand JT, Rech D, Dickens O, Frade S, Loolpapit M, et al. (2012) Systematic monitoring of the male circumcision scale-up in Eastern and Southern Africa (SYMMACS): Interim report on results from Kenya, South Africa, Tanzania and Zimbabwe. July. USAID \| Project Search: Research to Prevention. monitoring of the male circumcision scale-up in Eastern and Southern Africa (SYMMACS): Interim report on results from Kenya, South Africa, Tanzania and Zimbabwe. July. USAID \| Project Search: Research to Prevention. 2. Bailey RC, Moses S, Parker CB, Agot K, Maclean I, et al. (2007) Male circumcision for HIV prevention in young men in Kisumu, Kenya: a randomised controlled trial. Lancet 369: 643–656. ( ) p y , , and Zimbabwe. July. USAID \| Project Search: Research to Prevention. 12. Mutabazi V, Kaplan SA, Rwamasirabo E, Bitega JP, Ngeruka ML, et al. (2012) HIV prevention: male circumcision comparison between a nonsurgical device to a surgical technique in resource-limited settings: a prospective, randomized, nonmasked trial. J Acquir Immune Defic Syndr 61: 49–55 10.1097/ QAI.1090b1013e3182631d3182669. 3. Gray RH, Kigozi G, Serwadda D, Makumbi F, Watya S, et al. (2007) Male circumcision for HIV prevention in men in Rakai, Uganda: a randomised trial. Lancet 369: 657–666. 4. Njeuhmeli E, Forsythe S, Reed J, Opuni M, Bollinger L, et al. (2011) Voluntary medical male circumcision: modeling the impact and cost of expanding male circumcision for HIV prevention in eastern and southern Africa. PLoS Med 8: e1001132. 13. Mutabazi V, Kaplan SA, Rwamasirabo E, Bitega JP, Ngeruka ML, et al. (2013) One-arm, open-label, prospective, cohort field study to assess the safety and efficacy of the PrePex device for scale-up of nonsurgical circumcision when performed by nurses in resource-limited settings for HIV prevention. J Acquir Immune Defic Syndr 63: 315–322. 5. UNAIDS (2013) UNAIDS World AIDS Day report 2012: results 2012. Geneva: UNAIDS. 14. World Health Organization (2013) WHO prequalification of male circumcision devices. Public report. Product: PrePex. Geneva: WHO. 6. Reed JB, Njeuhmeli E, Thomas AG, Bacon MC, Bailey R, et al. (2012) Voluntary medical male circumcision: an HIV prevention priority for PEPFAR. J Acquir Immune Defic Syndr 60 Suppl 3: S88–95. 15. Author Contributions Conceived and designed the experiments: EN KK KH JR DE JJ DC SF. Performed the experiments: KK. Analyzed the data: KK. Contributed reagents/materials/analysis tools: KH SX OM. Wrote the paper: EN KK KH JR DE JJ DC SF SX OM. May 2014 \| Volume 9 \| Issue 5 \| e82533 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 6 Cost of Integrating PrePex into VMMC References World Health Organization Technical Advisory Group on Innovations Circumcision (2013) Evaluation of two adult devices. Geneva: WHO. J q y pp 7. World Health Organization (2013) Progress in scaling up voluntary medical male circumcision for HIV prevention in East and Southern Africa. Geneva: WHO. 16. Obiero W, Young MR, Bailey RC (2013) The PrePex device is unlikely to achieve cost-savings compared to the forceps-guided method in male circumcision programs in sub-Saharan Africa. PLoS ONE 8: e53380. 8. Morris BJ, Eley C (2011) Male circumcision: An appraisal of current instrumentation. In: Fazel PR, editor. Biomedical engineering: from theory to applications. New York: InTech. 17. Duffy K, Galukande M, Wooding N, Dea M, Coutinho A (2013) Reach and cost-effectiveness of the PrePex device for safe male circumcision in Uganda. PLoS ONE 8: e63134. 9. Wamai RG, Morris BJ, Bailis SA, Sokal D, Klausner JD, et al. (2011) Male circumcision for HIV prevention: current evidence and implementation in sub- Saharan Africa. J Int AIDS Soc 14: 49. 18. Sarley D, Baruwa E, Tien M (2010) Zimbabwe: supply chain costing of health commodities. Arlington, Va.: USAID \| DELIVER PROJECT, Task Order 1. 19. Bertrand JT, Njeuhmeli E, Forsythe S, Mattison SK, Mahler H, et al. (2011) Voluntary medical male circumcision: a qualitative study exploring the challenges of costing demand creation in eastern and southern Africa. PLoS One 6: e27562. 10. Bitega JP, Ngeruka ML, Hategekimana T, Asiimwe A, Binagwaho A (2011) Safety and efficacy of the PrePex device for rapid scale-up of male circumcision for HIV prevention in resource-limited settings. JAIDS J Acquir Immune Defic Syndr 58: e127–e134 110.1097/QAI.1090b1013e3182354e3182365. May 2014 \| Volume 9 \| Issue 5 \| e82533 7 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 7
https://openalex.org/W2915925300	https://chesterrep.openrepository.com/bitstream/10034/622051/2/article.pdf	English	null	Nanodiamond based surface modified screen-printed electrodes for the simultaneous voltammetric determination of dopamine and uric acid	Mikrochimica acta	2,019	cc-by	5,588	Abstract The electroanalytical detection of the neurotransmitter dopamine (DA) in the presence of uric acid (UA) is explored for the first time using commercially procured nanodiamonds (NDs). These are electrically wired via surface modification upon screen- printed graphite macroelectrodes (SPEs). The surface coverage of the NDs on the SPEs was explored in order to optimize electroanalytical outputs to result in well-resolved signals and in low limits of detection. The (electro)analytical outputs are observed to be more sensitive than those achieved at bare (unmodified) SPEs. Such responses, previously reported in the academic literature have been reported to be electrocatalytic and have been previously attributed to the presence of surface sp2 carbon and oxygenated species on the surface of the NDs. However, XPS analysis reveals the commercial NDs to be solely composed of nonconductive sp3 carbon. The low/negligible electroconductivity of the NDs was further confirmed when ND paste electrodes were fabricated and found to exhibit no electrochemical activity. The electroanalytical enhancement, when using NDs electronically wired upon SPEs, is attributed not to the NDs themselves being electrocatalytic, as reported previously, but rather changes in mass transport where the inert NDs block the underlying electroactive SPEs and create a random array of graphite microelectrodes. The electrode was applied to simultaneous sensing of DA and UA at pH 5.5. Figures of merit include (a) low working potentials of around 0.27 and 0.35 V (vs. Ag/AgCl); and (b) detection limits of 5.7 × 10−7 and 8.9 × 10−7 M for DA and UA, respectively. Keywords Detection . Dopamine . Uric acid . Nanodiamonds . Screen-printed electrodes . Micro-electrode array . Electrochemistry . Electrocatalysis Keywords Detection . Dopamine . Uric acid . Nanodiamonds . Screen-printed electrodes . Micro-electrode array . Electrochemistry . Electrocatalysis Nanodiamond based surface modified screen-printed electrodes for the simultaneous voltammetric determination of dopamine and uric acid Received: 26 November 2018 /Accepted: 11 February 2019 # The Author(s) 2019 Received: 26 November 2018 /Accepted: 11 February 2019 # The Author(s) 2019 Nanodiamond based surface modified screen- printed electrodes for the simultaneous voltammetric determination of dopamine and uric acid. Item Type article Authors Baccarin, Marina; Rowley-Neale, Samuel J.; Cavalheiro, Éder T. G.; Smith, Graham C.; Banks, Craig E. Citation Mikrochimica acta, volume 186, issue 3, page 200 Rights Licence for this article: cc by Download date 24/10/2024 05:53:09 Link to Item http://hdl.handle.net/10034/622051 Nanodiamond based surface modified screen- printed electrodes for the simultaneous voltammetric determination of dopamine and uric acid. Microchimica Acta (2019) 186:200 https://doi.org/10.1007/s00604-019-3315-y ORIGINAL PAPER GINAL PAPER Introduction Dopamine (DA) is responsible for a number of functions in the human body correlated with movement, memory, emo- tional control, sleep and attention [16, 24]. It has been shown that abnormal levels of DA is a key indicator associated with certain neurological diseases, such as Parkinson’s, depression, and Alzheimer’s. As a result of this, research has focused upon finding ever increasingly accurate and rapid DA detection methods [20]. There are numerous studies within the literature that report the electroanalytical detection for the quantification of DA within biological and in-vivo samples [1, 9, 10, 13]. Electrochemical detection techniques are favoured by re- searchers due to their low cost, portability and analytical sen- sitivity towards a range of target analytes. There is however, an inherent problem faced using electroanalytical techniques towards the sensing of DA, as real samples usually coexist Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00604-019-3315-y) contains supplementary material, which is available to authorized users. 4 Faculty of Science and Engineering, Department of Natural Sciences, University of Chester, Thornton Science Park, Pool Lane, Ince, Chester CH2 4NU, UK * Craig E. Banks c.banks@mmu.ac.uk; http://www.craigbanksresearch.com China.) which were shown to enhance the voltammetric outputs of ND modified gold electrodes towards the redox couples Ru(NH3)6 3+/2+ and Fe(CN)6 4−/3–. These reported electrocatalytic/electronic properties suggest that NDs are with uric acid (UA), which exhibits an electrochemical signal that overlaps with DA. Consequently, DAs analytical determi- nation can become convoluted; researchers are constantly ex- ploring different techniques and materials that are able to sep- arate the analytical signals. Various approaches have been utilised in order to overcome this issue. For example Song et al. [27] reported that Fe3O4 nanoparticle/graphene oxide modified glassy carbon electrodes (GCE) were able to differ- entiate the peak signal outputs for DA and UA within urine, blood and brain tissue samples using differential pulse volt- ammetry (DPV), displaying low limits of detection (LODs) in pH 7 phosphate buffer for DA and UA of 5.3 × 10−8 and 4.1 × 10−7 M, respectively. Han and co-workers [6] utilised a mod- ified GCE with a dispersion of chitosan-graphene for the si- multaneous detection of ascorbic acid (AA), DA, and UA, which under optimal conditions, via DPV; the LODs were reported to correspond to 5.0 × 10−5 M, 1.0 × 10−6 M, and 2.0 × 10−6 M for AA, DA and UA, respectively (in pH 7 phosphate buffer). Another approach has been to utilise nanodiamond (NDs). NDs were initially synthesized in the 1960s [11], and have since been used for a plethora of applications, such as the manufacture of micro abrasives, the production of cosmetics [31], delivery of drugs [32], and the development of electro- chemical sensors; the latter is overviewed within Table 1. For example, Simioni et al. [26] modified a GCE with an aqueous ND dispersion (note the NDs utilized in Simioni et al. [26] study were commercially procured from Sigma-Aldrich and are the same type of NDs utilized within this study) and reported the modified electrode to exhibit improved heteroge- neous electron transfer kinetics (k°) and a lower LOD (2.2 × Table 1 Comparison of different electrodes modified with NDs used for the detection of a range of target analytes Modifier material Bare electrode Analyte LOD (M) Reference PAA/N-NCD/GOx gold electrode Glucose 5.0 × 10−6 Zhao et al. [34] Chit/UND/Hb GCE H2O2 4.0 × 10−7 Zhu et al. [35] ND-NS(HRP) GCE H2O2 5.9 × 10−5 Gopalan et al. [5] QAS-ND/Mb GCE H2O2 3.5 × 10−6 Xiao-Ling et al. * Craig E. Banks c.banks@mmu.ac.uk; http://www.craigbanksresearch.com * Craig E. Banks c.banks@mmu.ac.uk; http://www.craigbanksresearch.com 1 Faculty of Science and Engineering, Manchester Metropolitan University, Chester Street, Manchester M1 5GD, UK 1 Faculty of Science and Engineering, Manchester Metropolitan University, Chester Street, Manchester M1 5GD, UK 2 Instituto de Química de São Carlos, Universidade de São Paulo, São Carlos, SP 13566-590, Brazil 3 Manchester Fuel Cell Innovation Centre, Manchester Metropolitan University, Chester Street, Manchester M1 5GD, UK 3 Manchester Fuel Cell Innovation Centre, Manchester Metropolitan University, Chester Street, Manchester M1 5GD, UK 4 Faculty of Science and Engineering, Department of Natural Sciences, University of Chester, Thornton Science Park, Pool Lane, Ince, Chester CH2 4NU, UK 4 Faculty of Science and Engineering, Department of Natural Sciences, University of Chester, Thornton Science Park, Pool Lane, Ince, Chester CH2 4NU, UK Page 2 of 9 Microchim Acta (2019) 186:200 10−7 M) towards the sensing of pyrazinamide than that achiev- able at an unmodified GCE; Simioni et al. [26] attributed this increase in analytical sensitivity due to the NDs increasing the electrode area. The studies described within Table 1 are thor- ough in their approach at studying the properties of ND but they lacking with regard to the transferability of their results to Binfield^ biomedical scenarios as they utilize traditional labo- ratory based electrodes, e.g. glassy carbon (GC), gold and boron-doped diamond (BDD) as underlying electrode sup- ports. All of these require several time consuming cleaning and polishing stages in between separate samples tests. The utilization of screen-printed electrodes (SPEs) as the underly- ing supporting electrode decreases the time necessary to per- form separate tests, since SPEs are comparatively cheap, tailorable and disposable. Therefore, SPEs allow for rapid and repeated on-site testing [14]. Previous studies utilizing SPEs as supporting platforms for electrochemical sensors have been highlighted in ESI Table 1. Many of the studies that present cases of ND catalysis are intriguing but yet coun- terintuitive [22, 28], as bulk diamond is an inherent insulating material with a relatively large band gap of 5.47 eV [29]. It has however, been theorized that NDs exhibit useful electrochem- ical properties due to the presence of unsaturated bonding, sp2-like carbon and the formation of oxygenated species within/upon their outer structure [7] and a large surface to mass ratio. Holt et al. [7] used commercially sourced NDs (procured from Shenzhen Jingangyuan New Material Co., P. R. Table 1 Comparison of different electrodes modified with NDs used for the detection of a range of target analytes * Craig E. Banks c.banks@mmu.ac.uk; http://www.craigbanksresearch.com [33] LOx/DNPs gold electrode Lactate 1.5 × 10−5 Briones et al. [2] Ni-NDs BDD Glucose 5.0 × 10−8 Dai et al. [4] ND-DHP GCE Codeine 5.5 × 10−8 Simoni et al. [25] ND GCE Pyrazinamide 2.2 × 10−7 Simioni et al. [26] ND SPE DA, UA 5.7 × 10−7, 8.9 × 10−7 This work Key: PAA/N-NCD/GOx poly(allylamine hydrochloride/ non-doped nanocrystalline diamond/ glucose oxidase, Chit/UND/Hb chitosan/ undoped nanocrystalline diamond/ hemoglobin, GCE glassy carbon electrode, ND- NS(HRP) nanodiamond-based sponges with entrapped horseradish peroxidase, QAS-ND/Mb quaternary ammo- nium salt-nanodiamond/myoglobin, LOx/DNPs Lactate oxidase /undoped diamond nanoparticles, Ni-ND nicklel- nanodiamond, BDD boron-doped diamond, ND-DHP nanodiamond-dihexadecyl phosphate, ND nanodiamond, DA dopamine, UA uric acid Page 3 of 9 200 Microchim Acta (2019) 186:200 and were vigorously degassed prior to electrochemical mea- surements with high purity, oxygen free nitrogen. potentially exciting for the basis of electrochemical sensing platforms. Consequently, we re-evaluate the electroanalytical performance of ND/SPEs for the simultaneous detection of DA and UA where enhanced voltammetric signals are ob- served using ND modified SPEs over bare SPEs; in contrary to the academic literature we provide a different insight into the reported and observed (in our work) the beneficial electro- analytical outputs. Electrochemical measurements were performed using an Ivium Compactstat™(Netherlands) potentiostat. All measure- ments were conducted, using a conventional three-electrode sys- tem utilizing a pseudo Ag/AgCl as a reference and a carbon ink formulation as a counter and working electrodes. The SPEs have a 3.1 mm diameter working electrode and were fabricated in- house with the appropriate stencils using a DEK 248 screen- printing machine (DEK, Weymouth, U.K.) [3]. A full descrip- tion of the SPE production methodology is found within the supporting information. The bare/unmodified SPEs fabricated display a heterogeneous electron transfer rate constant, k0, as measured using the Nicholson’s [17], and Lavagnini et al. [12] methods with [Ru(NH3)6]3+/2+ which is found to correspond to 4.2 × 10−3 cm∙s−1. The SPEs were modified with NDs via drop- casting. This procedure entials, 1 mg of NDs (optimized value) were solubilized in 1.0 mL deionized water and then, from this Fig. 1 Typical cyclic voltammograms using bare screen printed electrodes (SPEs) (a) and a ca. 140 ng∙cm−2 ND/ SPE (c) over a range of DA con- centrations from 50 to 400 μM in pH 7.4 phosphate buffer . Scan rate: 50 mV∙s−1. The analysis of voltammetric peak current as a function of concentration is shown in the respective (b) and (d) Electroanalytical performance of the ND/SPEs Initially the electrochemical oxidation of DA (Fig. 1) and UA (Fig. 2) was explored using bare/unmodified SPEs and ND sur- face modified SPEs (ND/SPEs) over a range of DA and UA concentrations. Note the use of SPEs as substrates for sensing devices has numerous advantages over more traditional carbon based electrodes as they are cheap, reproducible and disposable, making them perfect for in-field use where a single shot sensor which has no memory effect, is desirable. Clear and distinct voltammetric profiles are evident at both the bare SPEs and the ND/SPEs. Figure 1c and 2c display that for DA and UA the analytical signals appear at +0.18 V and + 0.31 V, respectively, giving an approximate peak separation of 130 mV, clearly dem- onstrating that the simultaneously detection of DA and UA is possible using the ND/SPEs. A closer inspection of the Experimental All chemicals (analytical grade or higher) were used as re- ceived from Sigma-Aldrich without any further purification, which includes the nanodiamonds (NDs) [23]. The NDs are electrochemically wired via surface modification (drop- casting) upon SPEs, (see below). All solutions were prepared with deionised water of resistivity not less than 18.2 MΩ cm Fig. 1 Typical cyclic voltammograms using bare screen printed electrodes (SPEs) (a) and a ca. 140 ng∙cm−2 ND/ SPE (c) over a range of DA con- centrations from 50 to 400 μM in pH 7.4 phosphate buffer . Scan rate: 50 mV∙s−1. The analysis of voltammetric peak current as a function of concentration is shown in the respective (b) and (d) Page 4 of 9 Microchim Acta (2019) 186:200 characterisation and equipment utilised can be found within the ESI (see Figure S1, S2 and S3). From the performed phys- icochemical analysis, we can be confident that the NDs utilized throughout this study are of both a high purity and quality. dispersion, 8 μL were deposited onto the SPEs surface. After 30 min, the solvent is completely evaporated (at ambient tem- perature) and the ND/SPEs are ready for use. Note that in addi- tion to the SPEs GC (3 mm diameter, BAS, USA), edge plane pyrolytic graphite (EPPG Le Carbone, Ltd. Sussex, UK; 4.9 mm diameter) and carbon paste electrodes (the technique by which they were fabricated is reported within the SI) were utilized. Fig. 2 Typical cyclic voltammograms using SPE (a) and a ca. 140 ng∙cm−2 ND/SPE (b) over a range of UA concen- trations from 60 to 500 μM in pH 7.4 phosphate buffer . Scan rate: 50 mV∙s−1. The analysis of voltammetic peak current as a function of concentration is shown in the respective (b) and (d) Physicochemical characterisation of the nanodiamonds (NDs) The physicochemical characterisation of the NDs was undertak- en in order to independently determine their quality and purity. This included Raman spectroscopy, scanning electron micro- scope (SEM), transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS) and X-ray diffraction (XRD). A full description of the performed physicochemical Fig. 2 Typical cyclic voltammograms using SPE (a) and a ca. 140 ng∙cm−2 ND/SPE (b) over a range of UA concen- trations from 60 to 500 μM in pH 7.4 phosphate buffer . Scan rate: 50 mV∙s−1. The analysis of voltammetic peak current as a function of concentration is shown in the respective (b) and (d) Fig. 2 Typical cyclic voltammograms using SPE (a) and a ca. 140 ng∙cm−2 ND/SPE (b) over a range of UA concen- trations from 60 to 500 μM in pH 7.4 phosphate buffer . Scan rate: 50 mV∙s−1. The analysis of voltammetic peak current as a function of concentration is shown in the respective (b) and (d) Page 5 of 9 200 Microchim Acta (2019) 186:200 NDs upon the supporting SPEs were explored and monitored as a function of analytical output verses ND coverage (ng∙cm−2), where increasing mass coverages of NDs result in larger analyt- ical peaks/signals until a critical mass coverage is achieved after which, further coverages reduce the achievable analytical signal. It was found that an optimal response is achievable at a coverage of NDs of ca. 140 ng∙cm−2 (see Table S2). voltammetric peak currents/signals of DA and UA are observed to be relatively increased using the ND/SPEs over that of the bare SPEs (see Figs. 1 and 2); this observation resonates with the academic literature (see Table 1). The detection parameters for DA and UA can be optimized by evaluating the pKa values and adjusting the pH of the electrolyte solution in accordance with the Henderson-Hasselbach equation [15]. Given the pKa of UA is 5.50 it can be expected to be a charge neutral species within the pH ca. 7 solutions, however at pH values of ca. 5, UA is expected to exist in its acidic form. DA which has pKa value of 8.9, will only exist in its acidic form when UA does not, as the combination of both molecules will affect the acid/base equilibrium. The electroanalytical response using the ND/SPEs is shown within Figure S4. Fig. 3 Differential pulse voltammograms (DPV) using ca. 140 ng∙cm−2 for: a DA concen- trations from 2 to 100 μM with 20 μM UA fixed; c UA concen- trations from 2 to 97 μM with 20 μM DA fixed and the respec- tive analytical curve for different additions of DA in (b) and for UA in (d). Each point is the average of three measurements and standard deviation. All measurements were performed in pH = 5.5 acetate buffer . Parameters of DPV: E- pulse = 20 mV; t-pulse = 200 ms; equivalent scan rate: 10 mV∙s−1 Physicochemical characterisation of the nanodiamonds (NDs) It is clear that the greatest separation between the DA and UA oxidation signals/ peaks was acquired utilizing a pH 5.5 solution. Figures S5 dem- onstrates that a pH 5.5 acetate buffer gives rise to larger analytical signals/peaks over that of a pH 5.5 phosphate buffer solution (see Figure S6). Using the optimized conditions, the performance of the ND/SPEs towards the analytical sensing of DA and UAwas evaluated. Note that a range of different surface modifications of The electroanalytical performance of the ND/SPE is shown in Fig. 3 where increasing concentrations of DA in the pres- ence of a fixed concentration of UA are explored and then the reverse is done, where the concentration of UA is fixed in a fixed concentration of DA. Clear separation of the DA and UA analytical signals/peaks. The analysis of the voltammetric peak currents as a function of DA and UA concentrations is shown in Fig. 3b and d with the following lines of best fit for the detection of DA and UA. In the case of DA the line equa- tion was I/A = 4.1 × 10−8 A + 0.02 AM−1; N = 5, R2 = 0.99 and I/A = 2.2 × 10−7 A + 0.01 AM−1; N = 5; R2 = 0.96. The initial linear range was used to calculate the LOD (3σ) of DA, which is found to be 0.57 μM. In the case of UA the line equations corresponded to I/A = 8.2 × 10−8 A + 0.030 AM−1; N = 5; Fig. 3 Differential pulse voltammograms (DPV) using ca. 140 ng∙cm−2 for: a DA concen- trations from 2 to 100 μM with 20 μM UA fixed; c UA concen- trations from 2 to 97 μM with 20 μM DA fixed and the respec- tive analytical curve for different additions of DA in (b) and for UA in (d). Each point is the average of three measurements and standard deviation. All measurements were performed in pH = 5.5 acetate buffer . Parameters of DPV: E- pulse = 20 mV; t-pulse = 200 ms; equivalent scan rate: 10 mV∙s−1 Fig. 3 Differential pulse voltammograms (DPV) using ca. Physicochemical characterisation of the nanodiamonds (NDs) 140 ng∙cm−2 for: a DA concen- trations from 2 to 100 μM with 20 μM UA fixed; c UA concen- trations from 2 to 97 μM with 20 μM DA fixed and the respec- tive analytical curve for different additions of DA in (b) and for UA in (d). Each point is the average of three measurements and standard deviation. All measurements were performed in pH = 5.5 acetate buffer . Parameters of DPV: E- pulse = 20 mV; t-pulse = 200 ms; equivalent scan rate: 10 mV∙s−1 Page 6 of 9 Microchim Acta (2019) 186:200 Fig. 4 Typical cyclic voltammograms recorded in 1 mM [Ru(NH3)6]3+/2+ / 0.1 M KCl for a bare unmodified (black line) GCE (a) and EPPG (b) modified with ca. 140 ng∙cm−2 NDs (red line). Scan rate: 100 mV∙s−1 R2 = 0.99 and I/A = 4. 9 × 10−7 A + 0.011 AM−1; N = 5; R2 = 0.96 m, respectively. The initial linear range was utilized in order to obtain the LOD (3σ) for UA of 0.89 μM. Note that the percentage standard deviation bars for the DA and UA signal can be observed in Fig. 3b and d, respectively. In both cases there was an increase in σ as concentration increased, the low percentage variation attests to the reproducibility of SPEs. electrochemically heterogeneous with two distinct zones that differ in their rate constants from fast to slow. This results in split peak voltammetry where two peaks would be observed rather than the expected redox process. Interestingly, SPEs do not display this secondary peak when modified with the ana- lytically optimal coverage of ca. 140 ng cm−2 NDs (see Table S2). This is likely due to the differing surface roughness [19], where SPEs have a relatively rougher surface than that of GCE or EPPG (see Figure S7 and SI), or the scan rate utilized Understanding the enhanced electroanalytical performances using ND/SPEs Fig. 5 Cyclic voltammograms for carbon paste electrodes (PE) with in- creasing amount of ND in 1 mM [Ru(NH3)6]3+/2+ / 0.1 M KCl; Scan rate: 50 mV∙s−1. Electrode compositions: (carbon black (60%): nujol (40%)) (small dotted line), 60: 40% (NDs: nujol) (solid line), and (carbon black (55%): NDs (5%): nujol (40%)) (large dotted line) for 1 mM [Ru(NH3)6]3+/2+ / 0.1 M KCl The ability of a ND modified SPE to simultaneously detect UA and DA suggests that the NDs are Belectrocatalytic^ to- wards DA and UA. Indeed the elegant work of Holt and co- workers [7, 8, 18, 21]. suggests that electron transfer can occur at the surface of undoped NDs due to the presence of a com- plex arrangement of sp2 and sp3 carbon, which facilitates elec- tron transfer between the NDs and the electrolyte [18]. In order to explore this further, the ND/SPEs were benchmarked using the near ideal outer-sphere probe, [Ru(NH3)6]3+/2+ and for comparative purposes, GCE and EPPG were also modified with NDs of varying coverages. Figure 4 shows the responses of the ND modified GCE and EPPGs, which clearly demon- strates some intriguing voltammetry, where in addition to the expected redox probe responses; two additional waves are seen prior to the main voltammetric redox response. The ini- tial reduction peak is observed at −217 mVand – 255 mV (vs. pseudo Ag/AgCl) for the ND/GCE and ND/EPPG, respective- ly. In both cases secondary voltammetric peaks are observed at ca. – 570 mV (vs. pseudo Ag/AgCl). Note that our exper- imental observations agree with the theory presented by Ward et al. [30] who demonstrate via numerical simulations that modifying an electrode surface with inert/less electrochemi- cally active materials the electrode surface becomes Fig. 5 Cyclic voltammograms for carbon paste electrodes (PE) with in- creasing amount of ND in 1 mM [Ru(NH3)6]3+/2+ / 0.1 M KCl; Scan rate: 50 mV∙s−1. Electrode compositions: (carbon black (60%): nujol (40%)) (small dotted line), 60: 40% (NDs: nujol) (solid line), and (carbon black (55%): NDs (5%): nujol (40%)) (large dotted line) for 1 mM [Ru(NH3)6]3+/2+ / 0.1 M KCl Page 7 of 9 200 Microchim Acta (2019) 186:200 was not sufficiently fast to display split peak volammetry in the case of SPEs. To further consider the Belectrocatalytic^ activity of the NDs (see above), carbon paste electrodes were fabricated using varying amounts of NDs. Understanding the enhanced electroanalytical performances using ND/SPEs Figure 5 shows the CVs pertaining to three carbon paste variants, those being; 60% carbon black and 40 nujol, 60% ND and 40% nujol, and 55% carbon black, 5% ND and 40% nujol. The differing voltammetric responses demonstrate that upon incorporation of an increasing amount of ND into the carbon paste electrode results in a decrease in electrochemical activity/ electron trans- fer and ultimately results in complete loss of electron transfer; such observations suggest that the NDs are non-conductive. As shown in Fig. 6 we also explore the effect of varying the coverages of NDs, via surface modification upon SPEs, where initially an improvement in electrochemical activity is ob- served, after which, the response decreases to a point where no electrochemical activity is observed. In all cases, a plot of peak height against the square root of scan rate yielded a linear relationship, thus implying the mass transport occurring at the bare/unmodified SPEs and ND modified SPEs is due to dif- fusional processes and not a thin layer effect. While the reports of Holt et al and others maybe plausible, we suggest, this is not credible, given the observed voltammetric profiles (i.e. to- wards the DA, UA, Ru(NH3)6]3+/2+ and incorporated within a carbon paste electrode) and that physicochemical character- isation, verifies that the NDs are solely composed of insulating Fig. 6 Cyclic voltammograms for a bare/unmodified SPE and then fol- lowing surface modification with ca. 140 ng∙cm−2 and ca. 1.4 μg∙cm−2 of NDs, in 1 mM [Ru(NH3)6]3+/2+ / 0.1 M KCl. Scan rate: 50 mV∙s−1. One can clearly see how the voltammetric response changes as the coverage of NDs is increased up to a point where the electrode surface is full cove blocked and electron transfer is not possible resulting in no voltamme signals. Note in reality the blocking NDs are randomly distributed u the electrode surface Fig. 6 Cyclic voltammograms for a bare/unmodified SPE and then fol- lowing surface modification with ca. 140 ng∙cm−2 and ca. 1.4 μg∙cm−2 of NDs, in 1 mM [Ru(NH3)6]3+/2+ / 0.1 M KCl. Scan rate: 50 mV∙s−1. One can clearly see how the voltammetric response changes as the coverage of NDs is increased up to a point where the electrode surface is full covered/ blocked and electron transfer is not possible resulting in no voltammetric signals. Conclusions 7. Holt KB, Ziegler C, Caruana DJ, Zang J, Millán-Barrios EJ, Hu J, Foord JS (2008) Redox properties of undoped 5 nm diamond nano- particles. Phys Chem Chem Phys 10:303–310 We have explored the use of ND modified SPE as the basis of an electrochemical sensing platform towards the sensing of DA and UA. We observe an electroanalytical enhancement using NDs over that of bare/unmodified SPEs, indicating a potential Belectrocatalysis^. However, further electrochemical and physicochemical characterization verifies that NDs are inert/non-conductive. Thus in summary, the observed benefi- cial electroanalytical response within the academic literature (see Table 1) is not due to the NDs being Belectrocatalytic^ but rather a change in mass transfer where the inert NDs facilitate the production of a random microelectrode array; the implica- tions for previous observations (i.e. Table 1) are justified. 8. Inel GA, Ungureau E-M, Varley TS, Hirani M, Holt KB (2016) Solvent–surface interactions between nanodiamond and ethanol studied with in situ infrared spectroscopy. Diam Relat Mater 61: 7–13 9. Jia D, Dai J, Yuan H, Lei L, Xiao D (2011) Selective detection of dopamine in the presence of uric acid using a gold nanoparticles- poly (luminol) hybrid film and multi-walled carbon nanotubes with incorporated β-cyclodextrin modified glassy carbon electrode. Talanta 85:2344–2351 10. Khan AF, Brownson DC, Randviir EP et al (2016) 2D hexagonal boron nitride (2D-hBN) explored for the electrochemical sensing of dopamine. Anal Chem 88:9729–9737 11. Krueger A (2008) Diamond nanoparticles: jewels for chemistry and physics. Adv Mater 20:2445–2449 Acknowledgements British Council Institutional Grant Link (No. 172726574) is acknowledged. The Manchester Fuel Cell Innovation Centre is funded by the European Regional Development Fund. MB would like to thank the Santander Bank and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001 for research funding. 12. 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Given the above, rather than simply attribute the beneficial electroanalytical responses of the ND/SPEs to false Belectrocatalysis^, the voltammetric responses coupled with the thorough physicochemical analy- sis indicate that instead, there is a change in the mass transport. Where the incorporation of insulating/non-conductive NDs, either in the bulk of, or on the surface, of an electrode, produce a randomly distributed graphite microelectrode array, since the inert/non-conductive NDs block the underlying electroactive electrode surface. 3. Choudry NA, Kampouris DK, Kadara RO, Banks CE (2010) Disposable highly ordered pyrolytic graphite-like electrodes: tailor- ing the electrochemical reactivity of screen printed electrodes. Electrochem Commun 12:6–9 4. Dai W, Li M, Gao S, Li H, Li C, Xu S, Wu X, Yang B (2016) Fabrication of nickel/nanodiamond/boron-doped diamond elec- trode for non-enzymatic glucose biosensor. Electrochim Acta 187: 413–421 5. 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https://openalex.org/W4387402397	https://www.nature.com/articles/s41467-023-41854-x.pdf	English	null	Drought and heat reduce forest carbon uptake	Nature communications	2,023	cc-by	4,504	Check for updates land-atmosphere feedbacks thatcan exacerbate heatwaves5, and forest management strategies in a changing climate6. Climate extremes threaten the land carbon sink and it is important to understand their impact in a changing climate. A recent study provides new insights on reduced forest carbon uptake during the severe 2022 drought and heatwave across Europe. Van der Woude et al.7 have taken advantage of recently available, near real-time data to show that the severe (i.e. intense and prolonged) drought and heatwave in 2022 reduced forest carbon uptake at local, regional and continental scales across Europe. These results are important because the drought and heatwave in 2022 was a recurrent event following those of 2003, 2010, 2015, 2018, 2019 and 2020. Recurrent events enable key insights for lagged ecosystem responses (or ‘legacies’)8 and for shifting risks in a warming climate that impact the forest carbon sink such as tree mortality, vulnerability to insect outbreaks and ﬁres, or shifts in species composition and forest structure6. During summer 2022, large areas of Europe experienced drought and heat that were among the strongest during the past 20 years. Large-scale droughts and heatwaves evolve from stationary, high pressure blocking patterns in atmospheric circulation, which inhibit cloud formation and precipitation, and increase available energy at the land surface9. Enhancing (i.e. positive) land-atmosphere feedbacks then further exacerbate extremely dry and hot conditions as water transpired by plants and evaporated from soils is reduced (see Fig. 1, blue arrows)5. Evaporative cooling thus becomes less efﬁcient Climate extremes such as drought and heatwaves are detrimental to society (e.g. food production, human health and energy resources) and to the functioning of terrestrial ecosystems. While droughts have been occurring for centuries, Europe has recently experienced an elevated incidence of these extremes1, and projections show an increased prevalence with climate warming2. This is concerning because drought and heat threaten ecosystem carbon uptake, which currently mitigates increases in atmospheric CO2 concentrations by offsetting one-third of anthropogenic fossil fuel emissions3. Although the link between drought and reduced carbon uptake is well estab- lished, important questions remain regarding the impact of recurrent droughts, the strength of seasonal and regional compensation effects4, Fig. 1 \| Impact of increasing drought stress on forest ﬂuxes. Drought stress evolves (left to right) from a combination of precipitation deﬁcits and heat. Check for updates This leads to declines in soil moisture and increases in atmospheric evaporative water demand, which is a combination of air temperature and relative humidity (denoted by thermometer and water drop, respectively). Compared to pre- drought conditions (a), drought stress evolves during singular drought (b, i.e. meteorological and agricultural drought) and further increases during recurrent or prolonged drought (c, i.e. hydrological drought). Green arrows show carbon uptake (by photosynthesis), beige arrows carbon release (by soil and plant respiration), brown arrows the net carbon storage in forest or release to the atmosphere, blue arrows the water vapor ﬂux (evapotranspiration), orange arrows the heat ﬂux (sensible heat), and yellow arrows the drought stress. As drought stress increases, trees become physiologically stressed and reduce photosynthesis (denoted by change in tree color). After prolonged drought stress or recurrent drought, partial or full crown mortality occurs. Respiration is initially reduced by drought stress but eventually increases from decomposing leaves and wood. As drought stress increases, there is a shift from water vapor ﬂux to more heat being released, enhancing the drought and heat conditions. (Source clip arts: Pixabay, modiﬁed) Fig. 1 \| Impact of increasing drought stress on forest ﬂuxes. Drought stress evolves (left to right) from a combination of precipitation deﬁcits and heat. This leads to declines in soil moisture and increases in atmospheric evaporative water demand, which is a combination of air temperature and relative humidity (denoted by thermometer and water drop, respectively). Compared to pre- drought conditions (a), drought stress evolves during singular drought (b, i.e. meteorological and agricultural drought) and further increases during recurrent or prolonged drought (c, i.e. hydrological drought). Green arrows show carbon uptake (by photosynthesis), beige arrows carbon release (by soil and plant respiration), brown arrows the net carbon storage in forest or release to the atmosphere, blue arrows the water vapor ﬂux (evapotranspiration), orange arrows the heat ﬂux (sensible heat), and yellow arrows the drought stress. As drought stress increases, trees become physiologically stressed and reduce photosynthesis (denoted by change in tree color). After prolonged drought stress or recurrent drought, partial or full crown mortality occurs. Respiration is initially reduced by drought stress but eventually increases from decomposing leaves and wood. As drought stress increases, there is a shift from water vapor ﬂux to more heat being released, enhancing the drought and heat conditions. (Source clip arts: Pixabay, modiﬁed) Comment Comment https://doi.org/10.1038/s41467-023-41854-x Drought and heat reduce forest carbon uptake Sebastian Wolf & Eugénie Paul-Limoges Check for updates Comment Increased mortality has been reported for ajor European tree species11, and the spatial distribution of these es regarding climatically suitable areas will be substantially ed by the end of this century12. For example, European beech is idered the most vulnerable broadleaf tree species to drought and 3, and substantial climate-induced growth declines are ected14. This is important because beech forests cover vast areas of ral and eastern Europe. To quantify carbon uptake, van der Woude and colleagues7 bined ‘bottom-up’ ecosystem ﬂux tower measurements in sts with ‘top-down’ approaches using satellite remote sensing biosphere model. Summer reductions of about 59 TgC were observed across the drought-affected area and resulted in 40 TgC reduced annual carbon uptake, which is equivalent to nearly one- quarter (23%) of the 2022 annual CO2 emissions of Germany – the European country with the largest emissions. Unlike in 2018 rela- ted to spring, only partial seasonal compensation was found from increased fall uptake due to a prolonged growing season. Similar carbon release from summer forest ﬁres (about 9 TgC) make the 2022 event comparable with 2003, when much higher (up to 500 TgC) annual net carbon release was reported10. However, van der Woude et al.7 emphasize that other regions were affected in 2022 compared to 2018 and 2003, which likely mediated the impacts on reduced carbon uptake because of differences in forest composition. While previous studies on the carbon cycle impact of climate extremes were delayed by data availability constraints4,10,15, van der Woude et al.7 demonstrate the importance of standardized ecosystem monitoring networks such as ICOS (European Inte- grated Carbon Observation System) and NEON (U.S. National Ecological Observatory Network) for providing timely informa- tion to stakeholders. The severe impacts of drought on the forest carbon sink need to be considered by governments for reaching net-zero goals. Besides reducing deforestation, tropical countries like Brazil and Indonesia are planning substantial changes in land use (e.g. reforestation) to compensate for greenhouse gas and more of the available energy heats the air (see Fig. 1, orange arrows). biosphere model. Summer reductions of about 59 TgC were observed across the drought-affected area and resulted in 40 TgC reduced annual carbon uptake, which is equivalent to nearly one- quarter (23%) of the 2022 annual CO2 emissions of Germany – the European country with the largest emissions. Comment In Europe, forest area has already been increasing for To quantify carbon uptake, van der Woude and colleagues7 combined ‘bottom-up’ ecosystem ﬂux tower measurements in forests with ‘top-down’ approaches using satellite remote sensing observations and atmospheric inversions coupled with a Introducon Vulnerability to Drought and Heat? Yes No Adaptaon Retain Previous Sink Migaon Increase Sink Forest Harvest Reduce Carbon Losses aer Logging Return to Carbon Neutrality Long-term Carbon Storage in Wood Emission Reducons in other Sectors Increasing Carbon Storage towards Net Zero Timber Monitor Reassess Forestry Wood Chain Fig. 2 \| Forest management to increase carbon storage towards net zero, despite drought and heat. Management options for the forestry wood chain (Forest →Harvest →Timber) to increase carbon storage (left to right). Wide arrows show carbon uptake or release, i.e. forest uptake through photosynthesis (green arrows), release from respiration or industry to the atmosphere (beige arrows), and changes in net forest storage (brown arrows). The stripped brown arrow denotes long-term carbon storage in wood materials. Forest management strategies based on the vulnerability to drought and heat could retain the current carbon sink by adaptation efforts (if vulnerable) to avoid forest carbon release, or increase the sink by mitigation efforts (if not vulnerable) to compensate for emissions from other sectors. After wood harvest, carbon losses from soil and wood residues can be reduced to support a return to carbon neutrality. Storing carbon in wood materials increases stable long-term storage by reducing emissions from constructions materials such as cement and steel. (Source clip arts: Pixabay, modiﬁed) nat re comm nications Increasing Carbon Storage towards Net Zero Vulnerability to Drought and Heat? Yes No Monitor Reassess Retain Previous Sink Forestry Wood Chain Increase Sink Long-term Carbon Storage in Wood Emission Reducons in other Sectors adaptation efforts (if vulnerable) to avoid forest carbon release, or increase the sink by mitigation efforts (if not vulnerable) to compensate for emissions from other sectors. After wood harvest, carbon losses from soil and wood residues can be reduced to support a return to carbon neutrality. Storing carbon in wood materials increases stable long-term storage by reducing emissions from constructions materials such as cement and steel. (Source clip arts: Pixabay, modiﬁed) Fig. 2 \| Forest management to increase carbon storage towards net zero, despite drought and heat. Management options for the forestry wood chain (Forest →Harvest →Timber) to increase carbon storage (left to right). Comment Unlike in 2018 rela- ted to spring, only partial seasonal compensation was found from increased fall uptake due to a prolonged growing season. Similar carbon release from summer forest ﬁres (about 9 TgC) make the 2022 event comparable with 2003, when much higher (up to 500 TgC) annual net carbon release was reported10. However, van der Woude et al.7 emphasize that other regions were affected in 2022 compared to 2018 and 2003, which likely mediated the impacts on reduced carbon uptake because of differences in forest composition. How does drought and heat affect forest carbon uptake? The net carbon balance of an ecosystem represents the difference between carbon uptake by photosynthesis and carbon release by respiration. Reduced forest carbon uptake during drought and heat comes from stress-related declines in photosynthesis (see Fig. 1, green arrows). Respiration from plants and soil is also reduced due to limitations in soil moisture, but this is typically to a lower extent than photosynthesis (see Fig. 1, beige arrows)10. These relative differences result in reduced net carbon uptake (see Fig. 1, brown arrows) or even net release, as e.g. reported by van der Woude et al.7 for some sites in France during summer 2022. The combined stress of intense drought and heat over prolonged periods (or recurrent events) leads to increased crown and eventually tree mortality (see Fig. 1c). Increased mortality has been reported for all major European tree species11, and the spatial distribution of these species regarding climatically suitable areas will be substantially altered by the end of this century12. For example, European beech is considered the most vulnerable broadleaf tree species to drought and heat13, and substantial climate-induced growth declines are projected14. This is important because beech forests cover vast areas of central and eastern Europe. While previous studies on the carbon cycle impact of climate extremes were delayed by data availability constraints4,10,15, van der Woude et al.7 demonstrate the importance of standardized ecosystem monitoring networks such as ICOS (European Inte- grated Carbon Observation System) and NEON (U.S. National Ecological Observatory Network) for providing timely informa- tion to stakeholders. The severe impacts of drought on the forest carbon sink need to be considered by governments for reaching net-zero goals. Besides reducing deforestation, tropical countries like Brazil and Indonesia are planning substantial changes in land use (e.g. reforestation) to compensate for greenhouse gas emissions16. nature communications (2023) 14:6217 \| 1 Comment Wide arrows show carbon uptake or release, i.e. forest uptake through photosynthesis (green arrows), release from respiration or industry to the atmosphere (beige arrows), and changes in net forest storage (brown arrows). The stripped brown arrow denotes long-term carbon storage in wood materials. Forest management strategies based on the vulnerability to drought and heat could retain the current carbon sink by Comment While previous studies on the carbon cycle impact of climate extremes were delayed by data availability constraints4,10,15, van der Woude et al.7 demonstrate the importance of standardized ecosystem monitoring networks such as ICOS (European Inte- grated Carbon Observation System) and NEON (U.S. National Ecological Observatory Network) for providing timely informa- tion to stakeholders. The severe impacts of drought on the forest carbon sink need to be considered by governments for reaching net-zero goals. Besides reducing deforestation, tropical countries like Brazil and Indonesia are planning substantial changes in land use (e.g. reforestation) to compensate for greenhouse gas emissions16. In Europe, forest area has already been increasing for Introducon Vulnerability to Drought and Heat? Yes No Adaptaon Retain Previous Sink Migaon Increase Sink Forest Harvest Reduce Carbon Losses aer Logging Return to Carbon Neutrality Long-term Carbon Storage in Wood Emission Reducons in other Sectors Increasing Carbon Storage towards Net Zero Timber Monitor Reassess Forestry Wood Chain ig. 2 \| Forest management to increase carbon storage towards net zero, espite drought and heat. Management options for the forestry wood chain Forest →Harvest →Timber) to increase carbon storage (left to right). Wide arrows how carbon uptake or release, i.e. forest uptake through photosynthesis (green adaptation efforts (if vulnerable) to avoid forest carbon release, or increase the sink by mitigation efforts (if not vulnerable) to compensate for emissions from other sectors. After wood harvest, carbon losses from soil and wood residues can be reduced to support a return to carbon neutrality. Storing carbon in wood materials more of the available energy heats the air (see Fig. 1, orange ws). How does drought and heat affect forest carbon uptake? The net on balance of an ecosystem represents the difference between on uptake by photosynthesis and carbon release by respiration. uced forest carbon uptake during drought and heat comes from s-related declines in photosynthesis (see Fig. 1, green arrows). iration from plants and soil is also reduced due to limitations in moisture, but this is typically to a lower extent than photosynthesis Fig. 1, beige arrows)10. These relative differences result in reduced arbon uptake (see Fig. 1, brown arrows) or even net release, as e.g. rted by van der Woude et al.7 for some sites in France during mer 2022. The combined stress of intense drought and heat over prolonged ods (or recurrent events) leads to increased crown and eventually mortality (see Fig. 1c). Comment nd more of the available energy heats the air (see Fig. 1, orange rrows). How does drought and heat affect forest carbon uptake? The net arbon balance of an ecosystem represents the difference between arbon uptake by photosynthesis and carbon release by respiration. Reduced forest carbon uptake during drought and heat comes from tress-related declines in photosynthesis (see Fig. 1, green arrows). Respiration from plants and soil is also reduced due to limitations in oil moisture, but this is typically to a lower extent than photosynthesis see Fig. 1, beige arrows)10. These relative differences result in reduced et carbon uptake (see Fig. 1, brown arrows) or even net release, as e.g. eported by van der Woude et al.7 for some sites in France during ummer 2022. The combined stress of intense drought and heat over prolonged eriods (or recurrent events) leads to increased crown and eventually ree mortality (see Fig. 1c). Increased mortality has been reported for ll major European tree species11, and the spatial distribution of these pecies regarding climatically suitable areas will be substantially ltered by the end of this century12. For example, European beech is onsidered the most vulnerable broadleaf tree species to drought and eat13, and substantial climate-induced growth declines are rojected14. This is important because beech forests cover vast areas of entral and eastern Europe. To quantify carbon uptake, van der Woude and colleagues7 ombined ‘bottom-up’ ecosystem ﬂux tower measurements in orests with ‘top-down’ approaches using satellite remote sensing bservations and atmospheric inversions coupled with a biosphere model. Summer reductions of about 59 TgC were observed across the drought-affected area and resulted in 40 TgC reduced annual carbon uptake, which is equivalent to nearly one- quarter (23%) of the 2022 annual CO2 emissions of Germany – the European country with the largest emissions. Unlike in 2018 rela- ted to spring, only partial seasonal compensation was found from increased fall uptake due to a prolonged growing season. Similar carbon release from summer forest ﬁres (about 9 TgC) make the 2022 event comparable with 2003, when much higher (up to 500 TgC) annual net carbon release was reported10. However, van der Woude et al.7 emphasize that other regions were affected in 2022 compared to 2018 and 2003, which likely mediated the impacts on reduced carbon uptake because of differences in forest composition. Acknowledgements di i k l d Funding support is acknowledged for S.W. by ETH Zurich and for E.P-L. by the MainWood project (ETH Domain Joint Initiatives 2023–2026). Funding support is acknowledged for S.W. by ETH Zurich and for E.P-L. by the MainWood project (ETH Domain Joint Initiatives 2023–2026). The study by van der Woude et al.7 shows that the reduced forest carbon uptake during the 2022 drought and heat might be no longer exceptional in a warming climate, revealing the vulnerability of the forest carbon sink to such climate extremes. While it might be too early to designate such conditions the “new normal”, there is clear evidence that these events have been increasing in frequency and intensity across most of Europe20, and are projected to further increase with climate warming during spring and summer2. It is becoming apparent that recurrent drought and heat challenges the net-zero goals of governments relying on forestry, and that forest management needs to be adapted to retain the forest carbon sink. nature communications (2023) 14:6217 \| 2 2 References Climate-change-driven growth decline of European beech forests. Commun. Biol. 5, 163 (2022). 14. Martinez del Castillo, E. et al. Climate-change-driven growth decline of European beech forests. Commun. Biol. 5, 163 (2022). 15. Bastos, A. et al. Direct and seasonal legacy effects of the 2018 heat wave and drought on European ecosystem productivity. Sci. Adv. 6, eaba2724 (2020). 15. Bastos, A. et al. Direct and seasonal legacy effects of the 2018 heat wave and drought on European ecosystem productivity. Sci. Adv. 6, eaba2724 (2020). 16. Grassi, G. et al. On the realistic contribution of European forests to reach climate objectives. Carbon Balance Manag. 14, 8 (2019). 16. Grassi, G. et al. On the realistic contribution of European forests to reach climate objectives. Carbon Balance Manag. 14, 8 (2019). 17. Churkina, G. et al. Buildings as a global carbon sink. Nat. Sustainability 3, 269–276 (2020). 17. Churkina, G. et al. Buildings as a global carbon sink. Nat. Sustainability 3, 269–276 (2020). 18. Toreti, A. et al. Drought in Europe June 2023. (Publications Ofﬁce of the European Union, Luxembourg, 2023). 17. Churkina, G. et al. Buildings as a global carbon sink. Nat. Sustainability 3, 269–276 (2020). 18. Toreti, A. et al. Drought in Europe June 2023. (Publications Ofﬁce of the European Union, Luxembourg, 2023). 18. Toreti, A. et al. Drought in Europe June 2023. (Publications Ofﬁce of the European Union, Luxembourg, 2023). 19. ECMWF. The European heatwave of July 2023 in a longer-term context. (2023). https:// climate.copernicus.eu/european-heatwave-july-2023-longer-term-context. 19. ECMWF. The European heatwave of July 2023 in a longer-term context. (2023). https:// climate.copernicus.eu/european-heatwave-july-2023-longer-term-context. 20. Seneviratne, S. I. et al. in Climate Change 2021: The Physical Science Basis. Contribution of Working Group I to the Sixth Assessment Report of the Intergovernmental Panel on Climate Change (eds Valérie Masson-Delmotte, Panmao Zhai, Anna Pirani, & et al.) 1513–1766 (Cambridge University Press, 2022). 20. Seneviratne, S. I. et al. in Climate Change 2021: The Physical Science Basis. Contribution of Working Group I to the Sixth Assessment Report of the Intergovernmental Panel on Climate Change (eds Valérie Masson-Delmotte, Panmao Zhai, Anna Pirani, & et al.) 1513–1766 (Cambridge University Press, 2022). Comment Sebastian Wolf 1 & Eugénie Paul-Limoges 2 1Department of Environmental Systems Science, Physics of Environmental Systems, ETH Zurich, Zurich, Switzerland. 2Swiss Federal Institute for Forest, Snow and Landscape Research (WSL), Forest Dynamics, Birmensdorf, Switzerland. e-mail: sewolf@ethz.ch decades (e.g. by 10% during 1990–2020) and land-use change (including reforestation) accounts for only 1% of the 2030 reduction target16. Essential in the European net-zero strategy is to retain the current forest carbon sink by adapting management practices, in particular because of the large uncertainty asso- ciated with climate change. Received: 8 September 2023; Accepted: 15 September 2023; Received: 8 September 2023; Accepted: 15 September 2023; To ensure resilience of the forest carbon sink, improvements of current management practices (Fig. 2, Forest box – Adaptation) should include shifts to species mixtures (ideally of uneven age) that are better adapted to future climate conditions12, while preserving local species and biodiversity. Managed regeneration can prepare for spe- cies transitions following disturbances and enhance carbon uptake. In forests already resilient to drought and heat, the net-zero focus can shift towards increasing carbon storage to compensate emissions from other sectors (Fig. 2, Forest box – Mitigation), for example, through sustainable short-rotations, higher tree densities, or the introduction of more productive species. References Atmospheric blocking and weather extremes over the Euro-Atlantic sector – a review. Weather Clim. Dynam. 3, 305–336 (2022). Forest management strategies in a changing climate need to combine adaptation and mitigation approaches to enable resilient carbon storage, potentially complemented with long-term storage in wood products to further increase the mitigation potential. If imple- mented adequately, this could ensure compliance with the net-zero goal and make future forests more resilient against climate extremes like the 2022 drought and heat. 10. Ciais, P. etal.Europe-wide reduction in primaryproductivity caused bythe heat anddrought in 2003. Nature 437, 529–533 (2005). 10. Ciais, P. etal.Europe-wide reduction in primaryproductivity caused bythe heat anddrought in 2003. Nature 437, 529–533 (2005). 11. George, J.-P. et al. Long-term forest monitoring reveals constant mortality rise in European forests. Plant Biol. 24, 1108–1119 (2022). 11. George, J.-P. et al. Long-term forest monitoring reveals constant mortality rise in European forests. Plant Biol. 24, 1108–1119 (2022). 12. Mauri, A. et al. EU-Trees4F, a dataset on the future distribution of European tree species. Sci. Data 9, 37 (2022). 12. Mauri, A. et al. EU-Trees4F, a dataset on the future distribution of European tree species. Sci. Data 9, 37 (2022). 13. Schuldt, B. & Ruehr, N. K. Responses of European forests to global change-type droughts. Plant Biol. 24, 1093–1097 (2022). 13. Schuldt, B. & Ruehr, N. K. Responses of European forests to global change-type droughts. Plant Biol. 24, 1093–1097 (2022). While the impacts of the 2022 event are still investigated, Europe is suffering yet another year with extreme temperatures and drought in 2023. Following a warm and dry winter, the Iberian Peninsula, southern France and northwestern Italy were affected by severe drought during late spring. Drought conditions then emerged in large parts of northern, central, and eastern Europe during June and July18, while record temperatures were observed along with a severe heatwave that peaked in late July19. The carbon cycle impact of this recent event remains to be determined, yet could provide further insights on legacy effects of recurrent drought and heat. The 2023 event might also rival the years 2018 and 2022 because of a combination of direct impacts (i.e. reduc- tions in uptake, see Fig. 1) in the most affected regions, carbon release from widespread wildﬁres in southern Europe, and from accumulated legacy effects related to tree mortality from previous drought years. 14. Martinez del Castillo, E. et al. References 1. Büntgen, U. et al. Recent European drought extremes beyond Common Era background variability. Nat. Geosci. 14, 190–196 (2021). 2. Spinoni, J., Vogt, J. V., Naumann, G., Barbosa, P. & Dosio, A. Will drought events become more frequent and severe in Europe? Int. J. Climatol. 38, 1718–1736 (2018). 3. Friedlingstein, P. et al. Global Carbon Budget 2022. Earth Syst. Sci. Data 14, 4811–4900 (2022). 4. Wolf, S. et al. Warm spring reduced carbon cycle impact of the 2012 US summer drought. Proc. Natl Acad. Sci. 113, 5880–5885 (2016). 4. Wolf, S. et al. Warm spring reduced carbon cycle impact of the 2012 US summer drought. Proc. Natl Acad. Sci. 113, 5880–5885 (2016). 5. Miralles, D. G., Gentine, P., Seneviratne, S. I. & Teuling, A. J. Land–atmospheric feedbacks during droughts and heatwaves: state of the science and current challenges. Ann. N. Y. Acad. Sci. 1436, 19–35 (2019). For long-term carbon storage, wood materials arise as an inter- esting option because the storage effect is magniﬁed by reducing large emissions from construction materials such as cement and steel17 (Fig. 2, Timber box). However, this option requires sustainable harvest management to minimize carbon release from deforested lands, such as by selective harvesting, supporting natural regeneration, and pro- tecting soil and understory plants (Fig. 2, Harvest box). 6. Anderegg, W. R. L. et al. Climate-driven risks to the climate mitigation potential of forests. Science 368, eaaz7005 (2020). 6. Anderegg, W. R. L. et al. Climate-driven risks to the climate mitigation potential of forests. Science 368, eaaz7005 (2020). Science 368, eaaz7005 (2020). 7. van der Woude, A. M. et al. Temperature extremes of 2022 reduced carbon uptake by forests in Europe. Nat. Commun. (2023). https://doi.org/10.21203/rs.3.rs-2841861/v1 8. Müller, L. M. & Bahn, M. Drought legacies and ecosystem responses to subsequent drought. Glob. Change Biol. 28, 5086–5103 (2022). 7. van der Woude, A. M. et al. Temperature extremes of 2022 reduced carbon uptake by forests in Europe. Nat. Commun. (2023). https://doi.org/10.21203/rs.3.rs-2841861/v1 7. van der Woude, A. M. et al. Temperature extremes of 2022 reduced carbon uptake by forests in Europe. Nat. Commun. (2023). https://doi.org/10.21203/rs.3.rs-2841861/v1 8. Müller, L. M. & Bahn, M. Drought legacies and ecosystem responses to subsequent drought. Glob. Change Biol. 28, 5086–5103 (2022). 9. Kautz, L. A. et al. Atmospheric blocking and weather extremes over the Euro-Atlantic sector – a review. Weather Clim. Dynam. 3, 305–336 (2022). 9. Kautz, L. A. et al. nature communications (2023) 14:6217 \| 3 Comment Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional afﬁliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2023 nature communications (2023) 14:6217 \| 4 4
https://openalex.org/W4308531372	https://www.researchsquare.com/article/rs-1358803/latest.pdf	English	null	Factors influencing adherence to antiretroviral therapy from the experience of people living with HIV and their healthcare providers in Sierra Leone: a qualitative study	BMC health services research	2,022	cc-by	7,736	Michael Lahai (  miclahisaac@gmail.com ) Faculty of Pharmaceutical Sciences, College of Medicine and Allied Health Scienc Sally Theobald Liverpool School of Tropical Medicine Haja R. Wurie Faculty of Pharmaceutical Sciences, College of Medicine and Allied Health Sciences, Freetown, Sierra Leone Sulaiman Lakoh Faculty of Pharmaceutical Sciences, College of Medicine and Allied Health Sciences, Freetown, Sierra Leone Mohamed Samai Faculty of Pharmaceutical Sciences, College of Medicine and Allied Health Sciences, Freetown, Sierra Leone Patrick O. Erah University of Benin Joanna Raven Liverpool School of Tropical Medicine Abstract Background: Antiretroviral therapy is the main drug for the treatment of Human Immunodeficiency Virus, slow disease progression, and reduce the spread of infection. HIV treatment is also known to require high level of adherence of over 90% to achieve good treatment outcomes and viral load suppression. In Sierra Leone, it is estimated that about 70% of PLHIV are non-adherent in their first year of treatment. Understanding the reasons behind this high-rate of non- adherence is critical for the development of strategies to improve adherence. This study identifies the barriers and facilitators influencing adherence to antiretroviral treatment in Sierra Leone. Methods: A qualitative study design using in depth interviews was conducted in two districts in Sierra Leone – Freetown and Bo. In-depth interviews were conducted with 4 health care workers and 16 people living with HIV. Results: Several facilitators and barriers to Antiretroviral therapy(ART) adherence at the personal, community, and health system levels were identified. The facilitating factors included perceived benefits of ART, family support, having a treatment partner, receiving free ART medicines and belonging to peer support groups. The barriers included denial and non-disclosure of HIV status, frequency of medication, use of traditional medicine, lack of money for food and transport, stigma and discrimination, work barriers, lack of medicines and test kits, limited health workers and long distance to clinics. Research Article Keywords: Adherence, Community, Health system, health workforce, peer support group, treatment partner, stigma, PLHIV, family support, Antiretroviral therapy Posted Date: March 28th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-1358803/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/15 Page 1/15 Page 1/15 Conclusions: The key emerging themes arising from different health system level in this study include Support from families, treatment partners and the need for peer support groups. Understanding the facilitators and barriers to antiretroviral therapy identified in this study can improve adherence and provide relevant information for more responsive and equitable program implementation in low and middle-income countries. There is the need for implementing adherence augmentation programs with implementation of activities that will improve community knowledge for prevention and reduction of stigma and the need for integrating HIV treatment services close to communities. Background The global commitment to Fast-Track the HIV response and end AIDS by 2030 is not on track(1). There were almost 700,000 deaths from AIDS-related causes and 1.7 million people with new HIV infections in 2019 (2). In Africa, despite improvements in HIV prevention, testing, and treatment, HIV/AIDS remains one of the leading causes of mortality, with more than 400,000 deaths on the continent in 2019. Of the estimated 25.8 million people in Africa living with HIV in 2019, 4.3 million were not diagnosed, and a further 3.4 million were not receiving antiretroviral treatment (ART) (1). In Sierra Leone in 2019, an estimated 78,000 people were living with HIV (PLHIV), with only 48% knowing their status and only 43% receiving ART (1). The mainstay for treatment of people living with HIV/AIDS is a combination of antiretroviral drugs. ART stops HIV from multiplying and can suppress HIV to undetectable levels in the blood, allowing a person’s immune system to recover, overcome infections, prevent the development of AIDs and reduce the risk of HIV transmission (2). Adherence describes how a person uses and receives treatment according to medical recommendations, including timing, dosing, and consistency. HIV treatment requires a high level of adherence of over 90% to achieve good treatment outcomes and viral load suppression (3)(4). Adherence can be challenging for example, in one hospital in Brazil, under 20% of people living with HIV did not stay on ART(5). There are many barriers and facilitators to adherence. Barriers include medication and health concerns, stigma, family responsibilities, and problems with schedule and routine(6). People with better socioeconomic status related to income, education, and employment status were more likely to adhere to Page 2/15 Page 2/15 treatment than patients with poor socioeconomic status(7). Expansion and decentralization of HIV/AIDS services allowed for implementation at the community level and increased collaboration through task sharing between health professionals, thereby increasing timely access to treatment for patients and overcoming limited human resources in hospitals (8). Interventions that focus on early ART initiation and groups of people who are more likely to drop out of treatment, can improve adherence(9). There are many challenges with ART services in Sierra Leone. Study Site The study was conducted in the two main referral hospitals in Sierra Leone: Connaught Hospital in the capital city of Freetown and the Bo government hospital in Bo, the second major city in Sierra Leone (see Fig. 1). Freetown and Bo districts were deliberately chosen as they are the two districts with the highest prevalence of HIV/AIDS in Sierra Leone, with rates of 2.7 and 1.8 in Freetown and Bo, respectively(12). Connaught hospital is a 300- bed hospital that houses an HIV/AIDS clinic and ward and serves as the main referral center for adult HIV/AIDS care in Freetown. Bo government hospital is a 500-bed hospital with a clinic and ward for people with HIV/AIDS. Background In 2011, a study revealed that 70% of people living with HIV remain on treatment for less than one year after initiation of ART with an estimated survival rate of 92%, but this study did not follow non-adherent patients and could not identify the factors affecting adherence to ART(10). A cohort study at the main tertiary hospital in Sierra Leone showed that 62% of eligible ART patients stopped care before initiating ART(11). The study also identified the main barriers to adherence to ART as a lack of understanding of the importance of treatment adherence due to poor relationships and communication between people living with HIV and healthcare professionals. The study recognized the need for context-specific interventions to support adherence that considers access to medicines, care, and support for people living outside the capital city of Freetown. HIV/AIDS research in Sierra Leone has mainly focused on describing ART adherence, and in one hospital, with little attention given to exploring the health systems and socioeconomic factors affecting adherence to ART. There is a gap in the evidence about the barriers and facilitators around adherence to ART in Sierra Leone. Understanding this from both people living with HIV and health care providers is critical to developing interventions that support adherence to ART. This study will provide relevant information for decision-makers, public health professionals, and clinicians on implementing more responsive and equitable ART programs that will improve adherence and the quality of life of people living with HIV. Study design and sample A qualitative study design employing in-depth interviews was used to explore the barriers and facilitators to adherence to and provision of ART (13). Qualitative interviews generate in-depth and contextual information about an individual’s experiences, beliefs, perceptions and explore reasons behind their answers through probing questions (14). In-depth interviews with four (4) healthcare workers and sixteen (16) people living with HIV were employed to explore the complexities of the issues related to adherence to ART. The research adapted a triangulation strategy by comparing patients’ and healthcare workers’ views to enhance the integrity of the findings (15). Purposive sampling was used to select participants based on features or characteristics that will enable a detailed understanding of the topic (16). In each hospital, the list of people living with HIV was reviewed to select patients that are adherent or non-adherent to treatment, who were receiving ART treatment and care at the health centre for at least Page 3/15 one year and attended the clinic at the time the research was conducted, was aged 18 years and above and spoke English or Krio. Healthcare workers (medical doctor, community health officer, the counsellors, and the nurse) involved in the care of people living with HIV were selected. Data collection Interviews with people living with HIV were conducted by the lead author, in Krio language, in a private room in the HIV clinic at Connaught Hospital and the Bo Government Hospital and lasted between 45 and 60 minutes. A topic guide was used to explore the background of participants, their experiences with ART, barriers and enablers to using ART services, and concerns about taking ART and other services related to ART. The lead author interviewed the health workers in English in the hospitals’ offices and lasted up to 60 minutes. Using a topic guide these interviews focused on the background of participants, their roles and responsibilities in ART provision, their perceptions and experiences of people’s adherence to ART, and their role in improving adherence to ART. Data analysis The recordings were transcribed verbatim. Where necessary, the recordings were transcribed in the local language (Krio) and then translated into English; and a sub-set was checked against the recordings for quality of transcription and translation. The interviews were analyzed using thematic framework analysis(15), and data were managed using NVivo 11 programme. A coding framework was developed based on themes emerging from the data. The coding framework was applied to transcripts of all interviews, charts were then developed for each theme, and these charts were used to describe the themes. Results Participant characteristics are included in tables 1 and 2. Sixteen PLHIV were interviewed – 8 from Bo and 8 from Freetown. The majority were female (11), adherent to ART (9) and employed (9). Four health workers were interviewed with two from each study site. Three health workers were female. Perceived benefits of ART Most participants were positive about the effects of ART especially when they were able to take the medication as prescribed and had regular appointments for follow-up. They explained that HIV care had improved their quality of life and health status. “I felt very good when I started taking the medicine at the time I was confirmed HIV positive. Currently, I’m not feeling good and I am losing weight[...] I was informed at the treatment centre that my blood is low and I believe it happened because I missed my appointment for medicine pick up. Now I have started treatment and I’m beginning to feel great again”. (Non-adherent Female, Bo) Facilitators and barriers to adherence to ART The study revealed several facilitators and barriers to ART adherence at person, community and health system levels (Figure 2). Table 1 characteristics of PLHIV in the interviews More female in Bo population than in Freetown and more employed participants in Freetown than Bo and more female among the Bo participants than in Freetown. Study site Adherence to ART Gender Age (years) Work Total Adherent Non- adherent Female Male 24- 35 >35- 45 >45 employed Unemployed Bo 5 3 7 1 3 3 2 3 5 8 Freetown 4 4 4 4 4 3 1 6 2 8 Total 9 7 11 5 7 6 3 9 7 16 More female in Bo population than in Freetown and more employed participants in Freetown than Bo and more female among the Bo participants than in Freetown. characteristics of health workers in the interviews characteristics of health workers in the interviews Study site Type of health worker Gender Age (years) Total Clinician Counsellor Female Male 20-30 30-40 Bo 1 1 2 - 1 1 2 Freetown 1 1 1 1 - 2 2 Total 2 2 3 1 1 3 4 (Adherent Female living with HIV, Bo). Many PLHIV reported not disclosing their status to other members in their family. Others spoke about being told not to speak about their HIV status with anyone as this would result in being harassed or ostracized. “you know as for our own health condition people normally hide it. It’s not like headache that you will easily disclose. It has to be kept secret. We were advised not to disclose it to anyone because if you disclose it to people they will start pointing fingers at you that you are HIV positive. That’s why we are always advised not to disclose our status and to continue taking our medications” (Adherent Female living with HIV, Bo) Health workers also reported that many PLHIV do not disclose their status in families, and some go to the extent of losing all their medication to keep their status a secret from their partner. “Most of our patients do not give us the correct information about their partner […] they usually say, my husband is not here, my husband has travelled[...]someone who is married and does not disclose their status to their partner […] when it is time for that person to take their medication it becomes a problem” (Female Health worker, Freetown). Family Support Support from family members is essential in providing hope for survival of PLHIV as highlighted by a healthcare worker: “so if there is strong support from the family, it will be very good. We need to pass the message to the family and relatives so that they will be able to support these people. If you stigmatize PLHIV, they will lose hope for survival. That’s why family is more important because they spend about 90-95 percent of their time with them” (Male Health worker, Freetown). A patient also noted that it was important to have a treatment partner that will provide additional support for adherence to ART medication. “I informed my small sister that the medication am using is for prevention and she must remember me in the morning and at night to take my medicine. She has been informing me on a daily basis.” Page 5/15 Medication issues Some PLHIV reported that the taste and size of the medicines deterred them from keeping to the treatment regime. Healthcare workers expressed concerns about the frequency of ART leading to difficulties in adherence to the regime. “it’s difficult to take a drug every day of your life especially with the kind of pills and the daily intake that is required coupled with other additional blood medicines” (Female Health worker, Bo). “it’s difficult to take a drug every day of your life especially with the kind of pills and the daily intake that is required coupled with other additional blood medicines” (Female Health worker, Bo). “For me it is the big tablet […] when I started taken the medicine at first, because of the taste I usually vomit” (Adherent Female living with HIV, Bo) Denial and non-disclosure of HIV status Many PLHIV reported that they were frightened of disclosing their HIV status to their partners for fear of their relationship ending. One woman explained that her husband gave her money and sent her back to her village: “My husband is a soldier so I was taken to the hospital where he works. He received the test and informed me that I am HIV positive. I did not believe him initially. He said we cannot continue the relationship. He informed me that it is possible that I may have contracted the disease from my work as a hairdresser (used needle and comb). He therefore asked me to return to Bo and gave me Le150,000 (£15 or $19) as hospital cost. At the hospital, I was tested and confirmed again to be HIV positive. I was encouraged to commence medication and advised to meet the requirement of monthly appointment” Money for food and transport Many PLHIV reported that they found it difficult to continue working and earning money when they were ill. This impacted on their ability to buy food which they need when they take ART and pay for transport to get to the health facilities. They relied on friends or relatives to provide food and help with transport, but often felt embarrassed to ask for help. “The little money I had is no more and there is nothing left at the moment […] if they will help with supply or money […]so that when I regain my health i will be able to start my business and support my children” (Non-Adherent Female living with HIV, Freetown). Healthcare workers also indicated that many PLHIV did not continue their treatment because of lack of food or money for transportation. They also reported that food was only provided for PLHIV who were underweight. “I’m not taking the medications because I do not have food […] others will say i do not have transport […] we have a system for providing food for people with low body mass index and that is all at the moment” (Female Health worker, Freetown). Male living with HIV, Freetown). Male living with HIV, Freetown). Use of herbal medicines Some PLHIV reported feeling better and stopped taking their ART with the belief that traditional herbal medications will help provide long-lasting solution but eventually became seriously ill. “It was last year that I stopped taking the medication. This year I was asked to take HIV test again when I started feeling sick. I informed the healthcare professionals that I was using herbal medication to treat HIV” (Non-adherent Page 6/15 Page 6/15 Treatment partner “initially I was taking my medication at 9 O’clock but then I noticed that it will give me problem at my work place […] I decided to take it before leaving my house.” (Non-adherent Female living with HIV, Freetown). Health care workers also reported many examples of PLHIV experiencing stigma and discrimination such as lack of support, fear of death and not sharing toilets and items such as cups and cutlery. This influenced how PLHIV were able to access and adhere to ART, as well as having profound effects on their mental health. “Look at this lady for example she came this morning crying […] she is staying with her mother. This is what she said, my mum said I’m going to die soon. She came crying and I told her to wait for me for a while and all these days she has been coming here saying that her mum hasn’t been supportive to her […] She said If my mum is not ready to support me how will I survive?” (Female Health worker, Freetown). Work barriers Most participants reported improvement in their health when they were taking their medication. However, many found it difficult to take the drugs regularly when they were working and often missed their medication during work hours. “well the medication is good because if you are on medication for a certain illness and you see improvement then it’s good [….] I miss my medication when I go out for work “(Adherent Female living with HIV, Bo). PLHIV faced discrimination in the workforce (as described above) and a woman spoke about losing her job because her employer observed her taking medication at the same time every day. “when I am taking the drug, they are watching me […] I was informed that If I know that I have a disease and I am taking a drug let me be careful […]so when it’s time to take the drug, I would think of my life and my only daughter […] So, I would usually take the drug. Later I was then dismissed from my duty” (Non-adherent female living with HIV, Freetown) Treatment partner Many PLHIV and healthcare workers recognized the importance of having a treatment partner to help with adherence to ART: “it is good for someone living with HIV to have a treatment partner” (Female Health worker, Freetown). Health workers and PLHIV also recognized the importance of acceptance of partner status and prescription refill by the treatment partner. “imagine the man is negative and the wife is positive but yet still he is here to collect the medication for the wife” (Female Health worker, Bo) “so my daughter normally comes for my medication” (Female Health worker, Bo) “so my daughter normally comes for my medication” (Female Health worker, Bo) Many PLHIV reported experiencing stigma and discrimination because of their HIV status from family, friends and community members. They explained that HIV/AIDs is still regarded as a taboo subject, that stopped them disclosing their status, discussing care and treatment and getting help. “Yes, initially I was staying with my husband’s relatives[...]when they discovered that I had the virus […] they started laughing at me […] they started throwing provocative words at me, they don’t even give me food to eat and they finally asked me out of the house (crying) […] Just look at me, my hair is not even neat because I don’t have someone to do it for me”. (Adherent Female living with HIV, Bo). Some PLHIV stated that frequent ailments and weight loss is associated with HIV by their community, and when they start ART and put weight on they are accepted, as one person explains here: Page 7/15 “Initially when I got the disease, my friends were not talking to me properly because I was constantly losing weight but now they feel free to talk to me and they come closer to me. Thanks to God for that […] I didn’t inform them about my condition […] they would have been afraid of me if I had informed them earlier of my status.” (Adherent female living with HIV, Bo). PLHIV also reported fear of discrimination in the workplace, and therefore did not disclose their HIV status to their employer or colleagues, and changed their times for medication: “initially I was taking my medication at 9 O’clock but then I noticed that it will give me problem at my work place […] I decided to take it before leaving my house.” (Non-adherent Female living with HIV, Freetown). Free medicines Healthcare workers emphasized received ART free and also mentioned the importance of ensuring that ART continue to be free for PLHIV in LMIC, as the high cost of these drug would prohibit uptake by PLHIV. “I think when it comes to the fight of HIV, I think the medication should be free. So, if the medication is free it will help most of our clients[...]the cost of their medication is too high” (Female Health worker, Freetown). Page 8/15 Page 8/15 Good relationships with health workers PLHIV reported the existence of good interaction with healthcare workers involved in their treatment. They mentioned that health workers encourage them to take their medications attend clinic and reassure them to take their medication to maintain good health and wellbeing. PLHIV reported the existence of good interaction with healthcare workers involved in their treatment. They mentioned that health workers encourage them to take their medications attend clinic and reassure them to take their medication to maintain good health and wellbeing. “They treat me well and they also encourage me. The first day that I came to the hospital I was so discouraged but the nurse talked to me and really encouraged me” (Adherent Female living with HIV, Bo) Lack of medicines and tests Healthcare workers reported a lack of ART medicines and test kits that are used in caring for PLHIV. They explained that drugs and test kits are provided by donor organizations, and that there are frequent delays in procurement. “…in terms of HIV care you go anywhere there is the issue of drug stock out and [….]test kits shortage are all the problems we encounter” (Female Health worker, Bo). Distance to health facilities PLHIV reported that they had long distances to travel to the ART clinics. In Bo, people would travel at least 30 minutes, and this would cost about £1. In Freetown, people would travel for an hour to reach the clinic, which would cost them about £2. The average weekly wage in Freetown is about £3.4 for workers that participated in this study or nothing for those without a job. They requested for ART services to be closer to their homes. “They need to bring the health facility closer to my community, if it is possible because there is no health facility where I live” (Adherent Female living with HIV, Freetown). Discussion The study revealed several facilitators and barriers to ART adherence at the personal, community, and health system levels. The facilitating factors included perceived benefits of ART, family support, having a treatment partner, receiving free ART medicines, and good relations with health workers. The barriers included denial and non-disclosure of HIV status, frequency of medication, use of traditional medicine, money for food and transport, stigma and discrimination, work barriers, lack of medicines and test kits, limited health workers and long distance to clinics. Here we discuss three key areas that have emerged. Limited numbers of skilled health workers Healthcare workers expressed concerns about the burden created by the small number of health staff working with PLHIV. They mentioned that this will allow them to spend more time in reviewing the wellbeing of patient, while allowing other staff to focus on drug-therapy related issues. “I will love to have another staff that would help us to assess patient (Female Health worker Bo). Community support to people living with HIV In addition, the participants expressed concerns of community stigmatization in which healthcare workers were involved in handling complaints from PLHIV against close family members and other community members. The study revealed several issues of stigmatization among couples, in-laws and close family members with divorce, house expulsion of a widow and provoking words from close relatives respectively. This showed the existence of stigmatization irrespective of the advice from healthcare providers to community members for acceptance of PLHIV. Other research findings suggested that there is an existing gap in perception between PLHIV, healthcare professionals and community individuals and emphasised the need for improvement of such interactions(24). Such can also be improved further by using the stepping stone approach in communities where PLHIV are being stigmatized. Some recommendations for the prevention and reduction of stigma in the community include the improvement of the knowledge of the community, mass media campaigns and direct contact or testimonies from PLHIV(25)(26). Support from families, treatment partners and peers is needed Page 9/15 Most healthcare workers and PLHIV revealed that support from close family members and having a treatment partner was very important in improving adherence among people living with HIV/AIDS(PLHIV). The likelihood for adherence and improvement in quality of life was more prominent among patients who informed their spouses of their status. Other studies revealed that non-disclosure of positive HIV-status can lead to HIV transmission, treatment discontinuity, poor health outcomes and difficulty in HIV prevention and control(17)(18)(19). Other studies clearly indicated the importance of including family members and other treatment partners in adherence augmentation programs by providing enough information that could aid long-term adherence(20)(21). Healthcare workers and patients also confirmed that the feeding program was available in communities among peer support groups to encourage patient interaction and adherence. Other studies also reaffirmed the importance of implementing adherence program for PLHIV in peer groups other than as individuals(22)(23). Therefore, it is important that adherence augmentation programs are implemented in communities as a means of ensuring that communities understand the management of HIV/AIDs also inform communities of their responsibility to their HIV spouses. Most healthcare workers and PLHIV revealed that support from close family members and having a treatment partner was very important in improving adherence among people living with HIV/AIDS(PLHIV). The likelihood for adherence and improvement in quality of life was more prominent among patients who informed their spouses of their status. Other studies revealed that non-disclosure of positive HIV-status can lead to HIV transmission, treatment discontinuity, poor health outcomes and difficulty in HIV prevention and control(17)(18)(19). Other studies clearly indicated the importance of including family members and other treatment partners in adherence augmentation programs by providing enough information that could aid long-term adherence(20)(21). Healthcare workers and patients also confirmed that the feeding program was available in communities among peer support groups to encourage patient interaction and adherence. Other studies also reaffirmed the importance of implementing adherence program for PLHIV in peer groups other than as individuals(22)(23). Therefore, it is important that adherence augmentation programs are implemented in communities as a means of ensuring that communities understand the management of HIV/AIDs also inform communities of their responsibility to their HIV spouses. Community support to people living with HIV Health systems support This study emphasized the need for improving adherence among PLHIV by ensuring the recruitment of additional skilled and motivated staff that can support treatment by providing interventions that will help improve drug therapy problems faced by PLHIV. Several studies have shown that Pharmacist intervention through appointments was vital in the prevention and reduction of drug related problems and in improving patient adherence to ART with subsequent increase in CD4 count(27)(28). The study also confirmed the need to ensure that medicines and test kits are free and always available for PLHIV. This would ensure patient confidence and reliance in the health sector so that patients do not miss doses or travel from long distances to get medications that may not be available at the health facilities. A study in Uganda also revealed that there was a significant association between missing doses and missing appointments. Therefore, it is important to ensure that strategies are designed that will help improve adherence to ART medications among PLHIV. Key to any strategy is to ensure that ART medications are free and available at all times considering the fact that about 60% of the population in Sierra Leoneans are poor(29) and may not be able to pay for treatment or ART medications. Adherence to ART can be improved by integrating treatment services by using accredited pharmacies(30) and clinics(31) close to communities. This will help reduce spending in transportation for PLHIV, reduce hospital visit for medication refill while improving adherence, monitoring, interaction and collaboration between the primary healthcare facilities and hospitals. Conclusions Our study revealed that several factors influence adherence. The factors identified as facilitators are the perceived benefit of ART by PLHIV, family support, treatment partner, free medicine, and good relations with health workers. The barriers include denial and non-disclosure of HIV status, frequency of medication, traditional medicines use, lack of money for food and transport, stigma and discrimination, lack of medicines and test kits, limited healthcare workers (medical doctors, pharmacists, and nurses), and the distance of PLHIV to the health facility. Strengthening the support from families, communities and the health system will help PLHIV to continue with ART and prevent or reduce stigmatization in the community. Limitations Page 10/15 It was a sensitive topic to explore, especially in an environment where stigma and discrimination towards PLHIV are common, and PLHIV may have been reluctant to express their ART experiences. By establishing a good rapport and sensitive questioning, we hope to have minimized this. In addition, PLHIV may have concealed information related to adherence because the interviews were conducted at the HIV clinic where they also receive treatment. This was minimized by ensuring that no one entered the room during the interviews and removed all personal identifiers from the transcripts and reports. It was a sensitive topic to explore, especially in an environment where stigma and discrimination towards PLHIV are common, and PLHIV may have been reluctant to express their ART experiences. By establishing a good rapport and sensitive questioning, we hope to have minimized this. In addition, PLHIV may have concealed information related to adherence because the interviews were conducted at the HIV clinic where they also receive treatment. This was minimized by ensuring that no one entered the room during the interviews and removed all personal identifiers from the transcripts and reports. Abbreviations PLHIV: People living with Human Immunodeficiency virus HIV/AIDs: Human Immunodeficiency Virus/Acquired Immunodeficiency Virus ART: Antiretroviral therapy CD4: Cluster of differentiation 4 LMIC: Low and middle-income country Ethics approval and consent to participate HIV/AIDS is a sensitive area of study, and people living with HIV are often stigmatized within their communities and were sometimes reluctant to talk about their illness and treatment. Several approaches were taken to support participation: building rapport with participants, sensitive probing, showing empathy and providing support if upset, ensuring private place for interviews, assuring that participation is voluntary and can withdraw at any time without fear of repercussion to treatment and care. Confidentiality of participants was vital for such a sensitive study and participants were informed to sign a consent form prior to participating in this study. All audio recordings and transcripts were coded and stored in a safe location. All methods were performed in accordance with the declaration of Helsinki and the study was approved by the Sierra Leone Ethics and Scientific review committee of the Ministry of Health and Sanitation. References Page 12/15 1. 2020_UNaids-data-book_en.pdf. 2. Tanser F, Bärnighausen T, Grapsa E, Zaidi J, Newell M-L. High Coverage of ART Associated with Decline in Risk of HIV Acquisition in Rural KwaZulu-Natal, South Africa. Science [Internet]. 2013 Feb 22 [cited 2020 Jul 21];339(6122):966–71. Available from: https://science.sciencemag.org/content/339/6122/966 3. Gardner EM, McLees MP, Steiner JF, del Rio C, Burman WJ. The Spectrum of Engagement in HIV Care and its Relevance to Test-and-Treat Strategies for Prevention of HIV Infection. Clin Infect Dis [Internet]. 2011 Mar 15 [cited 2020 Jul 21];52(6):793–800. Available from: https://academic.oup.com/cid/article/52/6/793/362421 4. Adherence to Antiretroviral Therapy by Human Immunodeficiency Virus—Infected Patients \| The Journal of Infectious Diseases \| Oxford Academic [Internet]. [cited 2020 Jul 21]. Available from: https://academic.oup.com/jid/article/185/Supplement_2/S143/888288 5. REMIEN RH, BASTOS FI, TERTO V, RAXACH JC, PINTO RM, PARKER RG, et al. Adherence to antiretroviral therapy in a context of universal access, in Rio de Janeiro, Brazil. AIDS Care [Internet]. 2007 Jul [cited 2021 Dec 13];19(6):740–8. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3539169/ 6. Genberg BL, Lee Y, Rogers WH, Wilson IB. Four types of barriers to adherence of antiretroviral therapy are associated with decreased adherence over time. AIDS Behav [Internet]. 2015 Jan [cited 2021 May 13];19(1):85– 1. 2020_UNaids-data-book_en.pdf. 1. 2020_UNaids-data-book_en.pdf. 2. Tanser F, Bärnighausen T, Grapsa E, Zaidi J, Newell M-L. High Coverage of ART Associated with Decline in Risk of HIV Acquisition in Rural KwaZulu-Natal, South Africa. Science [Internet]. 2013 Feb 22 [cited 2020 Jul 21];339(6122):966–71. Available from: https://science.sciencemag.org/content/339/6122/966 3. Gardner EM, McLees MP, Steiner JF, del Rio C, Burman WJ. The Spectrum of Engagement in HIV Care and its Relevance to Test-and-Treat Strategies for Prevention of HIV Infection. Clin Infect Dis [Internet]. 2011 Mar 15 [cited 2020 Jul 21];52(6):793–800. Available from: https://academic.oup.com/cid/article/52/6/793/362421 4. Adherence to Antiretroviral Therapy by Human Immunodeficiency Virus—Infected Patients \| The Journal of Infectious Diseases \| Oxford Academic [Internet]. [cited 2020 Jul 21]. Available from: https://academic.oup.com/jid/article/185/Supplement_2/S143/888288 5. REMIEN RH, BASTOS FI, TERTO V, RAXACH JC, PINTO RM, PARKER RG, et al. Adherence to antiretroviral therapy in a context of universal access, in Rio de Janeiro, Brazil. AIDS Care [Internet]. 2007 Jul [cited 2021 Dec 13];19(6):740–8. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3539169/ 5. REMIEN RH, BASTOS FI, TERTO V, RAXACH JC, PINTO RM, PARKER RG, et al. Adherence to antiretroviral therapy in a context of universal access, in Rio de Janeiro, Brazil. AIDS Care [Internet]. 2007 Jul [cited 2021 Dec 13];19(6):740–8. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3539169/ 6. Genberg BL, Lee Y, Rogers WH, Wilson IB. Authors' information Not Applicable Funding This study was funded by RECAP-SL Project. Authors’ contributions All authors have read and approved the manuscript. ML and JR developed the concept and proposal of the study. ML, JR and ST provided guidance on research methods. ML, JR and HRW analysed the dataset. ML and JR prepared documents and framework for ethical approval and consent for the study. ML, JR, HRW, SL, ST, MHS and POE provided expert review. ML trained the study assistants on use of the study and on transcribing for data collection and prepared the write-up. ML and ST and JR finalised the manuscript. Acknowledgements The authors express their sincere thanks and appreciation to all those who participated in this research. Their participation helped in understanding and providing solutions to the factors influencing adherence to treatment among people living with HIV/AIDS. We express our thanks and appreciation to the HIV/AIDS coordinators and staff of the HIV/AIDS clinics at Connaught Hospital and the Bo Government hospital. Consent for publication Not applicable Availability of data and materials Not applicable Availability of data and materials Page 11/15 Page 11/15 All data and materials are available, and can be provided upon reasonable request to the corresponding author. Competing interests The authors declare no competing interests whatsoever. 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Available from: https://doi.org/10.1007/s11904-016-0335-7 24. Stutterheim SE, Sicking L, Brands R, Baas I, Roberts H, van Brakel WH, et al. Patient and Provider Perspectives on HIV and HIV-Related Stigma in Dutch Health Care Settings. AIDS Patient Care STDs [Internet]. 2014 Dec 1 [cited 2021 Nov 15];28(12):652–65. Available from: https://www.liebertpub.com/doi/full/10.1089/apc.2014.0226 24. Stutterheim SE, Sicking L, Brands R, Baas I, Roberts H, van Brakel WH, et al. Patient and Provider Perspectives on HIV and HIV-Related Stigma in Dutch Health Care Settings. AIDS Patient Care STDs [Internet]. 2014 Dec 1 [cited 2021 Nov 15];28(12):652–65. Available from: https://www.liebertpub.com/doi/full/10.1089/apc.2014.0226 25. Bradley JE, Bhattacharjee P, Ramesh BM, Girish M, Das AK. Evaluation of Stepping Stones as a tool for changing knowledge, attitudes and behaviours associated with gender, relationships and HIV risk in Karnataka, India. BMC Public Health [Internet]. 2011 Jun 24 [cited 2021 Nov 16];11(1):496. Available from: https://doi.org/10.1186/1471-2458-11-496 25. Bradley JE, Bhattacharjee P, Ramesh BM, Girish M, Das AK. Evaluation of Stepping Stones as a tool for changing knowledge, attitudes and behaviours associated with gender, relationships and HIV risk in Karnataka, India. BMC Public Health [Internet]. 2011 Jun 24 [cited 2021 Nov 16];11(1):496. Available from: https://doi.org/10.1186/1471-2458-11-496 26. Creel AH, Rimal RN, Mkandawire G, Bose K, Brown JW. Effects of a mass media intervention on HIV-related stigma: ‘Radio Diaries’ program in Malawi. Health Educ Res [Internet]. 2011 Jun 1 [cited 2021 Nov 17];26(3):456– 65. Available from: https://academic.oup.com/her/article-lookup/doi/10.1093/her/cyr012 26. Creel AH, Rimal RN, Mkandawire G, Bose K, Brown JW. Effects of a mass media intervention on HIV-related stigma: ‘Radio Diaries’ program in Malawi. Health Educ Res [Internet]. 2011 Jun 1 [cited 2021 Nov 17];26(3):456– 65. Available from: https://academic.oup.com/her/article-lookup/doi/10.1093/her/cyr012 27. Figure 1 Map of Sierra Leone showing the selected study sites Map of Sierra Leone showing the selected study sites Figures Figures Page 14/15 Figure 1 Map of Sierra Leone showing the selected study sites Figure 1 Map of Sierra Leone showing the selected study sites Figure 1 Figure 2 Facilitators and barriers to ART adherence (+ indicates facilitators; - indicates barriers) Page 15/15
https://openalex.org/W3161351454	https://pure.ulster.ac.uk/files/90764975/fendo_12_689678.pdf	English	null	Proglucagon-Derived Peptides as Therapeutics	Frontiers in endocrinology	2,021	cc-by	28,911	General rights Th i ht General rights The copyright and moral rights to the output are retained by the output author(s), unless otherwise stated by the document licence. General rights The copyright and moral rights to the output are retained by the output author(s), unless otherwise stated by the docum Unless otherwise stated, users are permitted to download a copy of the output for personal study or non-commercial research and are permitted to freely distribute the URL of the output. They are not permitted to alter, reproduce, distribute or make any commercial use of the output without obtaining the permission of the author(s). If the document is licenced under Creative Commons, the rights of users of the documents can be found at https://creativecommons.org/share-your-work/cclicenses/. If the document is licenced under Creative Commons, the rights of users of the documents can be found at https://creativecommons.org/share-your-work/cclicenses/. Proglucagon-derived peptides as therapeutics Lafferty, R., O'Harte, F., Irwin, N., Gault, V. A., & Flatt, PR. (2021). Proglucagon-derived peptides as therapeutics. Frontiers in Endocrinology, 12, 689678. Article 689678. https://doi.org/10.3389/fendo.2021.689678 Link to publication record in Ulster University Research Portal Document Licence: CC BY Document Licence: CC BY General rights The copyright and moral rights to the output are retained by the output author(s), unless otherwise stated by the document licence. Link to publication record in Ulster University Research Portal DOI: 10.3389/fendo.2021.689678 Document Version Publisher's PDF, also known as Version of record Edited by: Edited by: Aylin Carla Hanyaloglu, Imperial College London, United Kingdom Imperial College London, United Kingdom Reviewed by: Sanjay Kalra, Independent researcher, Karnal, India Cornelius Krasel, University of Marburg, Germany Correspondence: Victor A. Gault va.gault@ulster.ac.uk Reviewed by: Sanjay Kalra, Independent researcher, Karnal, India Cornelius Krasel, University of Marburg, Germany Reviewed by: Sanjay Kalra, Independent researcher, Karnal, India Cornelius Krasel, University of Marburg, Germany Correspondence: Victor A. Gault va.gault@ulster.ac.uk Specialty section: This article was submitted to Gut Endocrinology, a section of the journal Frontiers in Endocrinology Received: 01 April 2021 Accepted: 05 May 2021 Published: 18 May 2021 Citation: Lafferty RA, O’Harte FPM, Irwin N, Gault VA and Flatt PR (2021) Proglucagon-Derived Peptides as Therapeutics. Front. Endocrinol. 12:689678. doi: 10.3389/fendo.2021.689678 Specialty section: This article was submitted to Gut Endocrinology, a section of the journal Frontiers in Endocrinology Specialty section: This article was submitted to Gut Endocrinology, a section of the journal Frontiers in Endocrinology Received: 01 April 2021 Accepted: 05 May 2021 Published: 18 May 2021 Proglucagon-Derived Peptides as Therapeutics Initially discovered as an impurity in insulin preparations, our understanding of the hyperglycaemic hormone glucagon has evolved markedly over subsequent decades. With description of the precursor proglucagon, we now appreciate that glucagon was just the ﬁrst proglucagon-derived peptide (PGDP) to be characterised. Other bioactive members of the PGDP family include glucagon-like peptides -1 and -2 (GLP-1 and GLP-2), oxyntomodulin (OXM), glicentin and glicentin-related pancreatic peptide (GRPP), with these being produced via tissue-speciﬁc processing of proglucagon by the prohormone convertase (PC) enzymes, PC1/3 and PC2. PGDP peptides exert unique physiological effects that inﬂuence metabolism and energy regulation, which has witnessed several of them exploited in the form of long-acting, enzymatically resistant analogues for treatment of various pathologies. As such, intramuscular glucagon is well established in rescue of hypoglycaemia, while GLP-2 analogues are indicated in the management of short bowel syndrome. Furthermore, since approval of the ﬁrst GLP-1 mimetic for the management of Type 2 diabetes mellitus (T2DM) in 2005, GLP-1 therapeutics have become a mainstay of T2DM management due to multifaceted and sustainable improvements in glycaemia, appetite control and weight loss. More recently, longer-acting PGDP therapeutics have been developed, while newfound beneﬁts on cardioprotection, bone health, renal and liver function and cognition have been uncovered. In the present article, we discuss the physiology of PGDP peptides and their therapeutic applications, with a focus on successful design of analogues including dual and triple PGDP receptor agonists currently in clinical development. Keywords: proglucagon, glucagon, GLP-1, GLP-2, oxyntomodulin, diabetes, obesity, multi-agonist Take down policy Take down policy The Research Portal is Ulster University's institutional repository that provides access to Ulster's research outputs. Every effort has been made to ensure that content in the Research Portal does not infringe any person's rights, or applicable UK laws. If you discover content in the Research Portal that you believe breaches copyright or violates any law, please contact pure-support@ulster.ac.uk Download date: 24/10/2024 REVIEW published: 18 May 2021 doi: 10.3389/fendo.2021.689678 REVIEW published: 18 May 2021 doi: 10.3389/fendo.2021.689678 INTRODUCTION Received: 01 April 2021 Accepted: 05 May 2021 Published: 18 May 2021 While the gut hormones secretin and gastrin were discovered almost two decades earlier (1, 2), it was the extraction, isolation and puriﬁcation of insulin from canine pancreatic extracts in Toronto in 1921, that truly signiﬁes the advent of peptide-based therapeutics (3). Indeed, the ﬁrst clinical use of animal-derived insulin began the following year. Continued innovation has led to the production of longer-acting formulations (4), as well as biosynthetic, recombinant DNA human insulins in the 1980’s (5). In this respect, it is incredible to think that a century later, insulin remains a vital mainstay in the management of Type 1 diabetes mellitus (T1DM). PROGLUCAGON – DISCOVERY AND PROCESSING We now understand that proglucagon is expressed in both alpha-cells of the pancreatic islets (21, 22), as well as neuroendocrine L-cells (23), primarily located in the distal ileum and colon ( (24); Figure 1). However, the PGDP proﬁle is not identical in the pancreas and gut, due to differential post- translational processing of proglucagon by tissue-speciﬁc enzymes termed prohormone convertases (PC) ( (25); Figure 1). Broadly speaking, it is accepted that pancreatic alpha-cells mainly possess PC2, which cleaves dibasic Lys-Arg sites within proglucagon to generate glicentin-related pancreatic peptide (GRPP), glucagon, intervening peptide-1 (IP-1) and major proglucagon fragment (MPGF) ( (26, 27); Figure 1). In contrast, in the L-cell, proglucagon is cleaved by PC1/3 at Arg- Arg sites to yield glicentin, GRPP, oxyntomodulin (OXM), GLP- 1, intervening peptide-2 (IP-2) and GLP-2 ( (23, 26); Figure 1). It is important to note that these distinctions are not totally sacrosanct, with a degree of crossover existing. As such, recent evidence has highlighted that the gut is a possible extrapancreatic source of glucagon ( (28); Figure 2), while local intra-islet GLP-1 production has also been established in alpha cells, particularly in times of beta-cell stress (29). Moreover, it is now understood that proglucagon-containing neurons are located in the solitary nucleus of the medulla oblongata (30), which utilises PC1/3 in a similar fashion to the gut to generate PGDP’s in the central nervous system (CNS) ( (31); Figure 1). These PGDP’s and their therapeutic exploitation will be discussed in due course. As its name suggests, GLP-1 is related to the glucose-elevating hormone, glucagon. Indeed, a family of glucagon-related peptides exists, all of which are derived from differential processing of a common prohormone, proglucagon (8). Whilst glucagon and its hyperglycaemic actions were discovered in 1922 (9), its amino acid sequence was not elucidated until 1957 (10). Furthermore, proglucagon went undiscovered until the early 1980’s, when its cDNA was initially identiﬁed in anglerﬁsh (11, 12), with discovery of a proglucagon equivalent in rat (13, 14), hamster (15), cow and human several years later (16). These discoveries were made possible with the advent of lab-scale cDNA cloning techniques, which made it feasible to accurately predict amino acid sequences of proteins by decoding the nucleotide sequences of cloned recombinant cDNA copies of mRNAs. Such experiments highlighted that glucagon and several peptides with a high degree of sequence homology were encoded by this prohormone (11, 12). Citation: Lafferty RA, O’Harte FPM, Irwin N, Gault VA and Flatt PR (2021) Proglucagon-Derived Peptides as Therapeutics. Front. Endocrinol. 12:689678. doi: 10.3389/fendo.2021.689678 Lafferty RA, O’Harte FPM, Irwin N, Gault VA and Flatt PR (2021) Proglucagon-Derived Peptides as Therapeutics. Front. Endocrinol. 12:689678. doi: 10.3389/fendo.2021.689678 Although insulin therapy is often indicated in poorly controlled Type 2 diabetes mellitus (T2DM), this condition is more often managed with diet plus an array of medications that augment May 2021 \| Volume 12 \| Article 689678 Frontiers in Endocrinology \| www.frontiersin.org Proglucagon-Derived Peptides as Therapeutics Lafferty et al. remaining endogenous insulin production and function. Indeed, peptide-based therapeutics have become important tools in the management of T2DM, emulating the success of insulin in T1DM. In particular, enzymatically stable analogues, based on the endogenous incretin hormone glucagon-like peptide 1 (GLP- 1), are now widely prescribed second- and third-line agents for T2DM (6). Furthermore, orally-available inhibitors of the enzyme dipeptidyl peptidase-4 (DPP-4), which degrades incretins including GLP-1, have been increasingly prescribed since their approval in 2007 (7). sequence homology with anglerﬁsh proglucagon was high, isolation of the ﬁrst mammalian proglucagon from hamster unveiled organisational differences, with the 158 amino-acid mammalian precursor containing three PGDP arranged in tandem, namely glucagon and what the authors termed, glucagon-like peptides 1 and 2 (GLP-1 and GLP-2) (15). The biological importance of these carboxy-peptides was initially unclear. Through a combined approach employing immunoassays, immunohistochemistry and chromatography of tissue extracts, it was established that GLP-1 and GLP-2 coexisted with glucagon in pancreatic islet cells and with oxyntomodulin in intestinal L-cells, where they are present at vastly greater concentrations than islets (20). PROGLUCAGON – DISCOVERY AND PROCESSING Interestingly, anglerﬁsh islets were demonstrated to express two separate proglucagon peptides, meaning a hybrid approach was taken to identify cDNA encoding the 29 amino acid (aa), anglerﬁsh glucagon (11, 12). From there, cDNA encoding for previously sequenced proteins, glicentin and oxyntomodulin was uncovered (17, 18), with glucagon located within the middle portion of this sequence (11). However, the proposed proglucagon sequence exhibited unexpected C-terminal elongation, containing an additional 34-residue glucagon- related carboxyl-terminal peptide, which exhibited structural similarity with another previously sequenced hormone, glucose-dependent insulinotropic polypeptide (GIP) (11, 19). Further study of anglerﬁsh proglucagon led to the characterisation of a second proglucagon cDNA, derived from a different mRNA and gene which encoded glucagon. This shared signiﬁcant homology with mammalian glucagons, but also a second C-terminal glucagon-related peptide, again comprised of 34 residues with signiﬁcant sequence homology to glucagon (12). Frontiers in Endocrinology \| www.frontiersin.org GLUCAGON promotes efﬁcient metabolism of protein, lipids and carbohydrate (favouring glycolysis) (38). Conversely, hypoglycaemia following fasting, or exercise is the most potent stimulus for glucagon secretion [(39, 40); Figure 2]. production of cyclic adenosine monophosphate (cAMP) and subsequent activation of protein kinase A (PKA). PKA stimulates the synthesis of transcription factors including cAMP response element-binding protein (CREB) in the nucleus, a promoter of gene expression. Simultaneously, GCGR activation of phospholipase C (PLC) and subsequent increase in inositol 1,4,5-triphosphate (IP3), facilitates release of calcium ions from the endoplasmic reticulum to inﬂuence CREB-regulated transcription co-activator (CRTC2), which enhances CREB-dependent gene expression (42). Importantly, glucagon is rapidly inactivated in the circulation by enzymes, including DPP-4, to generate inactive glucagon (3–29) (48); Figure 2). The hyperglycaemic action of glucagon is well-established, being demonstrated as early as its discovery, with the hormone’s name reﬂecting this; glucagon – “the glucose agonist” (9). Hyperglycaemic actions of glucagon are mediated through promotion of glycogenolysis and gluconeogenesis in liver, whilst also inhibiting glycolysis and glycogenesis (41). Furthermore, in times of limited carbohydrate availability, glucagon promotes non-carbohydrate energy formation in the generation of lipids and ketone bodies or through the breakdown of fatty acids to acetyl-coenzyme A (42). Further research into the actions of glucagon has demonstrated a role in satiety, with acute administration in humans diminishing hunger and reducing food intake (43), whilst also stimulating energy expenditure and cardiac contractility (44, 45). While considered for many years as solely a consequence of insulin deﬁciency, in the 1970’s the “bihormonal hypothesis”, proposed by Roger Unger, highlighted the role of an imbalance in the complex interplay between glucagon and insulin in instigating diabetic hyperglycaemia (35). Indeed, the rationale behind this longstanding hypothesis inspired research into the development of dual pump systems, sometimes termed “dual- hormone artiﬁcial pancreas”. Such pumps are regulated by a glucose sensor to deliver insulin or glucagon, as necessary, from independent pumps and are thought to be possibly more efﬁcacious than insulin-only pumps (49), although none have successfully reached the clinic to date. We now understand that T2DM is characterised by elevated fasting glucagon levels (50), while glucose suppression following a glucose challenge is stunted (51). Furthermore, it has been suggested that postprandial hyperglucagonaemia and impaired glucagon response to hypoglycaemia are features of T1DM (52). GLUCAGON The 29 aa polypeptide hormone glucagon (Table 1) is the most widely recognised PGDP (9, 10), produced by PC2-mediated cleavage of proglucagon in pancreatic alpha cells ( (26, 27); Figure 1). Discovered shortly after insulin (9), glucagon and insulin are intrinsically linked, with the major metabolic actions of glucagon counteracting those of insulin (35). As such, insulin secretion from pancreatic beta-cells is stimulated largely by elevated glucose concentrations, reducing circulating glucose levels via inhibition of glycogenolysis and gluconeogenesis, accompanied by stimulation of glycogen synthesis in the liver (36). Furthermore, insulin stimulates glucose uptake via GLUT-4 translocation in adipose and muscle (37), which in turn Whilst work in anglerﬁsh provided an excellent starting point, particularly in highlighting the presence of these carboxy glucagon-related peptides (11, 12), it was the elucidation of the structure of mammalian proglucagon which truly sparked interest in proglucagon-derived peptides (PGDP’s). While May 2021 \| Volume 12 \| Article 689678 Frontiers in Endocrinology \| www.frontiersin.org 2 Proglucagon-Derived Peptides as Therapeutics Lafferty et al. A B FIGURE 1 \| A schematic overview of tissue-speciﬁc proglucagon processing in the gut/brain (A) and in the pancreas (B). The proglucagon gene, located on chromosome 2 and comprised of 6 exons, is transcribed to generate proglucagon messenger RNA (mRNA). Proglucagon mRNA is subsequently translated to yield the 158 residue, precursor protein, proglucagon. In enteroendocrine L-cells of the ileum and colon (A) proglucagon is processed by prohormone convertase 1/3 (PC1/3) to generate glicentin, oxyntomodulin, glucagon-like peptides-1 and -2 (GLP-1, GLP-2) and intervening peptide-2 (IP-2). Conversely, in pancreatic alpha-cells (B), post-translational modiﬁcation by prohormone convertase 2 (PC2) is responsible for the generation of the major proglucagon fragment (MPGF), glucagon, glicentin-related pancreatic polypeptide (GRPP) and intervening peptide-1 (IP-1). FIGURE 1 \| A schematic overview of tissue-speciﬁc proglucagon processing in the gut/brain (A) and in the pancreas (B). The proglucagon gene, located on chromosome 2 and comprised of 6 exons, is transcribed to generate proglucagon messenger RNA (mRNA). Proglucagon mRNA is subsequently translated to yield the 158 residue, precursor protein, proglucagon. In enteroendocrine L-cells of the ileum and colon (A) proglucagon is processed by prohormone convertase 1/3 (PC1/3) to generate glicentin, oxyntomodulin, glucagon-like peptides-1 and -2 (GLP-1, GLP-2) and intervening peptide-2 (IP-2). Conversely, in pancreatic alpha-cells (B), post-translational modiﬁcation by prohormone convertase 2 (PC2) is responsible for the generation of the major proglucagon fragment (MPGF), glucagon, glicentin-related pancreatic polypeptide (GRPP) and intervening peptide-1 (IP-1). Frontiers in Endocrinology \| www.frontiersin.org GLUCAGON There is some debate over the receptor interactions at play in some of these biological actions, for example: given that circulating glucagon concentrations rise following a period of fasting, its involvement in food reduction seems counter- intuitive, suggesting cross-reactivity with the GLP-1 receptor (GLP-1R) (42). In the context of this article, we will consider glucagon actions mediated through agonism of its own speciﬁc G protein-coupled receptor (GPCR) the glucagon receptor (GCGR). This receptor is widely expressed, particularly in the liver, but is also found in the adrenal glands, heart, adipose tissue, GIT, and pancreas (46, 47). Binding with the receptor activates adenylyl cyclase that leads to intracellular May 2021 \| Volume 12 \| Article 689678 Frontiers in Endocrinology \| www.frontiersin.org 3 Proglucagon-Derived Peptides as Therapeutics Lafferty et al. A B FIGURE 2 \| An overview of PGDP actions and secretion from pancreatic alpha-cells (A) and enteroendocrine L-cells (B)). A fall in circulating glucose concentration sees a reduction in intracellular adenosine triphosphate (ATP) levels and resultant closure of ATP-sensitive K+ channels to depolarise the plasma membrane and trigger the inﬂux of Ca2+ ions, the primary stimulus for glucagon release (A). Glucagon is subject to N-terminal dipeptide removal by dipeptidyl-peptidase 4 (DPP-4). Glucagon(1-29) agonises glucagon receptors (GCGR) to evoke protein kinase A (PKA) activation and subsequent mobilisation of cyclic adenosine monophosphate (cAMP). Enteroendocrine L-cells of the distal gut are an open-type cell, rich in chemoreceptors and respond to digestion products of dietary carbohydrate, free fatty acids (FFA) and amino acids (AA’s) to release a number of PGDP’s into circulation (B). Glicentin(1-69) is an agonist for GCGR, GLP-1R and GLP-2R, although with less afﬁnity than their primary hormonal ligands. Additionally, glicentin may serve as a precursor to glucagon in the gut, facilitated enzymatic degradation by enzymes such as carboxypeptidases-B and -E (CP-B, CP-E). Oxyntomodulin (OXM) is a dual agonist for GCGR and GLP-1R, but shows bias towards GLP-1R. It is cleaved by DPP-4 to yield inactive OXM(3-37). Bioactive glucagon-like peptide 1 (GLP-1(7-36)) agonises target GLP-1R to evoke PKA-mediated rises in cAMP, while activation of b-arrestin is also implicated in insulin secretion. DPP-4 cleaved GLP-1(9-36) is inactive. Glucagon-like peptide 2 (GLP-2) agonises target GLP-2R to evoke rises in PKA/cAMP. It is inactivated by DPP-4 to generate GLP-2(3-23). Enzymes are indicated by yellow boxes/arrows. Receptor interactions are indicated by dashed lines, with afﬁnity indicated by increasing thickness of the arrow. GLUCAGON Major tissues expressing receptors are also provided. A B B A FIGURE 2 \| An overview of PGDP actions and secretion from pancreatic alpha-cells (A) and enteroendocrine L-cells (B)). A fall in circulating glucose concentration sees a reduction in intracellular adenosine triphosphate (ATP) levels and resultant closure of ATP-sensitive K+ channels to depolarise the plasma membrane and trigger the inﬂux of Ca2+ ions, the primary stimulus for glucagon release (A). Glucagon is subject to N-terminal dipeptide removal by dipeptidyl-peptidase 4 (DPP-4). Glucagon(1-29) agonises glucagon receptors (GCGR) to evoke protein kinase A (PKA) activation and subsequent mobilisation of cyclic adenosine monophosphate (cAMP). Enteroendocrine L-cells of the distal gut are an open-type cell, rich in chemoreceptors and respond to digestion products of dietary carbohydrate, free fatty acids (FFA) and amino acids (AA’s) to release a number of PGDP’s into circulation (B). Glicentin(1-69) is an agonist for GCGR, GLP-1R and GLP-2R, although with less afﬁnity than their primary hormonal ligands. Additionally, glicentin may serve as a precursor to glucagon in the gut, facilitated enzymatic degradation by enzymes such as carboxypeptidases-B and -E (CP-B, CP-E). Oxyntomodulin (OXM) is a dual agonist for GCGR and GLP-1R, but shows bias towards GLP-1R. It is cleaved by DPP-4 to yield inactive OXM(3-37). Bioactive glucagon-like peptide 1 (GLP-1(7-36)) agonises target GLP-1R to evoke PKA-mediated rises in cAMP, while activation of b-arrestin is also implicated in insulin secretion. DPP-4 cleaved GLP-1(9-36) is inactive. Glucagon-like peptide 2 (GLP-2) agonises target GLP-2R to evoke rises in PKA/cAMP. It is inactivated by DPP-4 to generate GLP-2(3-23). Enzymes are indicated by yellow boxes/arrows. Receptor interactions are indicated by dashed lines, with afﬁnity indicated by increasing thickness of the arrow. Major tissues expressing receptors are also provided. Glucagon Therapeutics and Hypoglycaemia hypoglycaemic-induced coma and death. Rather than requiring a potentially lengthy wait for arrival of a qualiﬁed healthcare professional to perform an i.v. infusion, glucagon emergency kits simply involve reconstitution of glucagon powder, which can be injected into the patient’s leg or abdomen (32, 55). Moreover, a ready-to use autoinjector preparation termed “Zegalogue®” has recently gained FDA approval for management of hypoglycaemia (33), further improving ease of use. I.v. dextrose may then be required to prevent rebound hypoglycaemia (34), a potential consequence of the rapid in vivo inactivation of administered native glucagon (48). Given glucagon’s ability to rapidly mobilise glucose from tissue stores, GCGR agonism has found valuable application in countering severe hypoglycaemia in T1DM patients, an adverse consequence of insulin therapy (53). Mild-to-moderate hypoglycaemia is deﬁned as an event that can be self-treated, irrespective of symptom severity, or an asymptomatic blood glucose measurement of ≤3.9 mmol/L (54). It is usually managed via ingestion of rapidly absorbed carbohydrates, such as drinks or foods high in glucose, whereas severe hypoglycaemia requires immediate, emergency intervention (32). While intravenous (i.v.) infusion of dextrose is an option, it is now more common for patients or carers to possess an injectable glucagon preparation, which can be administered subcutaneously (s.c.) or intramuscularly (i.m.) (55). Such intervention is reliable and faster than the dextrose method, greatly reducing the risk of Longer-acting, DPP-4 resistant analogues are in development that may address the issue of rebound hypoglycaemia. Two such analogues are the fatty-acid incorporating, NNC9204-0043 currently listed at Novo Nordisk ((34); Table 1), and dasiglucagon, which employs several amino acid substitutions TABLE 1 \| Glucagon and related analogues in the management of hypoglycaemia in T1DM. Peptide Name Primary Sequence Development Stage Reference Native glucagon HSQGTFTSDYSKYLDSRRAQDFVQWLMNT s.c. & i.n. formulations approved (32–33) NNC9204-0043 HSQGTFTSDYSKYLDSKKAQEFVQ(2xOEG-gGlu-C18diacid)WLLNT Preclinical (Novo Nordisk) (33) Dasiglucagon HSQGTFTSDYSKYLD-X-ARAEEFVKWLEST Approved 2021Phase III (Zealand Pharma) (34) Amino acid sequences are provided in their single-letter abbreviation format. Modiﬁcations from native sequences are highlighted by red lettering. Current development stages are provided for each, as are holding companies (in brackets). “OEG-gGlu-C18 diacid” represents a fatty acid inclusion. “X” indicates an unnatural a-Aminoisobutyric acid residue. TABLE 1 \| Glucagon and related analogues in the management of hypoglycaemia in T1DM. Peptide Name Primary Sequence Development Stage Reference Native glucagon HSQGTFTSDYSKYLDSRRAQDFVQWLMNT s.c. & i.n. Glucagon Therapeutics and Hypoglycaemia While not entirely novel, having been in development since the 1990’s (34), the ﬁrst such product was only approved in 2019 (58). Termed Baqsimi®, the ready-to-use i.n. formulation has been proposed to lead to resolution of hypoglycaemia up to four times faster than injectable glucagon kits (59). The single-use preparation simply requires the user to administer one spray into either nostril, which is reported to deliver a 3 mg dose of glucagon (57). Glucagon Therapeutics and Hypoglycaemia formulations approved (32–33) NNC9204-0043 HSQGTFTSDYSKYLDSKKAQEFVQ(2xOEG-gGlu-C18diacid)WLLNT Preclinical (Novo Nordisk) (33) Dasiglucagon HSQGTFTSDYSKYLD-X-ARAEEFVKWLEST Approved 2021Phase III (Zealand Pharma) (34) Amino acid sequences are provided in their single-letter abbreviation format. Modiﬁcations from native sequences are highlighted by red lettering. Current development stages are provided for each, as are holding companies (in brackets). “OEG-gGlu-C18 diacid” represents a fatty acid inclusion. “X” indicates an unnatural a-Aminoisobutyric acid residue. May 2021 \| Volume 12 \| Article 689678 Frontiers in Endocrinology \| www.frontiersin.org 4 Proglucagon-Derived Peptides as Therapeutics Lafferty et al. release inﬂuenced by the composition of each meal ingested; in particular, meals that are rich in fat and carbohydrate are known to be the primary physiological stimulus for GLP-1 secretion (78–81). Additionally, GLP-1 secretion can be triggered, not only by mixed nutrient load, but also via individual nutrients and bile acids. For example, oral administration of glucose alone has been shown to stimulate GLP-1 secretion in humans (82), as well as amino acids such as glutamine (83). Sodium-glucose transporter 1 (SGLT1) plays a glucose-sensing role on the L-cell surface, and although a contributor, is thought to play a lesser role than glucose transporters (GLUT) in relation to GLP-1 release (84). GLP-1 secretion is biphasic, with an early phase occurring 10- 15 min after ingestion of nutrients and a second, more prolonged phase occurring 30-60 min after ingestion (81). Given the distal location of L-cells in the gut, it is unlikely that direct nutrient contact with these cells can be the sole mechanism initiating GLP-1 secretion. Thus, the autonomic nervous system, in particular the vagus nerve (which innervates a signiﬁcant portion of the gut), is thought to play a role in this early phase of release, with nutrient content being more important for the second phase (85). to infer improved stability [(56); Table 1]. The former has only shown promise in in vitro settings (34), whereas dasiglucagon has very recently gained FDA approval in T1DM (56). Indeed, dasiglucagon is the active component of Zegalogue, and beyond application in preﬁlled injector pens, is currently in phase 3 trials as a subcutaneous infusion for treating congenital hyperinsulinaemia, and in phase 2 trials as part of a bihormonal artiﬁcial pancreas pump system alongside insulin (57). Glucagon emergency kits have been further improved with the development of intranasal (i.n.) glucagon. GLUCAGON-LIKE PEPTIDE-1 While GLP-1 (9–31, 35–39) is considered a weak antagonist of beta-cell GLP-1R (88), there is evidence suggesting that this metabolite may reduce inﬂammation in cardiac tissue following myocardial infarction (89). GLP-1 (9– 31, 35–39) has also been demonstrated to promote cardiac glucose uptake similar to GLP-1 (7–31, 35–39)-amide (90), so the descriptor “inactive” may not be entirely accurate. Additionally, a recent study suggests that GLP-1 (9–31, 35– 39)-amide may indirectly inﬂuence glycaemia through antagonism of GCGR on alpha-cells to inﬂuence the glucagonostatic effects of GLP-1 (91). However, the implications of any GLP-1 (9–31, 35–39) effects on glycaemia are thought to be relatively inconsequential in comparison to GLP-1 (7–31, 35–39)-amide (92). are well established (105–108). Culture of DPP-4 resistant, N- acetyl-GLP-1 (Table 2) with pancreatic ductal-cells has also been shown to induce expression of genes indicative of a transition to a beta-cell like phenotype (61, 109), but translation to humans requires further study. Advances in cell-lineage tracing technology have seen the development of transgenic animal models that employ ﬂuorescently tagged alpha- or beta-cells to identify such islet cell transitioning events in the in vivo setting (110, 111). Recent studies have shown that administration of liraglutide to such mice with diabetes can prevent beta- to alpha- cell transdifferentiation (112), whilst also actively driving alpha- to beta-cell conversion to help restore beta-cell mass (113–115). GLP-1 also inhibits glucagon secretion and exerts additional extra-pancreatic actions of therapeutic value including inhibition of gastric acid secretion and gastric emptying (Figure 3), which help reduce post-prandial spiking of blood glucose by slowing transit of nutrients from the stomach to the small intestine (81). In addition to locally produced GLP-1 (116), GLP-1 crosses the blood-brain barrier to agonise GLP-1R within hypothalamic CNS centres, where ingestive behaviour and satiety is dictated [(117); Figure 3]. Increased satiety reduces food intake, with resultant weight loss being an important beneﬁt in overweight or obese-T2DM patients. Moreover, the widespread tissue presence of GLP-1R has witnessed new physiological roles for GLP-1 beyond glycaemia and satiety such as cardioprotection ( (118, 119); Figure 3), enhancing bone mass and strength in preclinical models of T2DM (120), and is thought to play an important role in enhancing cognition ( (121); Figure 3). Additionally, a possible role for GLP-1 in resolution of hepatic steatosis (122– 124) through reduction in fatty acid accumulation by activation of both macroautophagy and chaperone-mediated autophagy (125), has attracted much interest. GLUCAGON-LIKE PEPTIDE-1 The next PC1/3-mediated (Figure 1), L-cell-derived PGDP to be discussed has become a mainstay of T2DM management, representing one of the principal modern success stories of peptide therapeutic development. GLP-1 is a 29-residue (Table 2), gut-derived incretin hormone (77). GLP-1 is released post-prandially from L-cells [(77–79); Figure 2], with The biologically active forms of GLP-1 are GLP-1 (7–36)- amide and GLP-1 (7–37) which are equipotent in terms of their incretin effects [(60); Table 2 and Figure 2]. However, they do not circulate equally, with GLP-1 (7–36)-amide accounting for ~80% (20, 82). Both forms of circulating GLP-1 are subject to rapid N-terminal degradation by DPP-4 (86, 87), cleaving after TABLE 2 \| GLP-1-based therapeutic peptides. Peptide Name AA Sequence Development Stage Reference GLP-1(1-37) HDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG N/A (60) GLP-1(1-36) HDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGR N/A (60) GLP-1(7-36) HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR N/A (60) N-acetyl GLP-1(7-36) Ac-HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR Preclinical (61) Exendin-4 (Exenatide) HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS Daily - Approved 2005, Weekly- Approved 2014 (d/c 2021), Phase II-AD/PD (AstraZeneca) (62–63) Lixisenatide HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPSKKKKKK Approved 2016-T2DM, Phase II- AD/PD (Sanoﬁ) (64) Liraglutide HAEGTFTSDVSSYLEGQAAK(Glu-hexadecanoyl-Glu-OH)EFIAWLVRGRG Approved 2010-T2DM, Approved 2019-Obesity, Phase II-AD/PD, CVD (Novo Nordisk) (65, 66) Albiglutide HGEGTFTSDVSSYLEGQAAKEFIAWLVKGR-{Human Albumin} Approved 2014 (d/c 2017)-T2DM, Phase II-CVD (GlaxoSmithKline) (67) Dulaglutide HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGG{Human IgG4- Fc} Approved 2014-T2DM, Phase II- CVD, Phase II-AD/PD (Eli Lilly) (68) Semaglutide HXEGTFTSDVSSYLEGQAAK(Glu-mPEG-17-carboxyheptadecanoyl-Glu-OH) EFIAWLVRGRG Approved 2017- T2DM, Filed 2021-Obesity, Phase II-CVD (Novo Nordisk) (69, 70) Oral Semaglutide (Rybelsus) HXEGTFTSDVSSYLEGQAAK(Glu-mPEG-17-carboxyheptadecanoyl-Glu-OH) EFIAWLVRGRG/SNAC Approved 2020-T2DM (Novo Nordisk) (71–72) D-Ala8GLP-1(Lys37) - pentasaccharide H(DA)EGTFTSDVSSYLEGQAAKEFIAWLVKGRK(Pentasaccharide) Preclinical (73, 74) [Gln28]exenatide HGEGTFTSDLSKQMEEEAVRLFIEWLKQGGPSSGAPPPS Preclinical (75) (Val8)GLP-1(GluPAL) HVEGTFTSDVSSYLEGQAAKEFIAWLVK(-Glu-PAL)GR Preclinical (76) TABLE 2 \| GLP-1-based therapeutic peptides. Amino acid sequences are provided in their single-letter abbreviation format. Modiﬁcations from native sequences are highlighted by red lettering. Current development stages, associated condition and holding companies (in brackets) are provided (where available) for each. FDA approval dates, and discontinuation date if applicable, are also provided where appropriate. “SNAC” represents formulation with sodium N-[8-(2-hydroxybenzoyl) amino caprylate, an absorption aid. “Ac” represents an N-terminal acetylation, “hexadecanoyl-Glu” and “carboxyheptadecanoyl-Glu” represent fatty acid attachments. “mPEG” indicates mini-polyethylene glycol addition. “PAL” indicates the addition of a palmitic acid chain. A “D” preﬁx before a residue indicates inclusion of the enantiomer for the naturally-occurring L form of the residue. May 2021 \| Volume 12 \| Article 689678 Frontiers in Endocrinology \| www.frontiersin.org Frontiers in Endocrinology \| www.frontiersin.org 5 Proglucagon-Derived Peptides as Therapeutics Lafferty et al. Ala2 to generate GLP-1 (9–31, 35–39) or (9–31, 35–40) metabolites (86, 87). Frontiers in Endocrinology \| www.frontiersin.org GLUCAGON-LIKE PEPTIDE-1 The GLP-1R is a family B, or secretin-like G-protein coupled receptor (GPCR) (93). A structurally identical GLP-1R has been identiﬁed in various tissues, for example: pancreatic tissue (alpha-, beta-, delta-cells), stomach, and intestine, as well as CNS regions including the hypothalamus and brainstem ( (81, 93); Figure 2). Binding of GLP-1 to its target-receptor on the beta-cell surface leads to activation of several intracellular transduction pathways (Figure 2). The hormone augments insulin secretion, mainly via stimulation of intracellular cAMP-mediated events and promotes glucose-induced biosynthesis of insulin, resulting in replenishment of insulin stores within beta-cells and reducing cell exhaustion (81, 94–96). Conversely, GLP-1 is known to suppress glucagon secretion from alpha-cells ( (97); Figure 3). The mechanisms behind this have been hotly debated, with it claimed to be an indirect effect mediated through increased insulin or somatostatin secretion (98, 99), while some have indicated the effect is more direct (100), especially given the presence, albeit at low expression (~10%), of GLP-1R on alpha-cells (101). Beyond this, activation of pancreas duodenum homeobox 1 (Pdx-1), a transcription factor essential for pancreatic development and beta-cell function (activated downstream from GLP-1R via cAMP activation), is thought to be a shared inﬂuence in these three processes (102). Prevention of beta-cell exhaustion may indirectly prevent cell death, but GLP-1 also directly inﬂuences proliferation by a number of proposed pathways including phosphatidylinositol 3-kinase (PI3-K) mediated rises in extracellular signal-related kinase (ERK) 1/2 and p38 mitogen-activated protein kinase (MAPK), as well as Pdx-1 (103). In keeping with this, exendin-4 has been shown to have no effect on proliferation or inhibition of apoptosis in beta-cell speciﬁc, Pdx-1 knockout (KO) mice (104). Since entry into the clinic, research on GLP-1 has continued, GLP-1 Therapeutics and Diabetes GLP 1 Therapeutics and Diabetes GLP-1 was not the ﬁrst incretin hormone to be discovered, with GIP being identiﬁed almost two decades previously in 1969 (126). However, with a proposed role for GIP in development of obesity coupled with a loss of insulinotropic effect in T2DM (127), therapeutic application did not follow such a straightforward path. Thus, when a preservation of the insulinotropic effects of GLP-1 in obesity was established (128), excitement surrounding the possible therapeutic application of this newly discovered incretin hormone began to grow. Furthermore, direct comparisons of analogues of these two incretins often resulted in more favourable outcomes for GLP- 1 compared to GIP (129). Nonetheless, current evidence regarding GIP-based therapy looks more promising in T2DM once glycaemic control has been re-established (130). This is perhaps evident with new compounds being developed that operate through combined activation of GLP-1R and GIPR (130), as discussed in more detail below. Since entry into the clinic, research on GLP-1 has continued, unveiling new mechanisms behind the various beneﬁts of GLP- 1R agonists, as well as possible new applications in other conditions. With regards to diabetes, it is now well established that chronic administration of GLP-1R mimetics not only enhances insulin secretion but also positively inﬂuences overall islet function, restoring normal morphology in even severe models of diabetes (105). Additionally, the ability of exogenous GLP-1R mimetics to maintain and promote beta-cell mass through reductions in apoptosis and increases in proliferation Initial therapeutic investigations into GLP-1 were promising, highlighting that delivery of exogenous, native peptide had the ability to improve overall glycaemia, insulin sensitivity, beta-cell function and reduce both appetite and food intake when administered by continuous s.c. infusion over a 6 week period May 2021 \| Volume 12 \| Article 689678 6 Lafferty et al. Proglucagon-Derived Peptides as Therapeutics FIGURE 3 \| An overview of the biological consequences for agonism of target receptors of major PGDP’s, namely glucagon receptor (GCGR) and glucagon-like peptide-1 and -2 receptors (GLP-1R, GLP-2R). Organ-speciﬁc actions are provided with arrows indicating up or downregulation of speciﬁc effects to highlight the therapeutic potential for multiagonism in relation to PGDP’s. As indicated by the key, the colour of arrow indicates the receptor interactions responsible. “GFR” indicates glomerular ﬁltration rate. FIGURE 3 \| An overview of the biological consequences for agonism of target receptors of major PGDP’s, namely glucagon receptor (GCGR) and glucagon-like peptide-1 and -2 receptors (GLP-1R, GLP-2R). Frontiers in Endocrinology \| www.frontiersin.org GLP-1 Therapeutics and Diabetes Organ-speciﬁc actions are provided with arrows indicating up or downregulation of speciﬁc effects to highlight the therapeutic potential for multiagonism in relation to PGDP’s. As indicated by the key, the colour of arrow indicates the receptor interactions responsible. “GFR” indicates glomerular ﬁltration rate. in patients with diabetes (131). Moreover, tachyphylaxis was not reported and the side-effect proﬁle was favourable (131). However, due to rapid inactivation by DPP-4 (132), continuous infusion was required, making it unsuitable for regular use in a “real world” setting. the peptide was proven to be a potent agonist for mammalian GLP-1R (62), effectively bringing about GLP-1R-mediated beneﬁts on glycaemia, body weight and appetite (133). Importantly, the substitution of Ala2 with Gly2 in exendin-4 conferred resistance to DPP-4, while further sequence variations rendered the peptide less susceptible to ectopeptidases like neprilysin (NEP) (134). Studies in anaesthetised pigs has shown that GLP-1 clearance involves multiple organs including hepatic, peripheral and renal extraction, whereas exendin-4 is subject solely to glomerular ﬁltration, which also appears to be With the discovery of exendin-4, an unexpected GLP-1R mimetic isolated from the saliva of the Gila monster lizard (Heloderma suspectum) (62), the tide began to turn. The ﬁrst 30 residues of this 39 aa peptide demonstrated 53% sequence identity with human GLP-1 (Table 2), but despite such variance, May 2021 \| Volume 12 \| Article 689678 Frontiers in Endocrinology \| www.frontiersin.org 7 Proglucagon-Derived Peptides as Therapeutics Lafferty et al. up to two-fold slower than native GLP-1 (134). This results in an in vivo action of ~5 hours (63), allowing for twice daily administration as opposed to continuous infusion. Synthetic exendin-4 reached full approval for therapeutic use in humans in 2005 (Byetta™), being prescribed under the generic trade name “exenatide” and has become a highly inﬂuential and widely prescribed second- and third-line agent in T2DM, generally following failure of metformin or metformin/sulphonylurea combination (135). Indeed, oral DPP-4 inhibitors, such as sitagliptin, were approved as second-line agents in 2007 (7), while a plethora of additional GLP-1 mimetics have since gained regulatory approval for diabetes in addition to exenatide, namely the longer-acting mimetics: liraglutide, semaglutide, albiglutide and dulaglutide (Table 2). In contrast, attempts to discover suitable bioactive small molecule agonists of GLP-1R have failed, despite considerable efforts, due to poor potency and allosteric alteration of receptor conformation (136, 137). Cardiovascular and Renal Beneﬁts The growing strength of the cardiovascular and renal beneﬁts of established GLP-1 mimetics add another string to their bow in the management of T2DM, with cardiovascular disease (CVD) being the number one cause of death in patients with T2DM (147). As demonstrated by long-term prospective cardiovascular outcomes trials (CVOTs), which have reported over the last four years, liraglutide (LEADER), semaglutide (SUSTAIN-6), albiglutide (HARMONY OUTCOMES) and dulaglutide (REWIND) have all shown signiﬁcant reductions in composite cardiovascular outcomes [(64, 119, 148); Table 2], indicating they may be the agents of choice when macrovascular complication risk is high in T2DM patients. These longer- acting GLP-1R mimetics elicit more favourable cardiovascular outcomes than shorter-acting agents like exenatide or its analogue lixisenatide (EXSCEL and ELIXA), which demonstrated non-inferiority, but no obvious cardiovascular beneﬁt [(64, 119, 148); Table 2]. Additionally, proposed renal beneﬁts of SGLT2 inhibitors have seen trials such as “DECLARE- TIMI 58” report reduced rates of hospitalisation due to heart failure in dapagliﬂozin-treated groups of T2DM patients (148). Thus, given the exploration of exenatide and dapagliﬂozin in the DURATION-8 and ENERGIZE trials (144–147), it may stand to reason that such a combination may be studied in relation to CVD, perhaps with a more favourable GLP-1R mimetic than exenatide. Indeed, the phase III FLOW trial is currently recruiting patients to assess the renoprotective actions of semaglutide. Thus, we await the results of this trial to determine whether semaglutide may be the GLP-1R mimetic of choice in this regard (149). GLP-1 Therapeutics and Diabetes There is also increasing interest in the therapeutic potential of combining currently available GLP-1R mimetics (Table 2) with other currently prescribed antidiabetic drugs. The combination of exenatide with the sodium–glucose co-transporter 2 (SGLT2) inhibitor, dapagliﬂozin, was investigated in the DURATION-8, phase III clinical trial which demonstrated a degree of synergy between the two agents, with improvements in short- and long- term glycaemia and weight loss exceeding either agent alone (144). Moreover, a 2-year follow-up demonstrated long-term efﬁcacy of this combination (145). An additional phase II trial, ENERGIZE, has sought to identify the mechanism behind the apparent synergy (146), the ﬁndings of which may inﬂuence whether such a combination is advanced further. Frontiers in Endocrinology \| www.frontiersin.org Bone Fragility d b f g y Increased bone fragility is a further complication associated with diabetes, with the aetiology suspected to be due to an increase in porosity of bone, impacting on bone quality (173). Bone fragility also appears to be a feature in both T1DM and T2DM (174–176). Like cardiovascular complications, effects on bone have the potential to limit physical activity in T2DM patients. Furthermore, a role for endogenous GLP-1 in the development of diabetes-associated bone fragility has been identiﬁed, with GLP-1R KO mice presenting with reduced bone mass through increased osteoclast activity (177, 178). Given the implication of GLP-1R involvement in the aetiology of bone fragility in diabetes, research has explored the possibility of GLP-1R agonist or DPP-4 inhibitor use in the management of the condition with favourable outcomes (175, 179). Exenatide has been shown to enhance bone strength by increasing trabecular bone mass, bone formation and trabecular microarchitecture, whilst also improving collagen maturity in rodent models of diabetes (180, 181). Similarly, liraglutide signiﬁcantly prevented deterioration of the quality of the bone matrix in a streptozotocin-induced, rodent model of T1DM (175). Importantly, GLP-1 is not the only incretin involved in the pathogenesis of bone fragility in diabetes, with single GIP receptor (GIPR) KO and dual GLP-1R/GIPR KO mice presenting with enhanced bone fragility (182, 183). Indeed, the unimolecular GIPR/GLP-1R/GCGR agonist, [D-Ala2]GIP–Oxm (Table 4), signiﬁcantly improved bone strength and mass at both organ and tissue levels in leptin receptor-deﬁcient, ob/ob obese diabetic mice (184). Possible translation of these ﬁndings from animals to humans is still required. Importantly, evidence suggests that the beneﬁcial effects of GLP-1 in relation to cognition may be independent from glycaemic improvement, with a study comparing metformin and the GLP-1 analogue (Val8)GLP-1(GluPAL) demonstrating that only the latter reversed memory impairment in DIO mice (76). This hypothesis is supported by the ﬁnding that GLP-1R agonists have also shown neuroprotective effects in non-diabetic patients with Alzheimer’s (AD) or Parkinson’s disease (PD) (161, 162). Long-term potentiation (LTP) of synaptic activity, the cellular correlate of memory (163), is impaired in diabetes. Liraglutide administration reversed diabetes-related LTP blockade and actively promoted LTP formation in DIO mice (157, 158), while rescuing hippocampal LTP loss in an ob/ob murine model of obesity-diabetes (164). Obesity Beyond glucose homeostasis, exciting research has highlighted extra-pancreatic beneﬁts and new applications for established GLP-1 mimetics, many of which are exciting prospects. For example, despite the enormous upsurge in the incidence of obesity and associated complications including T2DM (138), existing drug therapies for obesity are grossly insufﬁcient, with bariatric surgery being far more effective (139). Against this background, in 2019 liraglutide became the ﬁrst GLP-1 analogue approved by the FDA, EMA and MHRA as a treatment option for obesity (65). Importantly, while glycaemic improvements undoubtedly inﬂuence weight loss, pharmacokinetic investigation in human participants suggested the effects of liraglutide on weight loss are primarily mediated through increased energy expenditure (66). Prior to regulatory approval, the “SCALE”, phase III trials demonstrated a sustained 2-year weight loss with liraglutide treatment as an adjunct to diet and exercise in non-diabetic participants (140, 141), strengthening the argument that effects are largely independent of glycaemic modulation. Additionally, 3-year follow-up demonstrated that liraglutide delayed diabetes development in patients with pre-diabetes, taking almost 3 times longer in patients receiving liraglutide (142). Given the successful application of liraglutide in this regard and the scale of the obesity problem, other GLP-1R mimetics are beginning to be touted as treatment options for obesity. Indeed, a phase III clinical programme assessing efﬁcacy and safety of once-weekly semaglutide (SUSTAIN) in T2DM was completed recently for s.c. semaglutide, manifesting a substantial average weight loss of 14.9% (-15.3 kg) following 68 weeks treatment (69). Additionally, a direct comparison between liraglutide and semaglutide indicated superior weight loss was attained with the latter (143). FDA approval has now been sought for semaglutide use in obesity, meaning we may be on the verge of witnessing a new treatment option available for obesity that rivals bariatric surgery. Cognition, Alzheimer’s, and Parkinson’s Disease Vascular deterioration in T2DM can also be linked to cognitive impairment, with growing evidence highlighting cross-sectional and prospective associations between T2DM and cognitive impairment and diminished memory and executive function (150). Clinical studies have concluded that T2DM is a signiﬁcant risk factor that can double the likelihood of developing dementia (151). It appears that a loss of insulin sensitivity in the brain (152), coupled with impaired insulin function (153), results in impaired growth factor secondary messenger cascades that are vital for cell growth, repair and synaptic function (154). Obesity GLP-1 receptor mimetics such as May 2021 \| Volume 12 \| Article 689678 Frontiers in Endocrinology \| www.frontiersin.org 8 Proglucagon-Derived Peptides as Therapeutics Lafferty et al. exendin-4 or liraglutide can reverse insulin desensitisation in the brain (155, 156). Key biomarkers for cognitive impairment such as phosphorylation of protein kinase B (AKT) and glycogen synthase kinase-3beta (GSK-3B), were reduced by liraglutide administration in diabetic rats in a time-dependent manner (153). In more practical terms, exendin-4 administration in a diet-induced obese (DIO) model reversed impaired memory formation in mice (157) and liraglutide normalised object recognition memory impairment in a similar model (158). Similar ﬁndings have been observed with DPP-4 inhibitors (159), although it is important to note that other gut hormones, particularly GIP (157), are also implicated here. Additionally, similar to CVD (145, 146), it appears that the combination of GLP-1 mimetic with SGLT-2 inhibitor may too be beneﬁcial with regards to cognition, with DIO/STZ- mice receiving liraglutide/SGLT-2 combination therapy presenting with improved recognition and hippocampal morphology (160). improvement in daily activity was apparent between treatment or placebo (171), although some scores of cognitive function were improved by liraglutide. Despite such disappointment, interest in GLP-1R mimetics in relation to cognitive function has not been perturbed, with a number of phase II trials recruiting in 2020 to study currently available GLP-1R mimetics in AD and PD (172). Notably, a common theme of these trials is an adjustment of treatment demographic towards patients with relatively recently diagnosed AD/PD (172). Frontiers in Endocrinology \| www.frontiersin.org Bone Fragility d b f While the close relation between GLP-1 and insulin signalling is undoubtedly important in cognition, it is crucial to highlight that beyond this mechanism, GLP-1R mimetics upregulated several neuroprotective growth factors such as: insulin-like growth factor 1 (IGF-1) (165), brain-derived neurotrophic factor (BDNF) (166), glia-derived neurotrophic factor (GDNF) (164), as well as vascular endothelial growth factor (VEGF) (157, 158). Indeed, preclinical work in rodents has illuminated both the associations between cognitive decline in AD/PD and T2DM, whilst implicating the potential of GLP-1R activation in curbing such decline (167). As such, exenatide was employed in small- scale, proof of concept, human trials in PD patients, with these trials of <100 participants indicating exenatide treatment elicited improved scores in tests of cognitive function over the course of 12 months treatment (168, 169). Moreover, a further 12 months after study conclusion, those patients receiving exenatide still achieved signiﬁcantly improved cognition scores than those receiving placebo (170). With such promising results, it is unsurprising that larger scale trials were conducted, such as the phase II, ELAD trial (171), which employed liraglutide in patients with moderate AD and associated dementia. Outcomes were disappointing, with it announced in late 2020 that no difference in cerebral glucose metabolic rate or GLUCAGON-LIKE PEPTIDE-2 The discovery of GLP-1 and GLP-2 occurred simultaneously following the cloning of cDNAs and genes encoding mammalian proglucagon in the early 1980s, with experiments unveiling the sequences of two novel glucagon-like peptides (15, 16). At that time, the biological functions had not been described for either hormone, with the insulinotropic actions of GLP-1 reported in 1987 (96). This delay was due to the lack of bioactivity of GLP-1 (1–37) (203), which hampered progress until the truncated peptide GLP-1 (7–36)-amide was uncovered (204). Perhaps, as a result of subsequent research focusing on the exciting prospect of exploiting GLP-1 as a potential antidiabetic agent, GLP-2 based research may be considered somewhat less intense, with the biological action as a growth promoter in gut not being uncovered until almost a decade after actions of GLP-1 (205). The development of the ﬁrst GLP-1/GLP-2 secreting GLUTag cell-line represents a starting point in the elucidation of the biological function of GLP-2. This cell line was produced via the creation of a transgenic mouse model with GLP-1/2 secreting tumours in the colon, from which L-cells could be extracted and immortalised (206). An observation was made that these animals all exhibited marked enlargement of the small bowel following tumour-induction, inspiring the hypothesis that a PGDP secreted by these tumours must have been responsible for the intestinotrophic activity (205). Interestingly, Bloom had reported the ﬁrst enteroglucagonoma patient with small intestinal villous hypertrophy, malabsorption, as well as colonic and jejunal stasis some 20 years earlier (207). However, the question remained as to which hormone, or hormones, were responsible. Initially, the intermediary peptide, glicentin, was identiﬁed to elicit intestinotrophic action (208). However, subsequent administration of synthetic GLP-2 into mice indicated that GLP-2-mediated increases in small bowel weight surpassed those seen with glicentin (209), making it the more likely instigator. In addition to this novel delivery method, there is growing interest in development of oral GLP-1 therapies, with preclinical data now describing bioactivity of orally delivered exendin-4 (198, 199), albeit requiring a considerably larger dose than intraperitoneal injection in mice. Most notable is a novel formulation of semaglutide that makes use of an absorption enhancer, sodium N-(8-[2-hydroxylbenzoyl] amino) caprylate (SNAC), designed to protect peptides from proteolytic degradation and promote absorption across the gastric mucosa [(71); Table 2]. Phase II trials comparing oral to s.c. Polycystic Ovary Syndrome Polycystic Ovary Syndrome There is increasing evidence in support of incretin-analogue use in polycystic ovary syndrome (PCOS) (185), an endocrine disorder which greatly impacts fertility in women, with over 10% of women of reproductive age affected by the condition (186). PCOS is a metabolic disorder that has overlap with T2DM, with patients often being overweight, and presenting with symptoms such as severe insulin resistance, hyperinsulinaemia and dyslipidaemia (187). The interrelation between PCOS and T2DM is further highlighted by the ability of bariatric surgery, speciﬁcally Roux-en-Y bariatric surgery (RYGB), to totally ameliorate both T2DM and PCOS (188, 189). Moreover, incretin function has been shown to be impaired in PCOS (187), thus application of GLP-1 mimetics in this condition is a hypothesis built on ﬁrm physiological reasoning. Although in May 2021 \| Volume 12 \| Article 689678 Frontiers in Endocrinology \| www.frontiersin.org 9 Proglucagon-Derived Peptides as Therapeutics Lafferty et al. -6.4 kg/-7.2%) (71). This therapeutic has recently gained FDA approval following successful phase III trials (PIONEER-7) in T2DM patients and provides signiﬁcantly better improvements in glycated haemoglobin (HbA1c) than sitagliptin in T2DM (200). Like previously available oral antidiabetics (7), oral semaglutide is taken once-daily as a tablet formulation, being prescribed under the brand name Rybelsus® (201). Moreover, as part of the PIONEER trial program, oral semaglutide was studied in patients with renal impairment and demonstrated favourable outcomes (202), possibly indicating that like s.c. semaglutide there was cardiovascular beneﬁt (118). However, when outcomes were assessed upon completion of PIONEER-6 non-inferiority compared to placebo was evident (72), but there was no obvious cardiovascular beneﬁt. These new ﬁndings are highly relevant and should lead to greater patient acceptability and compliance in treatment of T2DM and other disorders, as compared to traditional injection route for peptide therapies. relative infancy compared to application in T2DM, the study of application of GLP-1 mimetics in PCOS has been overwhelmingly positive (190). Liraglutide was shown to normalise irregular menstrual bleeding in PCOS patients (191), whilst improving conception rates when used at low dosage in combination with metformin (192). Indeed, it has been suggested that in obese PCOS patients with concurrent insulin resistance, GLP-1 analogues may be a better treatment option than metformin (193). Possible application of PGDPs in female fertility is worthy of further exploration. Innovations in Formulation and Delivery of GLP-1 Therapeutics G p Since the approval of exendin-4 for T2DM, increasingly longer acting formulations of GLP-1 analogues have been developed. The ﬁrst, liraglutide, a mammalian GLP-1 analogue employing conjugation to a palmitic acid chain via a linker coupled to the Lys26 residue was approved in 2010 [(194); Table 2]. This modiﬁcation increased half-life to ~12 h, through promoting non-covalent binding to albumin and reduced renal clearance, permitting once daily administration (195). Indeed, further longer-acting analogues were developed employing several strategies. The conjugation of the native GLP-1 analogue, D- Ala8GLP-1(Lys37), to an antithrombin III (ATIII)-binding pentasaccharide, known as CarboCarrier®, produced a peptide with potential for once-weekly dosing [(73, 74); Table 2], while a once-weekly exenatide preparation (Bydureon™) which employs microspheres to form a slowly released, peptide-depot gained regulatory approval in 2014 [(196); Table 2]. Additionally, the once weekly preparations albiglutide and dulaglutide employ covalent interactions to attach the peptide to human albumin or a tail fragment of an IgG 4 antibody respectively, which impedes clearance (67, 68), while semaglutide achieves the same pharmacokinetic proﬁle with non-covalent interaction with albumin (70). Such advancement has continued with a once- monthly, hydrogel preparation utilising the analogue [Gln28] exenatide currently undergoing development (75), while a novel osmotic minipump, termed Itca 650, is currently in phase III clinical trials (FREEDOM-1) (197). This pump administers a constant infusion of exenatide following subcutaneous implantation, reported to last for up to 12 months before requiring replacement (197). Frontiers in Endocrinology \| www.frontiersin.org GLUCAGON-LIKE PEPTIDE-2 semaglutide in diabetes management revealed comparable improvements in glycaemia when compared to placebo, but notably oral treatment attained slightly greater weight loss over the 26 week study (-6.9 kg/-7.6%, compared to May 2021 \| Volume 12 \| Article 689678 Frontiers in Endocrinology \| www.frontiersin.org 10 Proglucagon-Derived Peptides as Therapeutics Lafferty et al. ability to manage the condition and reduce the need for PN was highly sought after. As their name suggests, both GLP hormones are closely related with both being synthesised by the action of PC1/3 and secreted from intestinal L-cells of the distal gut (Figure 1) (25, 210). Following liberation from proglucagon, the 33 residue GLP-2 is secreted post-prandially in a biphasic fashion from nutrient-sensing L-cells (Figure 2), particularly in response to carbohydrates and lipids contained within luminal contents [(211); Figure 2]. Notably, the distal location of these cells indicates a neural pathway must be involved, given plasma GLP-2 levels (along with other L-cell-derived hormones) are shown to rise rapidly following ingestion (212). In support of GLP-2 use in SBS, endogenous levels have been shown to rise following excision of bowel (220), while preclinical data showed promising improvements in bowel mass in rats receiving GLP-2 infusion following 75% removal of the mid jejuno-ileum (221). Moreover, infusion of GLP-2 in patients in whom the terminal ileum and colon had been resected, improved intestinal absorption and nutritional status (222). Thus, GLP-2R has clear application in treatment of the condition. As is the case with GLP-1(7–36), GLP-2 is rendered inactive by enzymatic N- terminal dipeptide (His1-Ala2) removal by DPP-4, producing the major fragment GLP-2 (3–33) (205). Thus, in order to be therapeutically viable, the native hormone must be modiﬁed to facilitate exogenous administration. GLP-2 exerts its actions through agonism of its own target receptor, a GPCR termed the GLP-2 receptor (GLP-2R) [(25); Figure 2]. The receptor is widely expressed throughout the entirety of the gut and is highly speciﬁc for GLP-2, with other PGDPs demonstrating relatively low afﬁnity (213). Similar to GLP-1, agonism of the GLP-2R evokes a rise in intracellular cAMP and subsequent PKA activation, however, intracellular calcium remains unchanged [(214); Figure 2]. Activation of the receptor directly reduces enterocyte apoptosis and increases crypt cell proliferation, which operates in tandem to increase microvilli height [(215); Figure 3]. The hormone has also been demonstrated to improve intestinal blood ﬂow, decrease gut motility and inhibit gastric acid secretion [(216); Figure 3]. GLUCAGON-LIKE PEPTIDE-2 There is some evidence that GLP-2 is produced in small functional amounts within pancreatic islets, but the alternative processing of proglucagon by PC1/3 in alpha-cells to give GLP-1 under conditions of cellular stress is likely much more signiﬁcant (217). Substitution of the penultimate Ala2 for Gly2 (as found in exendin-4) enabled the development of [Gly2]GLP-2 (Table 3), a DPP-4 resistant, long-acting GLP-2 mimetic (214). The peptide employed single amino acid substitution and presented a more speciﬁc approach than blanket DPP-4 inhibition (222). The analogue was later named “teduglutide” and demonstrated early promise in a dose-range pilot study in human SBS patients (227). Subsequent phase III clinical trials conﬁrmed beneﬁcial effects in several cohorts of SBS patients, manifesting in improved intestinal morphology, renal function as well as a favourable side-effect proﬁle (223, 228). Furthermore, treatment reduced reliance on PN in many patients (223), while a portion of previously dependent patients was able to completely discontinue PN (229). Teduglutide was subsequently approved by the FDA in 2012 and is prescribed under the trade names Gattex® in the USA and Revestive® in Europe (Table 3). GLP-2 Therapeutics and Short Bowel Syndrome Following the success of teduglutide, further GLP-2 analogues are currently in development, with research aimed to improve the ~5 h circulating half-life of teduglutide (230). Apraglutide ([Gly2, Nle10, D-Phe11, Leu16]-GLP-2) employed further substitutions (Table 3), identiﬁed through structure-activity relationship studies of lipophilic amino acid substitutions in positions 11 and 16 of teduglutide, and has been shown to prolong in vivo bioactivity through reduced renal clearance in rodents (224). Similar ﬁndings were observed in monkey and mini-pig (225), whilst exhibiting excellent speciﬁcity and potency for the GLP-2R. The peptide was more efﬁcacious than both teduglutide and another GLP-2 analogue in development, glepaglutide [(226); Table 3], and has started The intestinotrophic properties of GLP-2 were an attractive prospect in development of therapeutics for conditions such as short-bowel syndrome (SBS), usually a consequence of surgical removal of a section of the bowel in Crohn’s disease (218). This condition is characterised by malabsorption as a result of chronic diarrhoea with further dehydration and weight loss, and depending on severity, the overall quality of life can be greatly impaired. The condition can be managed by parenteral nutrition (PN) and hydration, however, in the long-term this increases the likelihood of infection and potentially sepsis (219). Additionally, patients have a strict reliance on PN which can impede mobility, further impacting on quality of life. Hence, a medication with the TABLE 3 \| GLP-2-based therapeutic peptides. Peptide Name AA Sequence Development Stage Reference Native GLP-2(1-33) HADGSFSDEMNTILDNLAARDFINWLIQTKITD N/A (205) [Gly2]GLP-2 HGDGSFSDEMNTILDNLAARDFINWLIQTKITD Preclinical (209, 214) Teduglutide HGDGSFSDEMNTILDNLAARDFINWLIQTKITD Approved 2012-SBS (Shire-NPS Pharmaceuticals) (222–223) Apraglutide HGDGSFSDE-Nle-(DF)TILDLLAARDFINWLIQTKITD Phase III-SBS (VectivBio) (224, 225) Glepaglutide HGEGTFSSELATILDALAARDFIAWLIATKITDKKKKKK Phase III-SBS (Zealand Pharma) (226) Amino acid sequences are provided in their single-letter abbreviation format. Modiﬁcations from native sequences are highlighted by red lettering. Current development stages, and associated condition, and holding companies (in brackets) are provided (where available) for each are provided for each. A “D” preﬁx before a residue indicates inclusion of the enantiomer for the naturally-occurring L form of the residue. “Nle” indicates the addition of a norleucine residue. May 2021 \| Volume 12 \| Article 689678 11 Frontiers in Endocrinology \| www.frontiersin.org Proglucagon-Derived Peptides as Therapeutics Lafferty et al. did not ascertain the involvement of other gut hormones. These ﬁndings are supported by more recent work in ovariectomised rats, an animal model replicating postmenopausal osteoporosis. It was established that 4 weeks s.c. Oxyntomodulin Oxyntomodulin (OXM) was discovered as a fragment of glicentin (243, 244), sharing substantial sequence homology and essentially the entire 29 amino acid glucagon molecule with an additional C-terminal octapeptide, IP-1, resulting in 37 residue OXM ( (245, 246); Figure 1 and Table 4). Like other gut-based PGDPs, OXM is released post- prandially from L-cells (254). OXM increases energy expenditure and physical activity, promotes weight loss and improves glycaemia in humans (254, 255). No speciﬁc OXM receptor is known to exist; rather, the peptide acts as a dual agonist for GCGR and GLP-1R (Figure 2), although it binds to both with lower afﬁnity than either of their primary ligands (256, 257). In the current thinking, OXM-mediated weight loss is believed to be elicited through activation of the GCGR, bringing about anorectic actions and increased energy expenditure [(258); Figure 3]. In contrast, GLP-1R agonism accounts for improved glucose homeostasis through augmented insulin secretion, overcoming the hyperglycaemic actions of GCGR activation [(259); Figure 3]. Mechanistic studies reveal that OXM behaves as a differential agonist depending on the receptor, acting as a full agonist in recruiting b-arrestin 2 to the GCGR, but partial agonist in recruiting b-arrestin 1 and 2 and GPCR kinase 2 to the GLP-1R (260). Furthermore, some data suggests that OXM is a GLP-1R-biased agonist relative to GCGR (260). However, unlike these related gut hormones, the role and indeed application of GLP-2 with respect to bone mass is more divisive. In initial studies of GLP-2 in SBS, an additional observation was made that, following 5 weeks treatment, patients presented with signiﬁcantly increased spinal areal bone mineral density (222). Subsequently, it was demonstrated that s.c. GLP-2 administration reduced bone resorption in post- menopausal women while not affecting bone formation (235). However, the ﬁndings in SBS patients were refuted, with a later study reporting that an intact bowel is required for exogenous GLP-2 administration to have such an effect (236). Additionally, unlike GIPR, an equivalent GLP-2R has not been identiﬁed on human osteoclasts (237), indicating that its actions are indirect, with inhibition of parathyroid hormone (PTH), mediated by activation of GLP-2R on PTH gland, suggested to be the mediator of its effects on bone resorption (236). Moreover, a small-scale trial in healthy males employed GIPR antagonists to conﬁrm the antiresorptive effects of GLP-2 are independent of this receptor (238). GLP-2 Therapeutics and Osteoporosis GLP-2 Therapeutics and Osteoporosis An additional similarity to GLP-1 research is the pursuit of new therapeutic applications. With the widespread expression of GLP-2R (213), it was postulated that GLP-2 may have application in the management of osteoporosis. Osteoporosis is a condition characterised by bone mass reduction and microarchitecture impairment caused by an imbalance in bone formation and resorption, increasing the risk of fractures (232). Moreover, the prevalence of osteoporosis continues to surge in accordance with an increasingly ageing population (233). A number of the widely-prescribed, anti-resorptive drugs, particularly bisphosphonates, are believed to possess unfavourable side-effect proﬁles (234), thus alternative treatment options are being sought. Indeed, the involvement of gut hormones in bone mass and formation has been widely researched, with the roles of GLP-1, as well as GIP (175), discussed above. Oxyntomodulin The mechanisms behind the bone actions of GLP-2 require further investigation to ﬁrmly establish a link. Despite this, several studies support the involvement and potential use of GLP-2 in bone formation in some capacity. In a study of postmenopausal women with concurrent T2DM, it was revealed that ingestion of a mixed nutrient meal saw a reduction in biomarkers for bone fragility, coupled with a rise in GLP-2 levels (239), indicating the importance of the gut. However, this study Frontiers in Endocrinology \| www.frontiersin.org GLP-2 Therapeutics and Short Bowel Syndrome administration of GLP-2 resulted in improvements of bone architecture and mass through both promotion of bone formation and a reduction in resorption (240). Interestingly, studies of GLP-2 effects on bone have all employed human GLP-2, as opposed to longer-acting analogues. Furthermore, i.v. administration of a high dose of GLP-2 was outperformed by lower doses of s.c. GLP-2 in terms of reducing bone resorption (241). Thus, given longer acting, s.c. teduglutide is currently available, as well as other enzyme resistant analogues in development, their potential use for therapy of osteoporosis is exciting. Moreover, given the involvement of several gut hormones in this gut-bone axis (242), coupled with the success of unimolecular multiagonists with relation to bone improvements (184), it stands to reason that incorporation of a GLP-2R agonising component may improve the efﬁcacy of such agents in promoting bone density. recruiting for phase III clinical trials in SBS patients (231). That said, glepaglutide has a reported half-life of 50 h and has also entered phase III clinical trials (226). It employs nine amino acid substitutions and a C-terminal tail of six Lys residues (Table 3). The analogue forms a subcutaneous depot at the injection site, from which glepaglutide and its active metabolites are gradually released into the circulation. Phase II trials indicated the analogue was well absorbed, effective and tolerated (226). Thus, apraglutide and glepaglutide may represent an exciting new step in development of GLP-2 analogues, emulating the success of long-acting GLP-1 analogues, which can be administered at less frequent intervals than currently available once-daily preparation, teduglutide. Oxyntomodulin Therapeutics and Obesity/Diabetes As alluded to above, the ability of OXM to effectively activate both GCGR and GLP-1R, thereby improving blood glucose and body weight, is attractive for the development of peptide May 2021 \| Volume 12 \| Article 689678 12 Proglucagon-Derived Peptides as Therapeutics Lafferty et al. TABLE 4 \| Oxyntomodulin-based therapeutic peptides. TABLE 4 \| Oxyntomodulin-based therapeutic peptides. Peptide Name AA Sequence Development Stage Reference Native OXM HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNKNNIA N/A (245) (D-Ser2)Oxm[mPEG-PAL] H(DS)QGTFTSDYSKYLDSRRAQDFVQWLMNTKRNKNNIA-[mPEG-PAL] Preclinical (247) Dogﬁsh OXM HSEGTFTSDYSKYMDNRRAKDFVQWLMSTKRNG Preclinical (248) Ratﬁsh OXM HTDGIFSSDYSKYLDNRRTKDFVQWLLSTKRNGANT Preclinical (248) [D-Ala2]GIP–Oxm YDAEGTFISDYSKYLDSRRAQDFVQWLMNTKRNRNNIA Preclinical (184) OX-SR Structure N/A Preclinical (249) LY3305677 Structure N/A Phase II-T2DM/Obesity (Eli Lilly) (250, 251) DualAG HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNKNNIA-Chol Preclinical (252) GLPAG HSEGTFTSDYSKYLDSRRAQDFVQWLMNTKRNKNNIA-Chol Preclinical (253) Amino acid sequences are provided in their single-letter abbreviation format. Modiﬁcations from native sequences are highlighted by red lettering. Current development stages, associated condition and holding companies (in brackets, where available) are provided for each. A “D” preﬁx before a residue indicates inclusion of the enantiomer for the naturally-occurring L form of the residue. “mPEG” indicates mini-polyethylene glycol addition. “PAL” indicates the addition of a palmitic fatty acid chain. “Chol” represents attachment of a human cholesterol fragment. “Structure N/A” represents a molecule for which the amino acid sequence has not been disclosed by authors. The therapeutic applicability of enzymatically stable OXM analogues is clear and a number of analogues are in various stages of development for potential use in T2DM therapy (Table 4). However, it has been demonstrated that a balance in GCGR/ GLP-1R agonism must be reached when designing OXM analogues, with a number of examples demonstrated to induce hyperphagia (266). OXM analogues with Glu3 substitution favour GLP-1R activation and do not exhibit an orexigenic effect (266), hence, it is assumed that such an effect must be mediated via GCGR agonism (266, 267). However, with the development of OX-SR, a sustained-release oxyntomodulin analogue which employs 5 central, depot-forming, amino acid substitutions between residues 16-27 of the human peptide (exact sequence not disclosed by authors), an OXM analogue capable of bringing about GCGR-mediated increases in energy expenditure was developed, and despite having an orexigenic effect actually elicited 2% weight loss following 3 days administration in rats (249). In this respect, while OX-SR was proven to agonise both receptors in vitro, the analogue showed greater afﬁnity for GCGR than GLP-1R (249). Oxyntomodulin Therapeutics and Obesity/Diabetes More prolonged studies, including those in models of diabetes are required to investigate the long-term consequences of such prolonged exposure to GCGR and GLP-1R activation by OX-SR, but the peptide does represent a potential once-weekly OXM formulation (249). Excitingly, regulatory approval of the ﬁrst OXM analogue may be on the horizon, with the long acting, fatty-acid derivatised analogue LY3305677 (sometimes termed IBI362) currently in separate phase II clinical trials investigating management of T2DM and obesity (250, 251). therapeutics for obesity/T2DM, provided an appropriate receptor balance is struck. Like all other PGDPs, OXM is subject to rapid inactivation by DPP-4 which targets cleavage after the N-terminal Ser2 residue (261). This rapid inactivation precludes use of the unmodiﬁed hormone as a therapeutic. Thus, while initial studies demonstrated that native OXM decreases food intake and enhances energy expenditure in both healthy and obese human volunteers, these employed undesirably frequent dosing of three- or four-times daily (262, 263). therapeutics for obesity/T2DM, provided an appropriate receptor balance is struck. Like all other PGDPs, OXM is subject to rapid inactivation by DPP-4 which targets cleavage after the N-terminal Ser2 residue (261). This rapid inactivation precludes use of the unmodiﬁed hormone as a therapeutic. Thus, while initial studies demonstrated that native OXM decreases food intake and enhances energy expenditure in both healthy and obese human volunteers, these employed undesirably frequent dosing of three- or four-times daily (262, 263). As with GLP-1, DPP-4 resistant forms of OXM are required therapeutically and given the sequence similarities between the two peptides, successful approaches taken with GLP-1 can be applied to OXM (261, 262). One example, (D-Ser2)Oxm[mPEG- PAL] (Table 4), employed substitution of the naturally occurring L-Ser2 with the enantiomer D-Ser2 to promote DPP-4 resistance, while further utilising C-16 palmitic acid conjugation via a mini- PEG linker at the C-terminus to reduce renal clearance and improve circulating half-life (247). The resulting peptide was fully resistant to DPP-4, whilst clearly retaining bioactivity: increasing cAMP in both GLP-1R and GCGR transfected cell lines, as well as enhancing insulin release from clonal pancreatic beta-cells (247). Additionally, daily administration of (D-Ser2) Oxm[mPEG-PAL] to ob/ob mice decreased food intake and body weight, whilst increasing plasma and pancreatic insulin and improving glucose tolerance (247). Several biomarkers of obesity were also improved, including increased adiponectin with reductions in both visfatin and triglyceride concentrations (247). Oxyntomodulin Therapeutics and Obesity/Diabetes The OXM analogue also exerted beneﬁcial effects on blood glucose control in STZ-diabetic mice, including elevations in total islet and beta-cell areas associated with an increase in the number of smaller-sized islets and enhanced islet proliferation (264). A follow-up study with (D-Ser2)Oxm[mPEG-PAL] in transgenic mice with ﬂuorescently tagged alpha cells also demonstrated highly favourable effects on islet cell transdifferentiation (265). Interestingly, another study employed dogﬁsh and ratﬁsh oxyntomodulin peptides (Table 4), which despite numerous sequence variations from human OXM, remained effective at mammalian GCGR and GLP-1R (248). This suggests a possible early advantage of such dual receptor actions in evolutionary terms. Frontiers in Endocrinology \| www.frontiersin.org MULTIAGONISTS terminal proglucagon fragment glicentin (243), which contained the entire glucagon sequence attached to an N-terminal portion later identiﬁed as GRPP (269, 270). The smaller fragment was essentially glucagon attached to a C-terminal octapeptide called intervening peptide-1 (IP-1) (271), later this C-terminally extended glucagon was denominated as oxyntomodulin. Unimolecular multiagonists represent an exciting future in the therapeutic application of PGDPs, with increasingly complex and experimental molecules being developed. As brieﬂy mentioned above, RYGB surgery induces rapid remission of T2DM in 70-80% patients (285). Importantly, secretion and action of a number of gut hormones, including the PGDPs GLP-1, GLP-2, OXM and glicentin, together with PYY, GIP, cholecystokinin (CCK), neurotensin (NT) and secretin, are positively modulated in concert following RYGB (286). These are thought to be major determinants in the improvements of appetite, body weight, glucose tolerance and insulin sensitivity demonstrated post-surgery (286). Thus, given high costs, limited availability and potential risks associated with surgical procedures, there is a current focus on designing multiagonist molecules with the ability to emulate the post-surgical, hormonal mechanisms of RYGB, which have the potential to be more widely available to patients than surgery. Additionally, they have the potential to evoke an array of positive actions within various organs (Figure 3), and such molecules could surpass advantages observed with individual peptides. g g y We now know glicentin is released post-prandially from L- cells of the distal ileum and colon, particularly in response to glucose, lipids and amino acids, especially arginine, entering the duodenum [(272–274); Figure 2]. The hormone elicits a number of physiological effects such as a paracrine role in promoting intestinal growth and regulating motility (275), as well as playing a role in glucose homeostasis through augmenting insulin secretion and inhibiting glucagon secretion (276). However, no glicentin receptor has yet been identiﬁed, but the hormone has been shown to agonise and elicit cAMP production following binding to glucagon, GLP-1 and GLP-2 receptors [(277, 278); Figure 2]. Additionally, earlier work with glicentin suggested that its actions were largely dependent upon the degradation of the hormone into smaller molecular fragments (279), possibly including carboxylase-mediated generation of glucagon (Figure 2). This may, in part, explain why there has been relatively little research exploring development of glicentin-based therapeutics. Furthermore, it is likely that a lack of commercialised detection methods for glicentin have hindered its overall investigation and therapeutic application (280). MULTIAGONISTS However, with increasing availability and affordability of capable assays and given the increasing interest in peptide therapeutics, we may see renewed interest in this PGDP (281). Moreover, it has recently been put forward that post-surgery rises in glicentin, along with OXM, are the best predictors of decreased in intake of energy-dense foods and weight loss following RYGB, more so than even GLP-1 (282). Whether this translates to functional involvement remains unclear, and instead it is postulated that increased glicentin levels are a useful indicator of improved overall L-cell function (282). Earlier research employing combinations of single gut hormones or analogues provided a sound basis for the application of multi-agonism in T2DM (287). Indeed, with the combination of liraglutide plus an acylated GIP analogue (288), synergy was demonstrated leading to improved glucose-lowering and insulinotropic actions in obese-diabetic mice compared to either of the individual incretin analogues alone. Furthermore, recent combination studies have further strengthened the idea that combined exogenous peptide administration can effectively emulate the beneﬁts of RYGB. As such, infusion of a multi- peptide preparation of GLP-1, OXM and PYY (3–31, 35–39) termed “GOP”, can replicate the postprandial levels of these hormones observed after RYGB, and can safely bring about 32% reduction in food intake in a standardised meal test (289). Moreover, continuous GOP infusion, delivered by pump over a 4-week period in obese patients with prediabetes or diabetes, resulted in improvements in glucose tolerance which surpassed those of RYGB (290). As discussed above, GRPP is one of the products of PC2 processing of proglucagon in pancreatic alpha-cells (25, 26). A 30 residue, N-terminal fragment of proglucagon (Figure 1), GRPP was discovered after glicentin using glicentin-speciﬁc antibodies in pancreatic extracts (269). Structural elucidation highlighted that the peptide was identical to the N-terminus of gut-derived glicentin, hence the name glicentin-related pancreatic peptide (269). Despite its discovery almost four decades ago, research on this PGDP is sparse, but earlier experiments in dogs suggest the peptide may inﬂuence glucose homeostasis through increasing plasma insulin and decreasing plasma glucagon (283). A more recent study utilised isolated-perfused pancreas and liver from rats to pursue a detailed investigation of the physiology of this peptide (284). Glicentin and Glicentin-Related Pancreatic Peptide Glicentin is a product of PC1/3 proglucagon processing, while GRPP glicentin-related pancreatic peptide (GRPP) is a product of PC2 processing in the pancreas [(22, 25); Figure 1]. Radioimmunoassay of gut extracts revealed substances with glucagon-like immunoreactivity that cross-reacted with antibodies directed towards the N-terminus of glucagon (268), with further investigation identifying two related proteins, one appearing to be a fragment of the other. Firstly, the 69 residue, N- May 2021 \| Volume 12 \| Article 689678 Frontiers in Endocrinology \| www.frontiersin.org 13 Proglucagon-Derived Peptides as Therapeutics Lafferty et al. Frontiers in Endocrinology \| www.frontiersin.org MULTIAGONISTS A “D” preﬁx before a residue indicates inclusion of the enantiomer for the naturally-occurring L form of the residue. “PAL” indicates the addition of a palmitic fatty acid chain, “PEG” indicates a polyethylene glycol linker. “Aib”, “Nle” and “NmeF” indicate the addition of an unnatural 2-aminoisobutyric acid, norleucine or N- methyl phenylalanine residues. “pE” indicates pyroglutamine. “K(Tac)” indicates inclusion of a side-chain substituted (o-tolyamino)carbonyl lysine residue. “(AEEAc‐AEEAc)” indicates a commonly employed linker molecule between peptide regions. “gE-PAL” represents a fatty acid attachment. with the cholesterol-conjugated OXM analogue DualAG and the glucagon, GLP-1 chimeric peptide “Aib2 C24 chimera 2 lactam 40K” both showing preclinical promise in murine, DIO models of obesity-diabetes [(252, 311); Table 4]. sequence contribution minimising hyperglycaemia whilst retaining weight loss (312). The effects on glycaemia were supported by acute administration studies in humans, however a slower dose titration was deemed necessary to avoid adverse effects on gastric emptying (291). y [( ) ] While a number of GLP-1/glucagon based peptides have been generated (Table 5), many have witnessed therapeutic pursuit abandoned. Currently, a molecule of particular promise is cotadutide (formerly MEDI0382). Cotadutide is a linear, chimeric peptide employing important residues from both glucagon and GLP-1 into its sequence (Table 5), with a palmitoyl FA attachment on Lys10 to prolong circulating half- life (253). The peptide is reported to be a balanced dual- agonist for GLP-1 and GCGR, which brought about signiﬁcant weight loss through improved glycaemia in DIO mice and non-human primates, being more effective than liraglutide alone (253). The concept of balance in respect to such molecules is crucial, as it is important to maximise weight loss whilst minimising the potential to cause hyperglycaemia, with as little as 10% relative GLP-1 When assessed in phase II trials in T2DM patients, slower titration of cotadutide was employed to reﬂect such ﬁndings (313). This study revealed that daily administration in patients with controlled T2DM improved overall glycaemic control, as measured by HbA1c, which was associated with sustained weight loss following 41 days daily administration (313). Subsequently, it was revealed that these positive effects on glycaemia were likely the result of improved gastric emptying and postprandial insulin response (292). Additionally, patients presented with signiﬁcant improvements in liver fat, with levels falling by 39% (313), which was notable given an equivalent fall in levels with liraglutide takes around 6 months (314). MULTIAGONISTS In contradiction of initial ﬁndings, this study demonstrated that while glucose output from the liver remained unaffected, GRPP brought about potent inhibition of glucose‐stimulated insulin secretion in perfused pancreas, with cAMP assay indicating that these actions were not mediated through either GLP-1R or GCGR, meaning an unidentiﬁed receptor may be at play (284). Given the lack of physiological data surrounding GRPP, it is unsurprising that no therapeutic exploration has been made on this PGDP. To date, a number of unimolecular double- and triple- agonists have been developed with several being actively pursued for clinical application (Table 5). The majority of these typically employing a GLP-1R agonist component combined with another gut hormone, often an incretin or other PGDP. Dual Agonism With GLP-1 and Glucagon As previously discussed, the notion of GCGR agonism in pursuit of a therapeutic for T2DM, or its related conditions, seems counterintuitive. However, given the surprising beneﬁcial effects of OXM agonists in T2DM, the beneﬁts of targeting these two receptors in tandem was clearly demonstrated (Figure 3). Additionally, the structural similarity between the two PGDPs was clearly demonstrated (Table 4). As such, this combination pioneered unimolecular PGDP-based research, May 2021 \| Volume 12 \| Article 689678 Frontiers in Endocrinology \| www.frontiersin.org 14 Proglucagon-Derived Peptides as Therapeutics Lafferty et al. TABLE 5 \| Multiagonists based on proglucagon-derived peptides in development. Peptide Name AA Sequence Target Receptors Development Stage Reference Dual Agonists Cotadutide HSQGTFTSDK-(Palmitoyl-E)SEYLDSERARDFVAWLEAGG GLP-1R/ GCGR Phase II-T2DM, NASH/ NAFLD (AstraZeneca) (291–292, 293) Eﬁnopegdutide Structure N/A GLP-1R/ GCGR Phase II-NASH/NAFLD (Merck & Co) (294–295) Tirzepatide Y-Aib-EGTFTSDYSI-Aib-LDKIAQK(C20 diacid g-E) AFVQWLIAGGPSSGAPPPS GLP-1R/GIPR Phase III-T2DM, Phase II- NASH (Eli Lilly) (296–297) NN9389 Structure N/A (GIP/Semaglutide Preparation) GLP-1R/GIPR Phase I-T2DM (Novo Nordisk) (298) CT-868 Structure N/A GLP-1R/GIPR Phase I-T2DM (Carmot Therapeutics) (298) TAK-094 Structure N/A GLP-1R/GIPR Phase I-T2DM (Takeda Pharmaceuticals) (298) (pGlu-Gln)-CCK-8/exendin-4 pEQDY-(SO3H)-MGWMDF-(AEEAc-AEEAc)- HGEGTFTSDLSKQMEEEAVRLFIEWLKN GLP-1R/ CCK1R Preclinical (299) C2816 HGEGTFTSDLSKQMEEEAVRLFIEWLKN-[PEG4]-Nle-GWK(Tac)D-NmeF GLP-1R/ CCK1R Preclinical (MedImmune/ Astrazeneca) (300) GUB06-046 HXEGTFTSDLSRLLEGAALQRFIQWLV GLP-1R/SCTR Preclinical (Gubra) (301) EP45 HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSRHYLNLVTRQRY GLP-1R/ NPY2R Preclinical (302) Exendin‐4/xenin‐8‐Gln HGEGTFTSDLSKQMEEEAVRLFIEWLKN‐(AEEAc‐AEEAc)‐HPQQPWIL GLP-1/NTSR1 Preclinical (303) Triple Agonists YAG-glucagon Y[DA]QGTFTSDYSIYLDSNVAQDFVQWLIGG GLP-1/GIPR/ GCGR Preclinical (304) Exendin‐4/gastrin/xenin‐8‐Gln HGEGTFTSDLSKQMEEEAVRLFIEWLKN‐(AEEAc‐AEEAc)‐YGWLDF ‐ (AEEAc‐AEEAc)‐HPQQPWIL GLP-1/ CCK2R/ NTSR1 Preclinical (305) Exendin‐4(Lys27g‐Glu‐PAL)/ gastrin/xenin‐8‐Gln HGEGTFTSDLSKQMEEEAVRLFIEWLK(g‐E‐PAL)N‐(AEEAc‐AEEAc)‐ YGWLDF ‐(AEEAc‐AEEAc)‐HPQQPWIL GLP-1/ CCK2R/ NTSR1 Preclinical (306) LY3437943 Structure N/A GLP-1/GIPR/ GCGR Phase I (Eli Lilly) (269) HM15211 Structure N/A GLP-1/GIPR/ GCGR Phase II (Hanmi Pharmaceuticals) (307–308) TA HXQGTFTSDK(gE-C16)SKYLDERAAQDFVQWLLDGGPSSGAPPPS GLP-1/GIPR/ GCGR Preclinical (309, 310) Amino acid sequences are provided in their single-letter abbreviation format. Frontiers in Endocrinology \| www.frontiersin.org MULTIAGONISTS The receptor targets for each molecule, as well as current stage of development and holding companies (in brackets, where available) are provided for each. A “D” preﬁx before a residue indicates inclusion of the enantiomer for the naturally-occurring L form of the residue. “PAL” indicates the addition of a palmitic fatty acid chain, “PEG” indicates a polyethylene glycol linker. “Aib”, “Nle” and “NmeF” indicate the addition of an unnatural 2-aminoisobutyric acid, norleucine or N- methyl phenylalanine residues. “pE” indicates pyroglutamine. “K(Tac)” indicates inclusion of a side-chain substituted (o-tolyamino)carbonyl lysine residue. “(AEEAc‐AEEAc)” indicates a l l d li k l l b t tid i “ E PAL” t f tt id tt h t HXQGTFTSDK(gE-C16)SKYLDERAAQDFVQWLLDGGPSSGAPPPS TA Amino acid sequences are provided in their single-letter abbreviation format. The receptor targets for each molecule, as well as current stage of development and holding companies (in brackets, where available) are provided for each. A “D” preﬁx before a residue indicates inclusion of the enantiomer for the naturally-occurring L form of the residue. “PAL” indicates the addition of a palmitic fatty acid chain, “PEG” indicates a polyethylene glycol linker. “Aib”, “Nle” and “NmeF” indicate the addition of an unnatural 2-aminoisobutyric acid, norleucine or N- methyl phenylalanine residues. “pE” indicates pyroglutamine. “K(Tac)” indicates inclusion of a side-chain substituted (o-tolyamino)carbonyl lysine residue. “(AEEAc‐AEEAc)” indicates a commonly employed linker molecule between peptide regions. “gE-PAL” represents a fatty acid attachment. Amino acid sequences are provided in their single-letter abbreviation format. The receptor targets for each molecule, as well as current stage of development and holding companies (in brackets, where available) are provided for each. A “D” preﬁx before a residue indicates inclusion of the enantiomer for the naturally-occurring L form of the residue. “PAL” indicates the addition of a palmitic fatty acid chain, “PEG” indicates a polyethylene glycol linker. “Aib”, “Nle” and “NmeF” indicate the addition of an unnatural 2-aminoisobutyric acid, norleucine or N- methyl phenylalanine residues. “pE” indicates pyroglutamine. “K(Tac)” indicates inclusion of a side-chain substituted (o-tolyamino)carbonyl lysine residue. “(AEEAc‐AEEAc)” indicates a commonly employed linker molecule between peptide regions. “gE-PAL” represents a fatty acid attachment. Amino acid sequences are provided in their single-letter abbreviation format. The receptor targets for each molecule, as well as current stage of development and holding companies (in brackets, where available) are provided for each. Dual Agonism With GLP-1 and GIP A literature search for dual agonists also reveals some slightly left-ﬁeld combinations with GLP-1, although importantly these involve other gut hormones shown to be upregulated by bariatric surgery (286). The combinations explored so far in preclinical studies all have the potential to elicit a range of additional effects on various systems in the body (Figure 3). For example, a long- acting GLP-1/CCK hybrid peptide has been developed which employs the key regions of (pGlu-Gln)-CCK-8, a stabilised form of CCK (320), and exendin-4 attached to one another via a linker molecule (Table 5). Through simultaneous activation of both GLP-1 and CCK-2 receptors, this co-agonist outperformed exendin-4 in terms of satiety and body weight reductions in obese-diabetic mice (299). A similar molecule, essentially reversing the conﬁguration of GLP-1 and CCK components [(300); Table 5], also highlights the potential of dual receptor activation in this regard, outperforming (pGlu-Gln)-CCK-8 in terms of body weight reduction following 10 weeks treatment in DIO mice (300). With synergy demonstrated by administration of liraglutide plus an acylated, enzyme resistant GIP analogue (288), the value of developing molecules targeting these two incretin receptors was evident. As such, a number of unimolecular GLP-1/GIP agonists have been developed and are at various stages of clinical testing (Table 5). One particular success story involves a molecule termed tirzepatide (formerly LY3298176) (296). The peptide is a linear, 39 aa peptide containing two unnatural residues and a C20 diacid fatty acid attached via a linker to Lys20 (Table 5), all of which contribute to a circulating half-life of ~5 days, which permits once weekly dosing (296). Tirzepatide may be considered a GIP-based analogue, sharing greater sequence homology with GIP than GLP-1 (particularly at the N- terminus) (Table 5), with GLP-1R agonism induced via aa substitution (296, 317). The peptide was shown to effectively lower blood glucose via insulinotropic actions at both receptors in preclinical studies in mice, while phase I trials revealed effective weight loss in T2DM patients and good tolerability (296). Interestingly, in vitro mechanistic studies suggest the peptide is biased towards the GIPR, activating with equipotency to native GIP whilst having 5-fold weaker afﬁnity than native GLP-1 at GLP-1R, with a preference to initiate cAMP mobilisation to enhance insulin secretion (317), which may be of particular beneﬁt in obesity-diabetes. MULTIAGONISTS These ﬁndings on liver fat have seen a refocus of research toward application in non-alcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH) (293), May 2021 \| Volume 12 \| Article 689678 Frontiers in Endocrinology \| www.frontiersin.org 15 Proglucagon-Derived Peptides as Therapeutics Lafferty et al. attributed to GIPR/GLP-1R agonism (318). Thus, while important synergy is likely to be occurring, conﬁrmation is required. both common consequences of uncontrolled T2DM (315). The study revealed that cotadutide’s actions on the liver to reduce lipid content, drive glycogen ﬂux and improve mitochondrial turnover and function are directly mediated through modulation of hepatic GCGRs, while metabolic improvements mediated via agonism of extrahepatic GLP-1Rs further enhanced improvement (293). A similar story is unfolding for the GLP- 1/GCGR agonist eﬁnopegdutide (formerly HM12525A), a longer acting agonist which employs modiﬁed exendin-4 conjugated to human IgG, facilitating once-weekly administration ( (294); Table 5). The peptide appealed as a treatment for T2DM due to promising preclinical results which demonstrated lipolytic and insulinotropic effects in diabetic mice (316). However, potent lowering effects on cholesterol and liver fat have seen this analogue also repurposed as a potential NAFLD/NASH medication (295). Tirzepatide has now progressed to phase III clinical trials in T2DM, and we await data from these studies with great anticipation. In similar fashion to the GLP-1/glucagon analogues discussed, tirzepatide has also found application in the treatment of NASH, with a follow-on study in T2DM patients revealing that several biomarkers of liver inﬂammation were reduced in patients receiving higher doses of the analogue (319). Indeed, a number of other analogues such as NN9389 (GIP and semaglutide combination), CT-868 and TAK-094 are all currently in phase I clinical trials as potential T2DM treatments (298), but any detailed literature on these analogues remains elusive at the time of writing. Frontiers in Endocrinology \| www.frontiersin.org Dual Agonism With GLP-1 and GIP These results were supported in phase II trials in T2DM patients with HbA1c reductions of 2%, highly impressive body weight reductions of 5-10% (max 11.3 kg) and signiﬁcant reductions in waist- circumference demonstrated following 12 weeks treatment (297). Moreover, comparison to the established GLP-1R mimetic, dulaglutide, proved tirzepatide to elicit more signiﬁcant reductions in body weight (-4.52 kg/6.4% compared to -1.3 kg/1.8% for dulaglutide after 4 weeks), with the authors concluding inclusion of GIPR agonism builds upon sole GLP-1R activation to enhance weight loss via modulating appetite and gastric emptying, with the antiemetic effect of GIPR also improving tolerability (296). It is likely further mechanistic investigation will be pursued to fully elucidate the biological processes at play, especially as no single effect could be entirely A GLP-1/secretin chimeric peptide, based on the sequence of secretin with GLP-1R activity induced via substitution of important GLP-1 residues (Table 5), has been developed. This peptide decreased food intake and body weight more effectively than liraglutide alone (301). Moreover, this analogue improved short-term glycaemic control (39% fall in fasting blood glucose), HbA1c (-1.6%) and promoted a 78% rise in beta cell mass following twice daily s.c. administration over an 8 week period in diabetic, db/db mice (301). Another successful, but seemingly counterintuitive pairing is the combination of GLP-1 and PYY. PYY is insulinostatic but holds therapeutic potential due to induction of beta cell rest, promotion of beta-cell mass, satiety and weight loss (321). Moreover, a synergistic effect between PYY and GLP-1 has been established (322), supporting their incorporation in a co- agonist. One such peptide, termed EP45, has been developed as a chimeric peptide employing PYY (25–36) incorporated with exendin-4(1–33); Table 5). Indeed, the peptide was demonstrated to effectively activate both GLP-1R and NPY2R in transfected cell lines (302), but in vivo application is yet to be published. May 2021 \| Volume 12 \| Article 689678 Frontiers in Endocrinology \| www.frontiersin.org 16 Proglucagon-Derived Peptides as Therapeutics Lafferty et al. aglycosylate Fc fragment which prolongs half-life to permit once weekly administration [(334); Table 5]. HM15211was more effective than daily administration of liraglutide in increasing energy expenditure, with improvements in weight loss and hepatic inﬂammation markers in rodent models (334), and is currently recruiting for phase II trials as a treatment for NASH (308). Dual Agonism With GLP-1 and GIP Finally, an enzyme resistant GLP-1/xenin dual-agonist, Exendin-4/xenin-8-Gln (303), has been developed with xenin, which is a regulatory peptide co-secreted postprandially with GIP from intestinal K-cells [(323); Table 5]. Xenin is known to potentiate the actions of GIP (324), and in addition to positive glycaemic outcomes, through reduced appetite and augmented insulin secretion, the peptide also restored GIP sensitivity (303) that is dampened in obesity (325). Consistent with these actions, Exendin-4/xenin-8-Gln induced substantial beneﬁts in DIO diabetic mice (303). Similar to single GLP-1 agonists, a tri-agonist termed TA is also ﬁnding application with regards to neuroprotection [(309); Table 5]. This hybridised GLP-1/GIP/glucagon activator was initially developed for management of obesity-diabetes and showed promising preclinical results in rodent models of diabetes-obesity (310). However, the more recent repurposing of this molecule towards management of AD is particularly exciting, with daily administration of the analogue in a murine model of AD over a 2 month period reversing memory deﬁcit, reducing pro-mitochondrial apoptosis markers and upregulating growth factors involved in synaptic function (309). The preclinical study has not been followed-up to date, but represents a potentially fruitful new avenue for the application of PGDP-based multiagonists. OTHER POSSIBLE GLUCAGON THERAPEUTICS Given the successful development of GLP-1/GIP and GLP-1/ glucagon dual-agonists (293, 296), the next obvious step was to develop triple-agonists based on these three gut hormones [(318); Table 5]. One such molecule, termed YAG-glucagon (Table 5), is an analogue based on human glucagon with a number of amino acid substitutions to impart GIPR and GLP-1R agonism (304). The DPP-4 resistant analogue was demonstrated to be an effective tri-agonist in vitro, while twice-daily administration in DIO mice manifested in improved blood glucose, circulating insulin and enhanced insulin sensitivity (304). While this molecule has not surpassed preclinical stage, a couple of examples appear to be progressing well at present. LY3437943, a reported tri-agonist is currently undergoing phase I trials in management of obesity-diabetes (307). More data is available for HM15211, a tri-agonist employing a GLP-1/GIP/ glucagon peptide (sequence not available) attached to a human The recognised role of hyperglucagonaemia in the pathophysiology of diabetes, and the effectiveness of concomitant activation of GCGR, alongside GLP-1R by oxyntomodulin, raises an apparently conﬂicting question: can glucagon antagonists or glucagon agonists be utilised as a diabetes or obesity therapy? A similar question exists for therapeutic GIP analogues (306). In fact, both aspects are being explored although neither is, as yet, fully understood, or nearing ﬁnal stages of development. Conjugation of GLP-1 and Nuclear Hormones Beyond the incorporation of GLP-1 with other gut hormones, there is also growing interest concerning the conjugation of GLP- 1 with nuclear hormones like oestrogen, thyroid hormone (T3) and dexamethasone (326). In particular, the conjugation of GLP- 1-estrogen allows selective targeting of oestrogen receptors (ER) in GLP-1R expressing cells. This reduces obesity and improves dyslipidaemia and hyperglycaemia more so than sole activation of either GLP-1R or ER (327). In relation to the metabolic effects of these conjugates, preclinical studies in rodents have demonstrated that these conjugates act on reward centres within the supramammillary nucleus to induce an anorectic effect (328), which positively inﬂuences glycaemia. Moreover, such conjugates were demonstrated to improve beta-cell function and survival (329), which in a study employing a combination of GLP-1-estrogen and insulin in a DIO model of diabetes, allowed for a 60% reduction in the insulin dose compared to a control group of animals receiving insulin monotherapy (330). While conjugation to GLP-1 proves an effective method to prevent the oncogenic and gynaecological actions of oestrogen (331), distinct differences in the hormonal aetiologies of obesity in males and females have demonstrated that administration of such agents in different sexes of mice elicit subtle differences in obesity-related inﬂammation pathways (332, 333). Thus, the impact of gender in relation to the applicability of these agents needs to be further explored. Interestingly, the aforementioned GLP-1/xenin combination has been exploited further with the development of the triple- agonist exendin‐4/gastrin/xenin‐8‐Gln (335), a direct descendent of the previously discussed dual-agonist (303). This incorporates the hexapeptide gastrin into its sequence (Table 5), evoking the ability to agonise GLP-1R, CCK2R and NTSR1 in tandem (335). Preclinical studies with this peptide were promising, eliciting improved glycaemic control when administered twice daily in DIO diabetic mice over 21 days, through elevation in circulating insulin levels, improved insulin and GIP sensitivity, with encouraging reductions in fat mass, triglycerides and cholesterol levels (335). Moreover, this analogue has been further modiﬁed via the covalent attachment of a hexadecanoyl fatty acid to improve circulating half-life and duration of effect (Table 5), with twice daily administration in obese-diabetic ob/ob mice recapitulating the metabolic beneﬁts attained with the non-acylated form (305). Frontiers in Endocrinology \| www.frontiersin.org Glucagon Antagonists The therapeutic potential for glucagon suppression is clear, especially given that a synthetic analogue of the glucagon May 2021 \| Volume 12 \| Article 689678 Frontiers in Endocrinology \| www.frontiersin.org 17 Proglucagon-Derived Peptides as Therapeutics Lafferty et al. suppressing hormone amylin, termed “Pramlinitide”, is currently prescribed in the USA as an adjunct to insulin therapy (336). However, pramlintide is not a speciﬁc inhibitor of glucagon secretion, as it also is well known to slow the rate of gastric emptying and induce satiety. Thus, direct glucagon receptor antagonism may represent a more speciﬁc alternative in this regard. While it is true that many small-molecule glucagon antagonists exist (337–339), these have been discounted due to undesirable pharmacokinetic properties which led to rapid renal clearance and diminished effects (337, 340). Moreover, off-target safety concerns were present, including activation of peroxisome proliferator-activated receptor-delta (PPAR-d) (337), a transcription factor which plays roles in inﬂammation and certain cancers (341). That said, a few small molecules such as Eli Lilly and Co’s GRA LY2409021 have made it as far as phase II trials (342). These demonstrated promising reductions in HbA1c but were ultimately let down by undesirable side-effect proﬁles, often eliciting potentially dangerous elevations in liver enzymes (343). Hence, a view was taken that development of glucagon peptide-based antagonists could herald better tolerated compounds with improved pharmacokinetic and safety proﬁles. receptor and palmitic acid (PAL) was attached via linker molecules to substituted Lys residues at differing positions (346), a means of prolonging circulating half-life (351). Indeed, both molecules, as well as their non-acylated counterparts, were shown to be resistant to DPP-4 (346, 353). The compounds possessed strong antagonist properties, dose-dependently reducing glucagon-mediated cAMP production and insulin secretion together with counteracting glucagon-mediated hyperglycaemia in vivo (346). Further related analogue development resulted in synthesis and characterisation of desHis1Glu9(Lys30PAL)-glucagon and desHis1Glu9-glucagon-[mPEG] [(299, 354); Table 6]. These peptides were resistant to DPP-4 degradation (344, 348, 355) and lacked adverse metabolic or islet morphological effects when administered twice daily to lean mice (347). Preclinical testing of desHis1Glu9-glucagon and desHis1Glu9(Lys30PAL)-glucagon in HFF obese mice reversed obesity-driven hyperinsulinaemia and insulin resistance together with improvements in lipid proﬁle, glucose tolerance and increased pancreatic insulin stores (345). Glucagon Antagonists These studies are typical of others that have led to development of peptidergic glucagon receptor antagonists for T2DM, in particular the ﬁrst reported antagonistic, glucagon analogue [l-N alphatrinitrophenylhistidine, 12-homoarginine]- glucagon, which elicited decreases in circulating glucose of up to 65% with continuous infusion in anaesthetised rats (356). However, despite this preclinical promise peptide-based agents were largely abandoned at this point possibly due to short half- life in pursuit of small-molecule antagonists (357). Logical design of such compounds took the sequence of native glucagon (Table 6), modifying the structure, paying particular attention to previously identiﬁed important residues for GCGR agonism, namely N-terminal His1, Gly4 and Asp9 residues (349– 353), whilst also ensuring that they are resistant to the actions of DPP-4 [(349); Figure 2]. Two such analogues termed desHis1Pro4Glu9(Lys12PAL)-glucagon and desHis1Pro4Glu9 (Lys30PAL)-glucagon [(346); Table 6], employed simple amino acid substitutions at residues 4 and 9, while His1 was deleted to produce compounds with the potential to effectively block the To date, no glucagon antagonist has reached regulatory approval, with previous safety concerns raised over hypoglycaemia (358), unfavourable alterations in serum lipid TABLE 6 \| Glucagon antagonist peptides for T2DM. Peptide Name AA Sequence Development Stage Reference Native glucagon HSQGTFTSDYSKYLDSRRAQDFVQWLMNT N/A (10) desHis1Glu9-glucagon SQGTFTSEYSKYLDSRRAQDFVQWLMNT Preclinical (344, 345) desHis1Pro4Glu9(Lys12PAL)-glucagon SQPTFTSEYSK(PAL)YLDSRRAQDFVQWLMNT Preclinical (346, 347) desHis1Pro4Glu9(Lys30PAL)-glucagon SQPTFTSEYSKYLDSRRAQDFVQWLMNTK(PAL) Preclinical (345, 346, 348) desHis1Glu9-glucagon-[mPEG] SQGTFTSEYSKYLDSRRAQDFVQWLMNT-[mPEG] Preclinical (346) Amino acid sequences are provided in their single-letter abbreviation format. Modiﬁcations from native sequences are highlighted by red lettering. Current development stages are provided for each. “mPEG” indicates mini-polyethylene glycol addition. “PAL” indicates the addition of a palmitic fatty acid chain. TABLE 7 \| Glucagon and related peptide analogues at preclinical stage for T2DM. Peptide Name AA Sequence Target Receptor Reference Native glucagon HSQGTFTSDYSKYLDSRRAQDFVQWLMNT CGCR (10) N-Acetyl-glucagon Ac-HSQGTFTSDYSKYLDSRRAQDFVQWLMNT GCGR (365) (D-Ser2)glucagon HDSQGTFTSDYSKYLDSRRAQDFVQWLMNT GCGR/GLP-1R (365) (D-Ser2)glucagon-exe HDSQGTFTSDYSKYLDSRRAQDFVQWLMNTPSSGAPPPS GCGR/GLP-1R (365) Dogﬁsh Glucagon HSEGTFTSDYSKYMDNRRAKDFVQWLMSTKRNG GCGR/GLP-1R (366, 367) (D-Ala2)dogﬁsh glucagon HDAEGTFTSDYSKYMDNRRAKDFVQWLMSTKRNG GCGR/GLP-1R (366, 367) (D-Ala2)dogﬁsh glucagon-exendin-4(31-39) HDAEGTFTSDYSKYMDNRRAKDFVQWLMSTKRNGPSSGAPPPS GCGR/GLP-1R (366, 367) (D-Ala2)dogﬁsh glucagon-Lys30-g-glutamyl-PAL HDAEGTFTSDYSKYMDNRRAKDFVQWLMSTK(*PAL)RNG GCGR/GLP-1R (366, 367) Paddleﬁsh glucagon HSQGMFTNDYSKYLEEKRAKEFVEWLKNGKS GCGR/GLP-1R (248) Amino acid sequences are provided in their single-letter abbreviation format. Modiﬁcations from native sequences are highlighted by red lettering. The receptor targets for each molecule are provided. A “D” preﬁx before a residue indicates inclusion of the enantiomer for the naturally-occurring L form of the residue. “Ac” represents an N-terminal acetylation. “mPEG” indicates mini-polyethylene glycol addition. “PAL” indicates the addition of a palmitic fatty acid chain. 5. Bell GI, Pictet RL, Rutter WJ, Cordell B, Tischer E, Goodman HM. Sequence of the Human Insulin Gene. Nature (1980) 284:26–32. doi: 10.1038/ 284026a0 6. Davies MJ, D’Alessio DA, Fradkin J, Kernan WN, Mathieu C, Mingrone G, et al. Management of Hyperglycemia in Type 2 Diabetes, 2018. a Consensus Report by the American Diabetes Association (ADA) and the European Association for the Study of Diabetes (EASD). Diabetes Care (2018) 41:2669–701. doi: 10.2337/dci18-0033 7. Wilkinson S, Douglas I, Stirnadel-Farrant H, Fogarty D, Pokrajac A, Smeeth L, et al. Changing Use of Antidiabetic Drugs in the UK: Trends in Glucagon Antagonists Amino acid sequences are provided in their single-letter abbreviation format. Modiﬁcations from native sequences are highlighted by red lettering. The receptor targets for each molecule are provided. A “D” preﬁx before a residue indicates inclusion of the enantiomer for the naturally-occurring L form of the residue. “Ac” represents an N-terminal acetylation. “mPEG” indicates mini-polyethylene glycol addition. “PAL” indicates the addition of a palmitic fatty acid chain. May 2021 \| Volume 12 \| Article 689678 Frontiers in Endocrinology \| www.frontiersin.org 18 Proglucagon-Derived Peptides as Therapeutics Lafferty et al. levels and liver enzymes (359, 360), as well as the potential for malignant hyperplasia of alpha-cells (361). Intriguingly, a similar tale is true for GLP-1R agonists, with a number of small molecule examples dotted through the literature (362), however none have managed to recapitulate the success of peptidergic agents. Preclinical data with peptide-based antagonists indicate more favourable side-effect proﬁles than small-molecules (345, 347). Thus, while work continues on small-molecule antagonists, such as RVT-1502, which has recently progressed through phase II trials, demonstrating reductions in HbA1c of up to 1% over 12 weeks treatment, concerns over liver function still remain, and the compound has not ascended to phase III trials (363). Such concerns may lead to an upsurge in interest for peptide-based glucagon antagonists. of diabetes [(248, 366, 367); Table 7]. Furthermore, studies using GLP1-R KO mice and cell lines indicated that beneﬁts on glucose tolerance, beta-cell function, insulin sensitivity and circulating triglycerides were mediated via dual GCGR and GLP-1R agonism (248, 366). Thus, in relation to management of T2DM, inclusion of GCGR agonist in multiagonist molecules appears to be where the future novelty lies for such agents. Glucagon Agonists Given the use of glucagon to rescue severe insulin-induced hypoglycaemia T1DM (53) and its ascribed role in the hyperglycaemia of diabetes (35, 51), the concept of using glucagon agonists therapeutically initially seems illogical. However, the surprising effectiveness of dual or triple agonism indicates that weight loss and increased energy expenditure associated with GCGR agonism can be exploited when the hyperglycaemic actions of the hormone are counteracted by the incretins GLP-1 and/or GIP (357, 364). Several approaches have been explored to generate such, potentially useful, enzyme-resistant GCGR agonists including (D- Ser2)glucagon, where a D-amino acid substitution has been employed to impart DPP-4 resistance more effectively than N- acetyl-glucagon [(365); Table 7]. Insulin-releasing activity was maintained, but when further modiﬁed to generate (D-Ser2) glucagon-exe, an analogue with the nine C-terminal amino acid residues of exendin(1–39) (Table 7), clear antidiabetic beneﬁts were induced (365). Importantly, inclusion of the C-terminal nonapeptide from exendin(1–39) in this molecule imparts the ability to agonise GLP-1R as well as GCGR, as demonstrated by reduced effectiveness in GLP-1R KO mice (365). Thus, twice daily administration of (D-Ser2)glucagon-exe in HFF mice improved glucose tolerance, insulin sensitivity and islet morphology, while improvements in energy expenditure, O2 consumption and physical activity together with reduced food-intake led to decreased body weight and inﬂuenced glycaemic improvement (365). AUTHOR CONTRIBUTIONS All authors contributed to the article and approved the submitted version. CONCLUDING REMARKS The application of proglucagon-derived peptides (PGDPs) in the management of conditions such as T2DM represents the pinnacle of a remarkable story in peptide discovery and rational drug design. It was shear perseverance which led to the elucidation of proglucagon almost six decades after that of glucagon (9, 11–16). Rapid discoveries followed of GLP-1 and GLP-2 (15, 16), both of which have been successfully exploited by peptide chemistry approaches to generate fully approved medications. While innovation has witnessed the production of increasingly long-acting agents, multi-action unimolecular agonists and novel delivery methods (67, 68, 70, 73–75, 195– 197), there is still a growing need for ever more effective agents to counter obesity, diabetes and a host of other degenerative diseases. Better understanding of the physiology of PGDPs and their various roles in the likes of cognition, bone turnover, cardiovascular function, fertility and liver function (157, 174, 185, 334, 368, 369), may herald important future uses for proglucagon-derived therapeutics. ACKNOWLEDGMENTS Research in the authors’ laboratories on gut peptide therapeutics has been generously supported over many years by Diabetes UK, European Foundation for the Study of Diabetes, Diabetes Research and Wellness Foundation, Invest Northern Ireland, Northern Ireland Department for Education, and Ulster University Strategic Funding. A number of naturally occurring, piscine-derived, glucagon peptides such as dogﬁsh glucagon (and its analogues) and paddleﬁsh glucagon have also been shown to possess potent antidiabetic/anti-obesity potential in cellular and animal models 1. Edkins JS. The Chemical Mechanism of Gastric Secretion. J Physiol (1905) 34:133–44. doi: 10.1113/jphysiol.1906.sp001146 2. 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https://openalex.org/W2812697226	https://europepmc.org/articles/pmc6135309?pdf=render	English	null	Rheumatic Heart Disease with Multiple Systemic Emboli: A Rare Occurrence in a Single Subject	Curēus	2,018	cc-by	3,685	Rheumatic Heart Disease with Multiple Systemic Emboli: A Rare Occurrence in a Single Subject Ravi R. Pradhan , Ashish Jha , Gaurav Nepal , Manju Sharma 1 2 3 4 1. Internal Medicine, Institute of Medicine, Tribhuvan University Teaching Hospital, Kathmandu, NPL 2. Medicine, Institute of Medicine ( Iom ), Tribhuvan University Teaching Hospital, Kathmandu, NPL 3. Maharajgunj Medical Campus, Tribhuvan University Institute of Medicine, Kathmandu, NPL 4. Cardiology, Manmohan Cardiothoracic Vascular and Transplant Center, Iom-Tuth, Kathmandu, NPL  Corresponding author: Ravi R. Pradhan , drravipradhan@iom.edu.np Disclosures can be found in Additional Information at the end of the article DOI: 10.7759/cureus.2964 Abstract Valvular heart disease is one of the more common diseases in low- and middle-income countries, when associated with atrial fibrillation (AF), carries a risk of multisystemic embolizations. We report a case of 37-year-old man with multiple systemic emboli consisting of acute ischemic stroke, acute myocardial infarction, and acute limb ischemia. This is a rare occurrence in a single subject. The patient had a background of rheumatic heart disease (RHD) involving severe mitral stenosis (MS) with AF, who was not compliant with his medications. A computed tomography (CT) scan of the head showed right-sided ischemic stroke involving more than one-third of the middle cerebral artery territory. An electrocardiogram (ECG) showed AF and ST-segment elevation in V4 to V6. Cardiac enzymes were elevated. A transthoracic echocardiogram demonstrated hypokinetic left ventricular anterolateral wall, severe MS, and a left atrial clot. An arterial Doppler of the right lower limb showed an occluding thrombus of the right common femoral artery and right popliteal artery with no flow in color Doppler. Patient adherence to medications in cases of RHD prevents devastating outcomes. Categories: Cardiac/Thoracic/Vascular Surgery, Cardiology, Internal Medicine Keywords: rheumatic heart disease, mitral stenosis, acute ischemic stroke, acute myocardial infarction, acute limb ischemia g / / g y, gy, Keywords: rheumatic heart disease, mitral stenosis, acute ischemic stroke, acute myocardial infarction, acute limb ischemia Open Access Case Report Open Access Case Report Received 06/29/2018 Review began 07/05/2018 Review ended 07/10/2018 Published 07/11/2018 © Copyright 2018 Pradhan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License CC-BY 3.0., which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. How to cite this article Pradhan R R, Jha A, Nepal G, et al. (July 11, 2018) Rheumatic Heart Disease with Multiple Systemic Emboli: A Rare Occurrence in a Single Subject. Cureus 10(7): e2964. DOI 10.7759/cureus.2964 Open Access Case Report Open Access Case Report Case Presentation A 37-year-old man from the Himalayan region of Nepal presented with swelling of the right leg for ten days and sudden onset weakness of the left half of the body for two days. The swelling of the right leg had an insidious onset and was gradually progressive. The pain disabled the patient to move his right leg. Meanwhile, the patient developed weakness of the left half of the body, predominantly in the upper left limb. The patient also developed slurring of speech and deviation of the face towards the right side. He denied any history of chest pain, diaphoresis, shortness of breath, loin pain, nausea or vomiting. He had decreased urine output and red colored urine and was a non-smoker and non-alcoholic. There was no history of hypertension, chronic kidney disease, and diabetes mellitus in this patient. On detail inquiry, the patient gave a history of recurrent throat infection during childhood, however, he was not medically managed then. Three years back, when he visited a cardiac centre with complaints of shortness of breath and palpitations, a diagnosis of RHD with severe MS and AF was made. On reviewing the past record of the patient, a significantly elevated serum antistreptolysin O (ASO) titer was seen. He was planned for per-cutaneous trans-mitral commissurotomy (PTMC) by his physician. However, he lost the follow-up and was non- compliant to his medications. On physical examination, his blood pressure measured 110/70 mm Hg; pulse rate was irregularly irregular at 77 beats per minute; respiratory rate was 16 breaths per minute, and body temperature measured 37.1 oC orally. The patient was alert, conscious, and cooperative. There were no appreciable pallor, icterus, clubbing, splinter hemorrhages, rashes and cyanosis. On cardiovascular systemic examination, the first heart sound (S1) was variable in intensity and second heart sound (S2) was normal. There was a low pitched rough rumbling mid-diastolic murmur at the apex heard best when the patient was lying on the left side with breath held in expiration using the bell of the stethoscope. There was a high pitched blowing pansystolic murmur at the left lower sternal border. Examination of the abdomen was unremarkable. On neurological examination, the patient had a left-sided central facial nerve palsy, muscle strengths in the left upper and lower limbs were 2/5 and 3/5 respectively, and there was an ipsilateral Babinski sign. A thorough eye examination along with fundoscopy was unremarkable. This case demonstrates a rare and atypical presentation of RHD with severe mitral stenosis (MS) and atrial fibrillation (AF) leading to multiple systemic emboli, that presented with acute ischemic stroke, acute myocardial infarction, and acute limb ischemia. Early recognition and aggressive management led to a satisfactory outcome in this case. Introduction Acute rheumatic fever (ARF) results from the body’s autoimmune response to a throat infection caused by Streptococcus pyogenes, a group A beta-hemolytic Streptococcus. Rheumatic heart disease (RHD) refers to the long-term cardiac damage caused by either a single severe episode or multiple recurrent episodes of ARF. RHD involving mitral valve causes inflammation and fibrosis with disruption of the atrial architecture. There is increased left atrial pressure that contributes to left atrial dilatation and increased wall stress predisposing to the development of atrial fibrillation leading to left atrial clot and subsequent systemic embolism. Received 06/29/2018 Review began 07/05/2018 Review ended 07/10/2018 Published 07/11/2018 © Copyright 2018 Pradhan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License CC-BY 3.0., which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. © Copyright 2018 Pradhan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License CC-BY 3.0., which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. RHD ranks among the important non-communicable diseases in low- and middle-income countries, and nearly eradicated in high-income countries. It is a sentinel of social inequality and a physical manifestation of poverty, and thus continues to be a substantial health care challenge in less privileged regions of the world [1]. Nevertheless, it is a cause of significant morbidity and mortality. 2018 Pradhan et al. Cureus 10(7): e2964. DOI 10.7759/cureus.2964 2018 Pradhan et al. Cureus 10(7): e2964. DOI 10.7759/cureus.2964 Case Presentation Local examination of the right leg revealed swelling below the level of the knee and blackish discoloration of toes and lower third of the leg on inspection. Vesicles could be seen over the lateral aspects of the leg and dorsum of the foot. The limb was cold to touch and tender on palpation. Dorsalis pedis and posterior tibial pulses were not palpable (Figure 1). 2 of 11 FIGURE 1: Right leg with swelling below the level of knee and blackish discoloration of toes and lower third of the leg Vesicles could be seen over the lateral aspects of the leg and dorsum of the foot. The limb was cold to touch and tender on palpation. Dorsalis pedis and posterior tibial pulses were not palpable. FIGURE 1: Right leg with swelling below the level of knee and blackish discoloration of toes and lower third of the leg FIGURE 1: Right leg with swelling below the level of knee and blackish discoloration of toes and lower third of the leg Vesicles could be seen over the lateral aspects of the leg and dorsum of the foot. The limb was cold to touch and tender on palpation. Dorsalis pedis and posterior tibial pulses were not palpable. On investigation, electrocardiogram (ECG) showed ST segment elevation of more than two millimetre (mm) in chest leads V4, V5 and V6, suggesting ST elevation myocardial infarction (STEMI) and AF with normal ventricular rate (Figure 2). Transthoracic echocardiography showed thickened, calcified mitral valve leaflets with mitral valve area (MVA) of 1.01 cm2 on planometry, mitral valve pressure half-time (PHT) of 344 millisecond, and mean pressure gradient of 9 mmHg (features suggestive of rheumatic severe mitral stenosis), left atrial enlargement and a left atrial clot measuring 12.8 mm x 13.2 mm, severe tricuspid regurgitation (TR) (pressure gradient of 57.8 mm Hg), hypokinesia of antero-lateral wall of the left ventricle with left ventricle ejection fraction of 35% to 40% (Figures 3-5). 3 of 11 FIGURE 2: Electrocardiogram showing atrial fibrillation with normal ventricular rate and ST-elevation from V4 to V6 FIGURE 3: Transthoracic echocardiography on short axis view showing thickened, calcified mitral valve leaflets with MVA of 1.01 cm2 on planometry 2018 Pradhan et al. Cureus 10(7): e2964. DOI 10.7759/cureus.2964 FIGURE 2: Electrocardiogram showing atrial fibrillation with normal ventricular rate and ST-elevation from V4 to V6 FIGURE 2: Electrocardiogram showing atrial fibrillation with normal ventricular rate and ST-elevation from V4 to V6 FIGURE 2: Electrocardiogram showing atrial fibrillation with normal ventricular rate and ST-elevation from V4 to V6 FIGURE 3: Transthoracic echocardiography on short axis view showing thickened, calcified mitral valve leaflets with MVA of 1.01 cm2 on planometry MVA: mitral valve area FIGURE 3: Transthoracic echocardiography on short axis view showing thickened, calcified mitral valve leaflets with MVA of 1.01 cm2 on planometry MVA: mitral valve area 4 of 11 2018 Pradhan et al. Cureus 10(7): e2964. DOI 10.7759/cureus.2964 2018 Pradhan et al. Cureus 10(7): e2964. DOI 10.7759/cureus.2964 FIGURE 4: Transthoracic echocardiography on parasternal long axis view showing left atrial enlargement, left atrial clot measuring 12.8 mm x 13.2 mm (arrow), and thickened and calcified mitral valve LV: left ventricle; LA: left atrium; RV: right ventricle; D: diameter. FIGURE 4: Transthoracic echocardiography on parasternal long axis view showing left atrial enlargement, left atrial clot measuring 12.8 mm x 13.2 mm (arrow), and thickened and calcified mitral valve LV: left ventricle; LA: left atrium; RV: right ventricle; D: diameter. LV: left ventricle; LA: left atrium; RV: right ventricle; D: diameter. 5 of 11 2018 Pradhan et al. Cureus 10(7): e2964. DOI 10.7759/cureus.2964 V: velocity; PG: pressure gradient. FIGURE 5: Transthoracic echocardiogram in four-chamber view with continuous wave (CW) mode showing severe tricuspid regurgitation with a pressure gradient of 57.8 mmHg V: velocity; PG: pressure gradient. FIGURE 5: Transthoracic echocardiogram in four-chamber view with continuous wave (CW) mode showing severe tricuspid regurgitation with a pressure gradient of 57.8 mmHg V: velocity; PG: pressure gradient. Chest radiography revealed cardiomegaly with cardiothoracic ratio 70%, straightening of left heart border, double right heart border, cephalization (upturned moustache sign or Antler sign), and widened carinal angle (more than 90o) typical of left atrial enlargement. The cardiac border was displaced laterally and downwards implying left ventricular enlargement (Figure 6). 6 of 11 2018 Pradhan et al. Cureus 10(7): e2964. DOI 10.7759/cureus.2964 FIGURE 6: Chest radiograph (posteroanterior view) showing cardiomegaly with cardiothoracic ratio 70%, straightening of left heart border (yellow line), double right heart border (white arrow), cephalization (white circle), and widened carinal angle typical of left atrial enlargement The cardiac border was displaced laterally and downwards implying left ventricular enlargement. FIGURE 6: Chest radiograph (posteroanterior view) showing cardiomegaly with cardiothoracic ratio 70%, straightening of left heart border (yellow line), double right heart border (white arrow), cephalization (white circle), and widened carinal angle typical of left atrial enlargement The cardiac border was displaced laterally and downwards implying left ventricular enlargement. A computed tomography (CT) scan of the head showed right-sided ischemic stroke involving more than one-third of the middle cerebral artery territory (Figure 7). A computed tomography (CT) scan of the head showed right-sided ischemic stroke involving more than one-third of the middle cerebral artery territory (Figure 7). 7 of 11 2018 Pradhan et al. Cureus 10(7): e2964. DOI 10.7759/cureus.2964 FIGURE 7: A computed tomography (CT) scan of the head showed right-sided ischemic stroke (white arrow) involving more than one-third of the middle cerebral artery territory FIGURE 7: A computed tomography (CT) scan of the head showed right-sided ischemic stroke (white arrow) involving more than one-third of the middle cerebral artery territory Arterial doppler of the right leg showed nearly occluding echogenic content in right femoral artery with minimal triphasic flow and occluding echogenic content in right popliteal artery with no flow in color and spectral Doppler study. No flow was noted in the right tibioperoneal trunk, anterior tibial artery, posterior tibial artery, and dorsalis pedis artery. Venous Doppler of the right leg demonstrated deep vein thrombosis (DVT) involving right femoral and popliteal vein. Abdominal ultrasonography was normal. 8 of 11 2018 Pradhan et al. Cureus 10(7): e2964. DOI 10.7759/cureus.2964 At workup, the patient had total creatine phosphokinase (CPK) 10000 unit per liter (U/L) (normal= 10 to 120 U/L), creatine phosphokinase-MB (CPK-MB) 3000 U/L (normal= 0-25 U/L), troponin I 40.6 nanogram per mililiter (ng/ml) (positive for > 0.12 ng/ml), total white cell count 18,210 per microliter (uL) (normal= 4000-11000 per uL), hemoglobin 15.4 gram per deciliter (g/dl), creatinine 3.28 milligram per deciliter (mg/dl) (normal= 0.6 to 1.1 mg/dl), sodium 122 milliequivalent per liter (mEq/L) (normal= 135 to 145 mEq/L), potassium 6.2 mEq/L (normal= 3.5 to 4.5 mEq/L), total billirubin 1.4 mg/dl (normal is < 1 mg/dl), alanine trasaminase (ALT) 516 U/L (normal= 30 to 65 U/L), aspartate trasaminase (AST) 2030 U/L (normal= 0 to 45 U/L), alkaline phosphatase 117 U/L (normal= 40 to 140 U/L), total protein 6.8 g/dl (normal= 6.4 to 8.2 g/dl), albumin 3.2 g/dl (normal= 3.8 to 4.9 g/dl), and prothrombin time (PT) was 13.3 second with international normalized ratio (INR) 1.1 (control= 12 second). Serum ASO titer was not done as there was no active rheumatic activity. A provisional diagnosis of RHD with severe MS and TR with AF leading to multiple systemic embolizations was formulated. Anticoagulation was started with low molecular weight heparin (enoxaparin 60 mg subcutaneous twice daily). The patient received aspirin 75 mg and digoxin 0.125 mg once daily. Hyperkalaemia was managed medically with intravenous calcium gluconate one gram over 10 minutes, 50 ml of 50% dextrose with regular insulin of 10 U and salbutamol nebulization. Subsequently, the patient underwent above knee amputation of the right lower limb. His liver and kidney function tests were monitored until they were normalized. He was discharged on warfarin 6 mg, aspirin 75 mg, spirinolactone 25 mg, enalapril 2.5 mg, digoxin 0.125 mg once daily and frusemide 20 mg twice daily. The patient was adviced for regular follow-up for monitoring his clinical status and adherence to medications. He paid two visits (one week apart) to the hospital after discharge and his clinical conditions were improving. 2018 Pradhan et al. Cureus 10(7): e2964. DOI 10.7759/cureus.2964 study (n=376) conducted by Jose VJ et al. [6], the overall prevalence of coronary artery disease in a group of patients with rheumatic heart disease undergoing valve surgery was 12.2%. In our case, the patient was diagnosed with STEMI based on ECG changes, cardiac biomarkers, and echocardiography, however, he didn’t give a history of chest pain. Primary percutaneous coronary intervention (PPCI) was not performed in this patient as there was no evidence of ongoing myocardial ischemia and no features suggestive of heart failure or cardiogenic shock. Acute peripheral arterial occlusion is characterized by "the six Ps": pain of sudden onset, pallor, pulselessness, paresthesias, paresis, and prostration with the symptoms of shock. Femoral bifurcation, the superficial femoral artery, and the popliteal artery are the most common sites for lodgment of embolus [7] and diseased heart is the main source of arterial emboli [8]. Our case is in accordance with the above-mentioned studies. Surprisingly, our patient also suffered from DVT in the right limb, which is an unusual finding. This might have arisen due to a restriction of movement in the right limb caused by ischemic pain. Apart from cerebral, coronary and peripheral arterial embolisms, such patients are also vulnerable for renal and mesenteric embolisms. Renal infarction mostly presents as abdominal pain and hematuria with an evident rise in serum lactate dehydrogenase [9]. Renal impairment was present in our case and the possible causes of renal impairment could be renal ischemia or rhabdomyolysis of the right ischemic limb. Rhabdomyolysis is more likely to be the cause of renal impairment in our case as there was evidence of hyperkalaemia, raised CPK and liver enzymes. In 50% of cases of rhabdomyolysis, urine could be normal in color, as with our case. Though the ultrasonography of the abdomen was normal, CT angiography of the abdomen to rule out renal ischemia was not performed in our case. After amputation of the ischemic limb, renal impairment and liver enzymes normalized. Rheumatic heart disease is effectively controlled by long-term antibiotic prophylaxis. Duration of therapy depends on the severity of cardiac involvement. Lifelong anticoagulation is the mainstay of prophylaxis for a cardioembolic event in patients of mitral stenosis with atrial fibrillation. This is, to the best of our knowledge, the first case report of it's kind in medical literature. Disclosures Human subjects: Consent was obtained 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. Discussion Of all native valvular diseases, rheumatic mitral valve disease carries the highest risk of systemic embolization. The incidence of AF in patients with RHD is 43.61% [2]. The presence of AF increases the risk of systemic embolization and mitral stenosis carries a higher risk of embolization compared with mitral regurgitation [2]. After the first episode of embolization, recurrent embolization occurs in 30%-65% of patients; with more than half of recurrences occurring during the first one year [3]. Similarly, in our case, the cardioembolic phenomenon associated with RHD was the prime cause of the patient's illness. The non-valvular atrial fibrillation (NVAF) increases the risk for ischemic stroke by five-fold, the risk is even greater among patients with mitral stenosis, increasing up to 17-fold [4]. Our patient had severe MS with AF and was at increased risk of cardioembolic stroke. Stroke in patients with AF is generally more severe and the outcome is markedly poorer than in patients with sinus rhythm. Studies in patients with both rheumatic mitral stenosis and atrial fibrillation have shown that warfarin is effective in preventing cerebral embolism [5]. Mitral valve repair and mitral valve replacement are also considered in the prevention of cerebral embolism for patients with hemodynamically significant rheumatic mitral stenosis or insufficiency [3]. When the afflicted patients are young, the tragic consequences for family, friends, and occupation are particularly catastrophic and unexpected as with our case. In a developing country like Nepal, where there is no facility for regular monitoring of PT/INR, anticoagulation with warfarin may not be feasible. However, the evidence regarding the use of newer oral anticoagulants like rivaroxaban and dabigatran in rheumatic valvular AF is yet to be proven. In this case, we discharged the patient with warfarin as an anticoagulant with provision for regular monitoring of PT/INR risking the adherence of the patient with it. RHD with mitral stenosis presenting for the first time as acute STEMI is rare. In a prospective RHD with mitral stenosis presenting for the first time as acute STEMI is rare. In a prospective 9 of 11 2018 Pradhan et al. Cureus 10(7): e2964. DOI 10.7759/cureus.2964 Conclusions Multiple systemic embolizations must always be considered in patients with valvular heart disease, especially in MS associated with AF. Clinical suspicion and follow up of such patients confirming adherence to prophylaxis can prevent adverse outcomes. 2018 Pradhan et al. Cureus 10(7): e2964. DOI 10.7759/cureus.2964 We would like to thank Dr. Smriti Shakya for her invaluable support. We would like to thank Dr. Smriti Shakya for her invaluable support. 2018 Pradhan et al. Cureus 10(7): e2964. DOI 10.7759/cureus.2964 Acknowledgements 10 of 11 2018 Pradhan et al. Cureus 10(7): e2964. DOI 10.7759/cureus.2964 References 1. Shrestha NR, Karki P, Mahto R, et al.: Prevalence of subclinical rheumatic heart disease in eastern Nepal: a school-based cross-sectional study. JAMA Cardiol. 2016, 1:89-96. 10.1001/jamacardio.2015.0292 2. Sharma SK, Verma SH: A clinical evaluation of atrial fibrillation in rheumatic heart disease . J Assoc Physicians India. 2015, 63:22-5. 3. Leary M, Caplan L: Cardioembolic stroke: an update on etiology, diagnosis and management . Ann Indian Acad Neurol. 2008, 1:52-63. 4. Wolf PA, Abbott RD, Kannel WB: Epidemiologic assessment of chronic atrial fibrillation and risk of stroke: the Framingham study. Arch Intern Med. 1987, 147:1561-4. 10.1001/archinte.1987.00370090041008 5. Whitlock RP, Sun JC, Fremes SE, et al.: Antithrombotic and thrombolytic therapy for valvular disease: antithrombotic therapy and prevention of thrombosis: American college of chest physicians evidence-based clinical practice guidelines. Chest. 2012, 141:576-600. 10.1378/chest.11-2305 5. Whitlock RP, Sun JC, Fremes SE, et al.: Antithrombotic and thrombolytic therapy for valvular disease: antithrombotic therapy and prevention of thrombosis: American college of chest physicians evidence-based clinical practice guidelines. Chest. 2012, 141:576-600. 10.1378/chest.11-2305 6. Jose VJ, Gupta SN, Joseph G, et al.: Prevalence of coronary artery disease in patients with rheumatic heart disease in the current era. Indian Heart J. 2004, 56:129-31. 7. Health Quality Ontario: Stenting for peripheral artery disease of the lower extremities: an evidence-based analysis. Ont Health Technol Assess Ser. 2010, 10:1-88. 8. Oneglia C, Rusconi C: Left atrial appendage thrombus as a source of peripheral embolism: TE evidence of direct relationship. Echocardiography. 2001, 18:389-90. 10.1046/j.1540- 8175.2001.00389.x 9. Hazanov N, Somin M, Attali M, et al.: Acute renal embolism. Forty-four cases of renal infarction in patients with atrial fibrillation. Medicine. 2004, 83:292-9. 10.1097/01.md.0000141097.08000.99 11 of 11
https://openalex.org/W3114946797	http://qspace.qu.edu.qa/bitstream/10576/17260/1/Hassani_2020_IOP_Conf._Ser.__Mater._Sci._Eng._991_012101.pdf	English	null	Formulation and antioxidant properties of curcumin gum Arabic nanoparticles for delivery to cancer cells	IOP conference series. Materials science and engineering	2,020	cc-by	2,562	IOP Conference Series: Materials Science and Engineering IOP Conference Series: Materials Science and Engineering PAPER • OPEN ACCESS PAPER • OPEN ACCESS This content was downloaded from IP address 89.211.163.77 on 24/12/2020 at 15:14 Formulation and antioxidant properties of curcumin gum Arabic nanoparticles for delivery to cancer cells To cite this article: A Hassani et al 2020 IOP Conf. Ser.: Mater. Sci. Eng. 991 012101 To cite this article: A Hassani et al 2020 IOP Conf. Ser.: Mater. Sci. Eng. 991 012101 Corresponding author: abdalmonemdoolaanea@yahoo.com;samni2004@yahoo.co.uk Abstract. Curcumin nanoparticles )Cur/GANPs( were formulated based on gum arabic )GA( as a stabilizer coatings for nanoparticles through efficient synthesis approach . The current study investigated the antioxidant properties and antihypertensive activity of curcumin )Cur( using various established in vitro assays, such as 1,1-diphenyl-2-picrylhydrazyl )DPPH( as well as angiotensin converting enzyme (ACE( inhibitory activity. The in vitro cytotoxicity of Cur/GANPs against human liver cancer )HepG2 (, and colon cancer )HT29 ( was investigated. The exposure of human cancer cells to Cur/GANPs )1.56-100 µg/ml ( using MTT )3-)4,5- dimethylthiazol-2-yl (2,5-diphenyl tetrazolium bromide( has revealed that the Cur/GANPs inhibited the growth of cell lines examined in a dose dependent manner. Hence, Cur/GANPs nanoparticles may have great potential to be applied for cancer treatment. Content from this work may be used under the terms of the Creative Commons Attribution 3.0 licence. Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI. Published under licence by IOP Publishing Ltd 1 To cite this article: A Hassani et al 2020 IOP Conf. Ser.: Mater. Sci. Eng. 991 012101 View the article online for updates and enhancements. This content was downloaded from IP address 89.211.163.77 on 24/12/2020 at 15:14 ICCEIB 2020 IOP Conf. Series: Materials Science and Engineering 991 (2020) 012101 IOP Publishing doi:10.1088/1757-899X/991/1/012101 Conf. Series: Materials Science and Engineering 991 (2020) 012101 doi:10.1088/1757-899X/991/1/0121 Formulation and antioxidant properties of curcumin gum Arabic nanoparticles for delivery to cancer cells A Hassani 1,2, S A Hussain2, H H Enezei3, S Saeed4,W N Ibrahim 5, M S Al- Qubaisi5, H Zentou2, R Rosli6, A A Doolaanea1* A Hassani 1,2, S A Hussain2, H H Enezei3, S Saeed4,W N Ibrahim 5, M S Al- Qubaisi5, H Zentou2, R Rosli6, A A Doolaanea1 A Hassani 1,2, S A Hussain2, H H Enezei3, S Saeed4,W N Ibrahim 5, M S Al- Qubaisi5, H Zentou2, R Rosli6, A A Doolaanea1 1Department of Pharmaceutical Technology, Faculty of Pharmacy, International Islamic University Malaysia, 25200, Kuantan, Malaysia 2Department of Chemical and Environmental Engineering, Universiti Putra Malaysia, Serdang 43400, Malaysia 3Department of Oral & Maxillofacial Surgery, Collage of Dentistry, University of Anbar, Ramadi-Iraq 4Department of Anaesthesiology and Critical Care, Faculty of Medicine, International Islamic University Malaysia, 25200, Kuantan, Malaysia 5 Department of Biomedical sciences, College of Health sciences, QU Health, Qatar University, Doha, Qatar 6UPM-MAKNA Cancer Research Laboratory, Institute of Bioscience, Universiti Putra Malaysia, Serdang 43400, Malaysia 1Department of Pharmaceutical Technology, Faculty of Pharmacy, International Islamic University Malaysia, 25200, Kuantan, Malaysia 2Department of Chemical and Environmental Engineering, Universiti Putra Malaysia, Serdang 43400, Malaysia 3Department of Oral & Maxillofacial Surgery, Collage of Dentistry, University of Anbar, Ramadi-Iraq 4 4Department of Anaesthesiology and Critical Care, Faculty of Medicine, International Islamic University Malaysia, 25200, Kuantan, Malaysia 5 Department of Biomedical sciences, College of Health sciences, QU Health, Qatar University, Doha, Qatar 6 6UPM-MAKNA Cancer Research Laboratory, Institute of Bioscience, Universiti Putra Malaysia, Serdang 43400, Malaysia 6UPM-MAKNA Cancer Research Laboratory, Institute of Bioscience, Universiti Putra Malaysia, Serdang 43400, Malaysia 6UPM-MAKNA Cancer Research Laboratory, Institute of Bioscience, Universiti Putra Malaysia, Serdang 43400, Malaysia *Corresponding author: abdalmonemdoolaanea@yahoo.com;samni2004@yahoo.co.uk 1. Introduction Nanomedicine has potentials to develop cancer therapies and diagnostics [1]. Nanoparticles made of chemotherapeutic hydrophobic drugs like curcumin have potential application in the treatment of solid tumors such us liver cancer and breast cancer [2-3]. Curcumin therapeutic efficacy towards several diseases is challenged with its poor solubility, rapid elimination, and low bioavailability [4-5]. The advancement in polymeric nanocarriers provides an effective approach to enhance the therapeutic efficiency of curcumin. The use of gum arabic as a polysaccharide in encapsulation process can play an important role in improving the antioxidant properties of curcumin due to its biodegradability, cost effectiveness, and biocompatibility with further modification in the presence of various functional groups. Previous studies mentioned that the incorporation of curcumin into nanoparticles systems improved its antioxidant properties and therapeutic viability [6]. 1 IOP Conf. Series: Materials Science and Engineering 991 (2020) 012101 doi:10.1088/1757-899X/991/1/012101 IOP Conf. Series: Materials Science and Engineering 991 (2020) 012101 doi:10.1088/1757-899X/9 Therefore, a novel nanoparticle system was developed using GA as a coating material to improve the therapeutic efficacy of curcumin (Cur/GANPs) against cancer cells. In this research, the Cur/GANPs have been prepared using the freeze-drying technique. The antioxidant activities of Cur/GANPs and GA were assessed by DPPH assays. The cytotoxicity of both pure curcumin and Cur/GANPs at various time intervals was individually determined based on the MTT assay. The main purpose of this research is to assess the antioxidant and antihypertensive properties of Cur encapsulated into GANPs. The in vitro cytotoxicity and therapeutic effect of Cur/GANPs against human cells was investigated. 2.4. Antihypertensive activity In this method 100 µL of curcumin and Cur/GANPs were reacted with ACE )25 µL, pH 8.3( for 5 min at 37 °C. To evaluate the ACE inhibition capacity, an addition of 10 µL of hippuryl-histidyl-leucine (3.5 mM) to the assay mixture was then made followed by incubation for 30, 60, and 90 min. In order to stop the reaction, 50 µL of 3 M HCI was added to the mixture. Ethyl acetate )1 mL( was added to extract hippuric acid formed. Finally, the evaluation of hippuric acid was performed with a measurement of absorbance at 228 nm. 2.5. Cell viability 200 µL of a 1×104 cells/mL suspension were seeded into each well of 96-well plate for 24 h. The cells were treated with Cur and Cur/GANPs of concentrations of )15.6 - 100 µg/mL( for 72 h. 20 µL of MTT solution )5 µg/mL( was added into each 96-well with fresh media then mixed gently and incubation for 4 hours at 37°C with 5% CO2. The MTT-including culture medium was replaced with 200 µL/ well of DMSO in order to dissolve the formazan crystals formed. The absorbance was determined at 570 nm. 2.1. Materials Gum Arabic was purchased from ENNASR company )Sudan(.Curcumin was bought from Biolutions Resources )China(. HepG2 and HT29 cells were bought from American Type Culture Collection (ATCC). Dexamethasone, DPPH )1,1-diphenyl-2-picrylhydrazyl(, TROLOX, Angiotensin-converting enzyme was purchased from R&M company )China. ( 2.2. Preparation of Cur/GANPs Cur/GANPs were prepared using the freeze-drying technique with slight modifications [7]. In the first step GA (0.70 g) was dissolved in 50 mL of deionized water. An aqueous solution of curcumin was prepared in ethanol at a concentration of 1 mg/mL then and added to the dispersion in a ratio of 1:4 then mixed using a homogenizer. The mixture was kept under mild agitation at room temperature for 72 h. The final suspension was subjected to a high pressure homogenizer at a pressure of 1000 bar for 10 cycles, which was then frozen at -80°C. The final product was freeze-dried for 24 h at -55°C. 2.3. The DPPH scavenging activity of Cur/GANPs This reaction has been related to the donating ability of the antioxidant occurred in the process [8]. Curcumin and Cur/GANPs nanoparticles were used as test samples. A serial dilution of 100 µg of trolox diluted in 1mL of methanol was used in the range of 50- 200 µg/mL. 200 µL of DPPH solution was then added to each well of 96-well microplate. The absorbance was determined at 517 nm. 2.4. Antihypertensive activity 3. Results and discussions The role of gum Arabic nanoparticles in drug delivery applications can be related to their unique properties, particularly high stability and low toxicity [2, 9] . Cur/GANPs displayed a particle size over the range of 20-260 nm as shown in Figure 1. The Cur/GANPs exhibited more DPPH scavenging activity due to its hydrogen-donating ability (Figure 2(. Therefore, ACE inhibition activity of Cur/GANPs appeared to be higher than that of free curcumin due to the antihypertensive effect of gum 2 ICCEIB 2020 IOP Conf. Series: Materials Science and Engineering 991 (2020) 012101 IOP Publishing doi:10.1088/1757-899X/991/1/012101 Conf. Series: Materials Science and Engineering 991 (2020) 012101 doi:10.1088/1757-899X/991/1/0121 arabic under specific in vitro conditions )Figure 3(. As a result, the encapsulation of curcumin in gum Arabic could improve the antihypertensive capacity of curcumin. The results denoted that the anticancer activity of curcumin loaded into gum Arabic nanoparticles (Cur/GANPs) was higher than that of free curcumin. Cur/GANPs have exhibited improved cytotoxicity and tumour targeting against HepG2 cells )Figure 4( [8,10 [ . These results were similar to the data from the literature, which suggest that curcumin might be a potential antitumor compound [11]. In a previous study, the mechanism of GA nanoparticles targeted to the liver has been explained based on the interactions between asialoglycoprotein receptors, the function of receptor-mediated endocytosis, and nanocarriers [8]. The role of gum Arabic nanoparticles in drug delivery applications can be related to their unique properties, particularly high stability and low toxicity. Consequently, the presence of the asialoglycoprotein receptors on the surface of hepatocytes, which interact with galactose moiety on gum Arabic have increased the anticancer activity of the Cur/GANPs. In a previous study, gum Arabic nanoparticles have also showed increased uptake caused by the malignant liver cancer cells [8]. Thus, this study indicates that Cur/GANPs have promising future to further develop a nanocarrier for cancer therapy. Figure 1. Particle size of Cur/GANPs. Figure 2. DPPH scavenging of free curcumin and Cur/GANPs. Figure 1. Particle size of Cur/GANPs. Figure 1. Particle size of Cur/GANPs. Figure 2. DPPH scavenging of free curcumin and Cur/GANPs. Figure 2. DPPH scavenging of free curcumin and Cur/GANPs. 3 60 0 60 80 100120 0 1.563 80 100120 1.563 Figure 3. ACE inhibition )%( for free curcumin and Cur/GANPs after 30, 60, and 90 minutes. Figure 3. ACE inhibition )%( for free curcumin and Cur/GANPs after 30, 60, and 90 minutes. (a) (b) Figure 4. 3. Results and discussions Cytotoxicity effect of curcumin, Cur/GANPs at different treatment concentrations on can cell lines (a) HepG2; and (b) HT-29) 0 20 40 60 80 100 120 0 1.563 3.125 6.25 12.5 25 50 100 Free curcumin Cur/GANPs Cell viability )%( Conc )µg/mL) 0 20 40 60 80 100 120 0 1.563 3.125 6.25 12.5 25 50 100 Free curcumin Cur/GANPs Cell viability )%( Conc )µg/mL) Figure 3. ACE inhibition )%( for free curcumin and Cur/GANPs after 30, 60, and 90 minutes. 90 minutes. (a) (b) Figure 4. Cytotoxicity effect of curcumin, Cur/GANPs at different treatment concentrations on cancer cell lines (a) HepG2; and (b) HT-29) 0 20 40 60 80 100 120 0 1.563 3.125 6.25 12.5 25 50 100 Free curcumin Cur/GANPs Cell viability )%( Conc )µg/mL) 0 20 40 60 80 100 120 0 1.563 3.125 6.25 12.5 25 50 100 Free curcumin Cur/GANPs Cell viability )%( Conc )µg/mL) 0 20 40 60 80 100 120 0 1.563 3.125 6.25 12.5 25 50 100 Free curcumin Cur/GANPs Cell viability )%( Conc )µg/mL) (b) 0 20 40 60 80 100 120 0 1.563 3.125 6.25 12.5 25 50 100 Free curcumin Cur/GANPs Cell viability )%( Conc )µg/mL) Figure 4. Cytotoxicity effect of curcumin, Cur/GANPs at different treatment concentrations on cancer cell lines (a) HepG2; and (b) HT-29) 3.125 ICCEIB 2020 IOP Conf. Series: Materials Science and Engineering 991 (2020) 012101 g doi:10.1088/1757-899X/991/1/012101 Conf. Series: Materials Science and Engineering 991 (2020) 012101 doi:10.1088/1757-899X/991/1/0121 4. Conclusion The purpose of this present study is to enhance the therapeutic efficacy and the antioxidant properties of curcumin encapsulated into gum arabic polymer. As a result of the antioxidant activity on DPPH, Cur encapsulated into gum Arabic nanoparticle had been considerably higher than its free counterpart. This study reveals that Cur/GANPs are a potential candidate compound for the evaluation of prevention and treatment of cancer cells. Extrapolation of the in vitro cytotoxicity effects of Cur/GANPs to in vivo anticancer effects demands further investigation in light of its application as a cancer chemotherapeutic agent. Acknowledgment The authors would like to acknowledge International Islamic University Malaysia and Universiti Putra Malaysia for awarding the funds to conduct this research under project number of P-RIGS18-026-0026 and GP-IPS/2016/9505500. [11] Jinhuan J, Hua J, Li L, Jing P and Fen, J C 2013 J. Scanning. Microsc 35 253–260 [1] Achmawati H R, Oraya I S, Urniati N Fand Ahma A R 2016 Sci Pharm 84 131–140 [2] Ahmed A A, Fedail J S, Musa H H, Musa T H and Sifaldin A Z 2016 Middle East Fertil. Soc. J. 21(2) 101–108 References [1] Achmawati H R, Oraya I S, Urniati N Fand Ahma A R 2016 Sci Pharm 84 131–140 [2] Ahmed A A, Fedail J S, Musa H H, Musa T H and Sifaldin A Z 2016 Middle East Fertil. Soc. J. 21(2) 101–108 [3] Ali M M and Peter N Z 2012 Postharvest Biol. Tec. 76 119–124 [4] Meiyanto E, Dewi D, Putri P and Susidarti R A 2014 Asian Pac. J. Cancer Prev. 22 1–7 [5] Yin H, Zhang D, Wu X, Huang X and Chen, G 2013 Asian Pac. J. Cancer Pre.v 14 409–412 [6] Noor SJ 2019 Iraqi J. Pharm. Sci. 28)2( 9-16 [7] Abdelwahed W, Ghania D and Serge S 2006 Adv. Drug Deliv. Rev. 58 1688–1713 [8] Sarika PR., James NR, Kumar PR, Raj DK and Kumary TV 2015 Carbohydr. Polym. 134 167– 174 [9] Qilong W, Xiaoying Z, Jingyao M, Zhong-Bin D and Xiaoyu X 2013 Nat. Commun. 12 14–26 [10] Rajesh K, Gangwar G B, Tomar V A and Smita Z 2013 J. Agric. Food Chem. 23 9632–9637 [11] Jinhuan J, Hua J, Li L, Jing P and Fen, J C 2013 J. Scanning. Microsc 35 253–260 References [1] Achmawati H R, Oraya I S, Urniati N Fand Ahma A R 2016 Sci Pharm 84 131–140 [2] Ahmed A A, Fedail J S, Musa H H, Musa T H and Sifaldin A Z 2016 Middle East Fertil. Soc. J. 21(2) 101–108 ( ) [3] Ali M M and Peter N Z 2012 Postharvest Biol. Tec. 76 119–124 [3] Ali M M and Peter N Z 2012 Postharvest Biol. Tec. 76 119–124 ] Meiyanto E, Dewi D, Putri P and Susidarti R A 2014 Asian Pac. J. Cancer Prev. 22 1–7 [5] Yin H, Zhang D, Wu X, Huang X and Chen, G 2013 Asian Pac. J. Cancer Pre.v 14 409– [6] N SJ 2019 I i J Ph S i 28)2( 9 16 [5] Yin H, Zhang D, Wu X, Huang X and Chen, G 2013 Asian Pac. J. Cancer Pre.v 14 [5] Yin H, Zhang D, Wu X, Huang X and Chen, G 2013 Asian Pac. J. 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https://openalex.org/W3120966751	https://ijbnpa.biomedcentral.com/track/pdf/10.1186/s12966-020-01076-6	English	null	The effect of behaviour change interventions on changes in physical activity and anthropometrics in ambulatory hospital settings: a systematic review and meta-analysis	The international journal of behavioural nutrition and physical activity	2,021	cc-by	13,645	REVIEW Open Access The effect of behaviour change interventions on changes in physical activity and anthropometrics in ambulatory hospital settings: a systematic review and meta-analysis Stephen Barrett1,2, Stephen Begg1, Paul O’Halloran3, Owen Howlett1,4, Jack Lawrence5 and Mic Stephen Barrett1,2, Stephen Begg1, Paul O’Halloran3, Owen Howlett1,4, Jack Lawrence5 and Michael Kingsley6,7* Barrett et al. International Journal of Behavioral Nutrition and Physical Activity (2021) 18:7 Barrett et al. International Journal of Behavioral Nutrition and Physical Activity (2021) 18:7 Barrett et al. International Journal of Behavioral Nutrition and Physical Activity (2021) 18:7 https://doi.org/10.1186/s12966-020-01076-6 https://doi.org/10.1186/s12966-020-01076-6 Abstract Background: The aim of this systematic review and meta-analysis was to investigate whether behaviour change interventions promote changes in physical activity and anthropometrics (body mass, body mass index and waist circumference) in ambulatory hospital populations. Methods: Randomised controlled trials were collected from five bibliographic databases (MEDLINE, Embase, CINA HL, The Cochrane Central Register of Controlled Trials (CENTRAL) and PsycINFO). Meta-analyses were conducted using change scores from baseline to determine mean differences (MD), standardised mean differences (SMD) and 95% confidence intervals (95% CI). The Grades of Recommendation, Assessment, Development and Evaluation approach was used to evaluate the quality of the evidence. Results: A total of 29 studies met the eligibility criteria and 21 were included in meta-analyses. Behaviour change interventions significantly increased physical activity (SMD: 1.30; 95% CI: 0.53 to 2.07, p < 0.01), and resulted in significant reductions in body mass (MD: -2.74; 95% CI: −4.42 to −1.07, p < 0.01), body mass index (MD: -0.99; 95% CI: −1.48 to −0.50, p < 0.01) and waist circumference (MD: -2.21; 95% CI: −4.01 to −0.42, p = 0.02). The GRADE assessment indicated that the evidence is very uncertain about the effect of behaviour change interventions on changes in physical activity and anthropometrics in ambulatory hospital patients. Conclusions: Behaviour change interventions initiated in the ambulatory hospital setting significantly increased physical activity and significantly reduced body mass, body mass index and waist circumference. Increased clarity in interventions definitions and assessments of treatment fidelity are factors that need attention in future research. PROSPERO registration number: CRD42020172140. Background Chronic diseases are leading causes of ill health world- wide [1]. Modifiable risk factors such as insufficient physical activity (PA), poor diet, and obesity are associ- ated with an increased risk for chronic disease [2] and, due to medical improvements, individuals are living with chronic disease for longer periods [3, 4]. Increased * Correspondence: M.Kingsley@latrobe.edu.au; Michael.Kingsley@Auckland.ac.nz 6Holsworth Research Initiative, La Trobe Rural Health School, La Trobe University, PO Box 199, Bendigo, Victoria 3552, Australia 7Department of Exercise Sciences, University of Auckland, Newmarket 1023, New Zealand Full list of author information is available at the end of the article Data sources and search strategies To avoid duplication, a search was undertaken in the Cochrane Database of Systematic Reviews, PubMed Clinical Queries and PROSPERO International prospect- ive register of systematic reviews to confirm that no similar systematic reviews or protocols have been con- ducted. Eligible studies were collected (from inception until May 2020) using computer-based searches in MEDLINE, Embase, CINAHL, Web of Science, Psy- cINFO and The Cochrane Central Register of Controlled Trials (CENTRAL) electronic databases. Database- specific search strategies were developed with the guid- ance of professional clinical librarians. The database searches were performed using three main concepts: am- bulatory secondary hospital care, lifestyle behaviour change interventions and outcomes (PA and anthropo- metric measures). For each main concept relevant re- lated terms and keywords were included in the sensitive search (search details for MEDLINE are presented in Additional file 2). In the inpatient setting, patients are removed from their home environments, often suffering from a serious condition, and are potentially confined to their bed or the hospital room [14]. Being hospitalised has been iden- tified as a major life event, increasing the likelihood of engaging in recommended care [15]. The inpatient en- vironment imposes unique constraints on individuals, including their perception of autonomy of their care [13]. Consequently, the decision to initiate health behav- iour change is potentially impacted by the inpatient set- ting [13]. Ambulatory hospital patients, on the other hand, en- gage in care under different circumstances. These indi- viduals are community-dwelling, and maintain more autonomy over their care, including decisions regarding the treatment plan, or when they can expect to see the doctor next [16]. The delivery of preventive health care in the ambulatory hospital setting should be targeted, patient-centred, and characterised by interventions that support people with chronic disease risk factors and should include self-management support wherever pos- sible [17]. Knowledge of the impact of behaviour change interventions on ambulatory hospital patients might allow prioritising preventive interventions in the ambula- tory hospital setting for the prevention and management of chronic disease. To the best of our knowledge, no Two additional steps were undertaken to ensure the comprehensiveness of our search. Firstly, searches were undertaken in clinical trial registries, including ClinicalTrials.gov, EU Clinical Trials Register, Australian New Zealand Clinical Trials Registry and the World Health Organization International Clinical Trial Registry Platform to source relevant ongoing and unpublished trials. Research question Do behaviour change interventions result in positive changes and maintenance in PA and anthropometrics in adults attending ambulatory hospital clinics? Data sources and search strategies Secondly, we performed a snowball search on ref- erence lists, and grey literature databases. © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Page 2 of 19 Barrett et al. International Journal of Behavioral Nutrition and Physical Activity Page 2 of 19 Page 2 of 19 survival amongst people with chronic disease results in a higher prevalence of morbidity, and lower quality of life [4]. As a result, secondary prevention has become im- portant for chronic disease management globally [5]. review has examined the effect of behaviour change in- terventions that address changes in PA and anthropo- metrics in non-admitted secondary care patients. Therefore, the aim of this review was to examine the ef- fect of behaviour change interventions on changes and maintenance on PA, and anthropometrics, initiated in the ambulatory hospital setting only. Secondary prevention aims to reduce the impact of chronic disease through early detection and treatment. Behaviour change as a secondary prevention strategy is emerging as a way to mitigate the impact of disease and slow down disease progression [6]. Hospitals are import- ant settings for the delivery of secondary prevention pro- grams given their unique access to members of the local community who might benefit [7]. Hospital attendees are not necessarily registered with a GP and may not be actively engaged with community health promotion ser- vices [7], but because their health is already compro- mised, these individuals can be readily motivated to engage with lifestyle behaviour changes [8]. Behaviour change interventions are advocated as the first-line ap- proach to behavioural risk factor management [9]. Methods A systematic review and meta-analysis was conducted according to the Preferred Reporting Items for System- atic Reviews and Meta-Analyses (PRISMA) [18] (Add- itional file 1). This review was registered with PROSPERO (registration ID: CRD42020172140). Results from recent meta-analyses indicate that sec- ondary prevention behaviour change interventions result in positive effects in PA [10, 11], anthropometrics [11] and cardiovascular health [12]. These reviews included studies from hospital settings, though many studies re- cruited patients from the inpatient setting [10, 11]. Con- textual differences exist in recruiting individuals for behaviour change interventions from the admitted ver- sus ambulatory hospital setting [13, 14]. Study selection Studies were entered into Review Manager (Version 5.3; The Cochrane Collaboration, Denmark) and duplicates were removed. Screening was carried out using Covi- dence (Covidence Systematic Review Software, Veritas Health Innovation, Melbourne, Australia). Two authors independently screened title/abstracts and full text. Studies were systematically excluded when they did not meet the pre-specified inclusion criteria. Disagreements between reviewers were resolved by discussion, or where required with consensus of a third reviewer. Statistical analysis Means and standard deviations of change scores for both intervention and control groups were included in one of the extracted studies [28]. Using these change data, the correlation coefficients were calculated for the interven- tion group (r = 0.81) and control group (r = 0.80), with an average r of 0.80 [28]. For all included studies, the standard deviation of change scores from baseline were calculated using a correlation coefficient of 0.8 [25], and entered directly into Review Manager 5.3 (Version 5.3; The Cochrane Collaboration, Denmark) for analysis. Analyses based on changes from baseline are more effi- cient and powerful than comparison of final values through the removal of between-person variability [25]. Study quality assessment The risk of bias of the included studies was assessed by two reviewers independently using the Cochrane Risk of Bias assessment tool [25]. The following methodological criteria were assessed: sequence generation; allocation concealment; blinding of participants, personnel and outcome assessors; incomplete outcome data; selective outcome reporting; and other potential threats to valid- ity [25]. Each of these criteria were judged and classified as ‘low risk’, ‘high risk’ or ‘unclear risk’ of bias. Studies were included that reported any of the follow- ing physical activity outcome measures: changes in daily steps, METs per week (METs/wk) or minutes per day/ week of moderate to vigorous physical activity (MVPA) measured subjectively (e.g., self-report) or objectively at baseline and post intervention. g The overall strength of the evidence was assessed using the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) [26] system through the GRADEpro 3.6 software (GRADEpro GDT: GRADEpro Guideline Development Tool [Software]; McMaster University, USA). Quality of evidence for meta-analyses began at the high level and was down- graded to lower levels of evidence when risk of bias, in- consistency, indirectness, imprecision or publication bias were present. Publication bias was examined by Egger test [27]. Eligibility criteria The term behaviour change interventions is used to de- fine coordinated activities designed to change specified behaviour outcomes [19]. For the purpose of this review, Barrett et al. International Journal of Behavioral Nutrition and Physical Activity (2021) 18:7 Barrett et al. International Journal of Behavioral Nutrition and Physical Activity Page 3 of 19 Page 3 of 19 (2021) 18:7 the number of individuals that were randomised and analysed); the professional background of the person de- livering the intervention); and outcome variables (out- come definition, unit of measurement, time points measured and reported). Continuous data including, means, standard deviations and the sample size numbers were extracted. When information was unclear, insuffi- cient or missing, the authors of trials were contacted for clarifications and additional results. Where standard de- viations were not available, measures of variance were estimated from the standard error of a mean, confidence intervals or p-values according to the Cochrane Hand- book for Systematic Reviews of Interventions the Cochrane Collaboration [19]. When data were presented as median and interquartile range, the mean and stand- ard deviation were estimated using the formula from Hozo et al. [24]. we included behaviour change interventions that specif- ically aimed to elicit changes in anthropometrics and/or PA changes through the use of behaviour modification components and strategies. Inclusion criteria to select studies were: 1) Study population: adult (aged 18 or older) ambulatory hospital patients; 2) Types of studies: peer-reviewed randomised controlled trials regarding a behaviour change intervention compared to a control intervention or usual care comparison group. The be- haviour change intervention could be a single interven- tion or a multi-component intervention, but needed to include at least one session that was delivered in a 1:1 format (delivered in person, via the phone or telehealth) because of the importance of an individualised approach to self-management [20]; 3) Primary outcomes: PA, an- thropometric measures – body mass, body mass index (BMI) and waist circumference (WC). Due to the clinical relevance of changes in body mass, BMI and WC, an a priori decision was made to undertake an meta-analysis on each outcome individually [21, 22]. Behavioural sci- ence highlights the need to draw the distinction between initial behaviour change and behaviour change mainten- ance [23]. To establish the maintenance effect of inter- ventions, studies that included a follow-up duration of less than 12 weeks were excluded. Data extraction Data were independently extracted by two reviewers. Data extraction was performed with the aid of a prede- signed and piloted data collection form. For each study, the reviewers extracted information with respect to study characteristics (type of study, population descrip- tion, focused disease or condition); study participants (sample size, demographics); methods (intervention dur- ation, type and frequency, fidelity blinding, amount of intervention groups, number of included participants, Barrett et al. International Journal of Behavioral Nutrition and Physical Activity (2021) 18:7 Barrett et al. International Journal of Behavioral Nutrition and Physical Activity (2021) 18:7 Page 4 of 19 Barrett et al. International Journal of Behavioral Nutrition and Physical Activity Page 4 of 19 Page 4 of 19 The mean differences with 95% confidence intervals (CIs) were calculated for anthropometric outcomes. For PA outcomes, standardised mean differences (SMD) with 95% CIs were calculated using Review Manager 5.3 as the mean difference divided by the pooled standard deviation [25]. Due to the heterogeneity in the study in- terventions and populations, meta-analyses were con- ducted using a random effects model [25]. In keeping with recommendations, an effect size of 0.2 was consid- ered small, 0.5 moderate, and 0.8 or more was consid- ered large [29]. The effect of heterogeneity of each summary effect size was quantified using a chi-squared test and the I2 statistic, in which the boundary limits 25, 50, and 75% were designated as a low, moderate, and high heterogeneity value, respectively [25]. to evaluate the effect of that study on the summary estimates. Subgroup analyses were performed to investigate the essential elements in designing effective behaviour change interventions in the ambulatory hospital setting. The subgroup analyses included the study population, follow-up duration, objective or self-reported measure- ments, the duration of intervention, and the dose of the intervention. The duration of intervention was classified as short term (≤3 months) or longer term if ≥4 months [30]. The reporting of the length of intervention sessions was poor in many of the included studies. As a result the intervention dose quantified in this review is through the number of sessions. This intervention dose was cate- gorised as low intensity (≤6 sessions), medium intensity (7–12 sessions) or high intensity (≥13 sessions) [31]. GRADE assessment The overall certainty of evidence for the effectiveness of behaviour change interventions for changes in PA and anthropometrics in adults attending ambulatory hospital clinics is presented in Table 2. The certainty of evidence for the meta-analysis stratified by follow-up duration and for studies with a low risk of bias are presented in Additional file 4. In addition, the GRADE quality assess- ments are presented in Additional file 5. The intervention components used in the included studies varied. All of the included studies had at least one component that was delivered 1:1. The underlying theory informing the behaviour change intervention and the behaviour change techniques used are detailed in Table 1. For nine studies, the main focus of the interven- tion was on increasing PA [28, 34, 38, 43, 47, 48, 51, 54, 58]. Changes in anthropometrics was the primary focus in four studies [40, 44, 52, 54]. Risk of bias The risk of bias assessment for all studies is detailed in Fig. 2. In trials involving behaviour change interventions the blinding of participants is extremely difficult to undertake. As a result, all studies were judged to have a high of risk of performance bias (lack of blinding of par- ticipants and personnel). Twelve studies were judged to have a high risk of attrition bias, and four studies were rated as unclear. Seven of the included studies reported blinding of the outcome assessors (detection bias), whereas the majority of the studies did not adequately report blinding of the outcome assessors (n = 18). Five of the included studies were judged as a high risk of se- lection bias due to the lack of detail regarding the alloca- tion concealment. Fifteen studies were judged to have an unclear risk of bias due to the lack of information pro- vided on the random sequence generation. The individ- ual risk of bias assessment is included in Additional file 3. Study characteristics The behaviour change interventions in the included studies varied in intervention duration from 4 to 416 weeks. With the exclusion of Sone et al. [55], which used a low grade intervention over 8 years, the adjusted inter- vention duration was 32 ± 24 weeks. The intervention duration was 26 weeks or greater in 66% of the included studies. Follow-up duration varied amongst the studies: 5 studies had a 3-month follow-up [38, 42, 47, 48, 56], 1 study had a 4-month follow-up [59], 10 studies had a 6- month follow-up [34, 35, 37, 40, 43, 46, 51, 52, 54, 57], 2 studies had a 9-month follow-up [28, 39], 4 studies had a 12-month follow-up, and the remaining 7 studies var- ied between 15 months and 8 years of follow-up [33, 44, 45, 49, 53, 55, 58]. Results All analyses were repeated with correlations set at lower (0.50) and higher (1.0) r values than the calculated value of 0.80. Sensitivity analyses were performed to assess heterogeneity of the studies and to evaluate the robust- ness of the results. Each study was individually removed Following de-duplication, 2984 studies were screened. The PRISMA diagram for the screening is shown in Fig. 1. Twenty-nine full-text articles fulfilled the inclu- sion criteria and were included in qualitative (n = 29) Fig. 1 PRISMA Barrett et al. International Journal of Behavioral Nutrition and Physical Activity (2021) 18:7 Page 5 of 19 Barrett et al. International Journal of Behavioral Nutrition and Physical Activity Page 5 of 19 (2021) 18:7 [36], dietitians [37, 38, 40, 50, 53, 55], exercise counsel- lors [51, 58], exercise scientists [48, 50], graduate level therapists [41], health professionals [32], health educa- tors [38, 39], lifestyle coaches [44], nurses [28, 33, 35, 45, 55], physicians [33, 55], physiotherapists [34, 53, 55], psychologists [46], researchers [56, 57, 59], and thera- pists [43]. and quantitative (n = 21) syntheses (Table 1) [28, 32– 59]. Included studies were published over a 17-year period from 2003 to 2020. The studies were performed in 14 different countries, with the largest representation from the United States (n = 6), Holland (n = 4) and Australia (n = 4). Populations within the included studies represented various health conditions including impaired glucose tolerance (IGT) or type 2 diabetes (T2DM) (n = 10) [32, 36, 37, 40, 48, 49, 53, 55, 56, 59], cardiovascular diseases (CVD) (n = 10) [28, 35, 38, 39, 41, 45–47, 50, 51], overweight/obesity (n = 5) [42, 44, 52, 54, 57], insuf- ficiently physically active (n = 1) [34], Chronic Obstruct- ive Pulmonary Disease (COPD) (n = 1) [58], cerebrovascular disease (n = 1) [33], and cancer (n = 1) [43]. Effects of behaviour change interventions on changes in physical activity For PA outcomes, objective measurement was used in 8 studies using accelerometers, pedometers and objective measurement of exercise capacity [34, 37–39, 41, 43, 51, 58]. Self-reported instruments were used in the other 18 studies [28, 33, 35, 36, 40, 41, 44–48, 50, 52–57]. The measures of anthropometrics in the studies included body mass [28, 32, 34, 35, 44, 52–54, 57], BMI [28, 32– 36, 39, 40, 50, 52–55, 57, 59] and WC [34, 35, 40, 50, 53]. Objective measurement of anthropometrics was used in 15 of the studies [32, 34, 39, 44, 46, 47, 49–57], with self-reported methods used in the remaining 3 studies [37, 45, 59]. Thirteen of the 29 included studies provided PA data for the intervention and control groups at the post- intervention follow-up, and were included in the meta- analysis. The meta-analysis for behaviour change inter- ventions versus standard care for change in PA demon- strated a significant effect in favour of the intervention (SMD: 0.96; 95% CI: 0.45 to 1.48, p < 0.01, Fig. 3) [28, 34, 38, 39, 41, 43, 44, 47, 50, 52, 54, 57, 58]. Subgroup analyses indicated that behaviour change in- terventions resulted in a significant increase in PA when the follow-up lasted for 6 months or less (SMD: 1.30; 95% CI: 0.53 to 2.07, p < 0.01, Fig. 3) [34, 38, 43, 44, 47, 52, 54, 57]. Behaviour change interventions with a follow-up of greater than 6 months demonstrated a non- The professional background of the persons delivering the interventions included community health workers Barrett et al. International Journal of Behavioral Nutrition and Physical Activity (2021) 18:7 Page 6 of 19 Barrett et al. Effects of behaviour change interventions on changes in physical activity International Journal of Behavioral Nutrition and Physical Activity Table 1 Characteristics of the included studies Study Country Study Population N (% male) Mean age (SD) Intervention delivery Underlying Theory a Behaviour change techniques a Length of intervention Length of follow- up Outcome measures Aas, 2005 [32] Norway Overweight patients with T2DM 38 (63%) 57 ± 6 14 x group education sessions; 2 x counselling sessions over 12 months, delivered in person; Group exercise sessions delivered twice a week Not stated Goal setting 12 months 12 months Anthropometric: Objective measurement Ahmadi, 2020 [33] Germany & Denmark Patients with cerebrovascular disease 2098 (33%) 67 ± 10 8 x individual counselling sessions over 24 months, delivered in person MI Feedback on behaviour; Feedback on outcome 2 years 3 years Anthropometric: NS; Physical Activity: Self-reported Alsaleh, 2016 [28] Jordan Patients with CVD 156 (53%) 58 ± 9 6 × 15–20 min counselling sessions over 6 months delivered via telephone; Educational text messages were provided 2 per week for first 3 months, and 1 per week for last 3 months Social Cognitive Theory; Self- Efficacy Theory Feedback; Goal setting; Self-monitoring; MI techniques 6 months 9 months Anthropometric: NS; Physical Activity: Self-reported using IPAQ Altenburg, 2014 [58] Netherlands Patients with COPD 155 (65%) 62 ± 4 5 × 30 min counselling sessions over 12 weeks delivered in person Goal setting and task performance Goal setting; MI techniques 3 months 15 months Physical Activity: Pedometer Barrett, 2018 [34] Australia Insufficiently physically active adults 72 (25%) 53 ± 8 1 x group education session; 8 × 30-min individual sessions over 12 weeks, delivered via telephone Integrated MI and CBT Goal setting, action planning, self- monitoring, personal feedback; relapse prevention 3 months 6 months Anthropometric: Objective measurement; Physical Activity: Actigraph Accelerometer Cakir, 2006 [35] Turkey Patients with hypertension 70 (58%) 52 ± 8 1 × 30-min group lecture; 4 × 60-min group education classes; 4 x individual counselling sessions, delivered in person Not stated Education; Stress management; Coping strategies 3 months 6 months Anthropometric: NS; Physical Activity: Self-reported using Health Promoting Lifestyle Profile. Carrasquillo, 2017 [36] USA Latinos with T2DM 300 (45%) 55 ± 7 4 x individual counselling sessions over 12 months, delivered in person; 12 x individual counselling sessions over 12 months, delivered via telephone; Intervention participants were invited to monthly educational groups and bimonthly exercise groups in parks located within a convenient proximity to their homes. Effects of behaviour change interventions on changes in physical activity Not stated MI skills; Education 12 months 12 months Anthropometric: NS; Physical Activity: Self-reported using IPAQ Cheung, 2019 [37] Australia Post-partum women with GD 60 (0%) 34 ± 4 2 × 30 min individual counselling sessions over 6 months, delivered in person; 1 x follow-up session, up to 12-weeks post- partum, delivered via phone Focused on the adoption phase of behaviour change Not stated 6 months 6 months Anthropometric: Self-reported; Physical Activity: Fitbit Dogru, 2019 [59] Turkey Patients with T2DM 60 (32%) NS 4 × 15-20 m individual counselling sessions, delivered once a month for 4-months via MI MI techniques 4 months 4 months Anthropometric: Self-reported Barrett et al. International Journal of Behavioral Nutrition and Physical Activity (2021) 18:7 Page 7 of 19 Barrett et al. International Journal of Behavioral Nutrition and Physical Activity Table 1 Characteristics of the included studies (Continued) Study Country Study Population N (% male) Mean age (SD) Intervention delivery Underlying Theory a Behaviour change techniques a Length of intervention Length of follow- up Outcome measures telephone Duscha, 2018 [38] USA Patients with CVD 25 (76%) 64 ± 8 24 × 30–60 min telephone coaching sessions over 12 weeks delivered in person; In addition, coaches sent educational material via email and sent text messages to remind them to practice healthy lifestyle habits. Effects of behaviour change interventions on changes in physical activity Health Coaching Planning; Motivation 3 months 3 months Physical Activity: Fitbit Elkoustaf, 2019 [39] USA Patients with CVD 79 (57%) 66 ± 9 1 x groups introduction session; 18 x group sessions over 6 months; 1:1 individual coaching sessions, delivered via phone (unspecified number) Wellness coaching Not stated 9 months 9 months Anthropometric: Objective measurement; Physical Activity: Objective functional measurement Fappa, 2012 [40] Greece Patients with Metabolic Syndrome 87 (42%) 49 ± 12 7 × 60-min counselling sessions over 6 months, delivered in person Goal setting theory Self-monitoring; Problem-solving tech- niques; Relapse prevention 6 months 6 months Anthropometric: Objective measurement; Physical Activity: Self-reported using Harokopio PA Questionnaire Freedland, 2015 [41] USA Patients with CVD 158 (54%) 56 ± 11 1 × 60-min counselling sessions weekly for the first 6 months, delivered in person; 4 × 30-min counselling sessions in the final 6 months, delivered via phone CBT Problem-solving; Goal setting 12 months 12 months Physical Activity: Objective functional measures Gade, 2014 [42] Norway Patients who were morbidly obese 102 (68%) 43 ± 10 4 x individual counselling session over 10 weeks delivered in person; 6 x individual counselling session over 10 weeks delivered via telephone CBT Psychoeducation; Homework; Self- monitoring; Relapse prevention 10 weeks 3 months Anthropometric: NS Goedendorp, 2010 [43] Netherlands Patients with cancer undergoing curative treatment 240 (34%) 57 ± 11 10 × 60-min counselling sessions over 6 months, delivered in person CBT Restructuring of cognitions and beliefs; education; Behavioural instructions 6 months 6 months Physical Activity: Actometer Goodwin, 2014 [44] Canada & USA Overweight postmenopausal women 338 (0%) 61 ± 7 19 × 30-60 m coaching sessions over 2 years delivered via telephone Not stated Lifestyle coaching; Motivation; Relapse prevention; Overcoming barriers 24 months 24 months Anthropometric: Objective measurement; Physical Activity: Self-reported using IPAQ Harting, 2006 [45] Netherlands Patients with CVD risk 1270 (69%) 61 ± 9 6 × 30–45 min counselling sessions over 4 months, delivered in person Health Counselling based on Not stated 4 months 18 months Anthropometric: Objective measurement; Barrett et al. International Journal of Behavioral Nutrition and Physical Activity (2021) 18:7 Barrett et al. Effects of behaviour change interventions on changes in physical activity International Journal of Behavioral Nutrition and Physical Activity Page 8 of 19 Table 1 Characteristics of the included studies (Continued) Study Country Study Population N (% male) Mean age (SD) Intervention delivery Underlying Theory a Behaviour change techniques a Length of intervention Length of follow- up Outcome measures stage of behavioural change Physical Activity: Self-reported using a ‘short validated survey’ Ijzelenberg, 2012 [46] Netherlands Patients with CVD 146 (77%) 60 ± 11 22 x group exercise sessions over 6 months; 3 x individual exercise sessions over 6 months; 7 x group counselling sessions over 6 months; Individually counselling sessions over 6 months, delivered in person (unspecified number) Lifestyle counselling Motivation; Goal setting; Stress management 6 months 6 months Anthropometric: Objective measurement; Physical Activity: Self-reported using the SQUASH survey Kim, 2019 [47] Korea Women at risk of CVD 58 (0%) 57 ± 6 12 x individual counselling session over 3 months, delivered in person; 1 x education text message delivered weekly for 3 months Theory of planned behaviour; Theory of self- regulation Education; goal setting, self- monitoring; feedback 3 months 3 months Anthropometric: Objective measurement; Physical Activity: Self-reported using the IPAQ Kirk, 2004 [48] UK Inactive patients with T2DM 70 (50%) 58 ± 8 2 × 30-min individual counselling sessions over 9 months delivered in person; 4 x individual counselling sessions over 9 months delivered via telephone Transtheoretical model Problem solving; Social support; Goal setting 9 months 12 months Physical Activity: Self-reported Kosaka, 2005 [49] Japan Men with IGT 458 (100%) NS 6 x individual counselling sessions over 12 months, delivered in person Not stated Education; Self- monitoring; Social support 12 months 48 months Anthropometric: Objective measurement. Lear, 2003 [50] Canada Patients with CVD 302 (82%) 64 ± 9 6 x group exercise sessions over 12 months; 2 x lifestyle and risk-factor assessments; 6 x individual counselling sessions over 12 months, delivered in person Counselling based on principles of behavioural change Feedback (outcomes); Counsel on lifestyle behaviours and risk factors 12 months 12 months Anthropometric: Objective measurement; Physical Activity: Self-reported using MLTPA questionnaire. Effects of behaviour change interventions on changes in physical activity Miura, 2004 [51] Japan Patients with HTN 57 (51%) 62 ± 10 6 x individual counselling sessions over 6 months, delivered in person Behaviour theory; Social cognitive theory Not stated 6 months 6 months Anthropometric: Objective measurement; Physical Activity: Actigraph Accelerometer O’Brien, 2018 [52] Australia Overweight patients with OA 120 (36%) 62 ± 12 1 x brief group education session; 10 x individual counselling session over 6- months, delivered in person MI; Self-regulation principles Problem solving; Goal setting 6 months 6 months Anthropometric: Objective measurement; Physical Activity: Self-reported using AAS Page 9 of 19 Barrett et al. International Journal of Behavioral Nutrition and Physical Activity (2021) 18:7 Barrett et al. International Journal of Behavioral Nutrition and Physical Activity Table 1 Characteristics of the included studies (Continued) Study Country Study Population N (% male) Mean age (SD) Intervention delivery Underlying Theory a Behaviour change techniques a Length of intervention Length of follow- up Outcome measures Oldroyd, 2006 [53] UK Patients with IGT 78 (50%) 58 ± 10 12 × 15–20 min individual counselling sessions over 24 months, delivered in person Stages of change model of behaviour change MI techniques; Action planning; Goal setting 24 months 24 months Anthropometric: Objective measurement; Physical Activity: Self-reported using a ‘lifestyle questionnaire’ Rimmer, 2009 [54] USA Women with morbid obesity & mobility issues 92 (0%) 59 ± 11 1 x individual counselling sessions each week over 6 months, delivered in person; Option to attend a monthly exercise support group. Not stated Goal Setting; Performance feedback; Overcoming barriers 6 months 6 months Anthropometric: Objective measurement; Physical Activity: Self-reported using a ‘lifestyle questionnaire’ Sone, 2010 [55] Japan Patients with T2DM 2033 (47%) 59 ± 7 1 x group education session; 2 × 15-min individual counselling session monthly over 96 months, delivered in person Not stated Feedback on behaviour; Feedback on outcomes 96 months 96 months Anthropometric: Objective measurement; Physical Activity: Self-reported using a ‘lifestyle questionnaire’ Wattanakorn, 2013 [56] Thailand Patients with T2DM and obesity 76 (16%) 50 ± 8 4 × 30–45 min individual counselling sessions over 1 month, delivered in person MI; Self-regulation theory. Effects of behaviour change interventions on changes in waist circumference Nine studies provided data on changes in body mass for the experimental and control groups at the post- intervention follow-up, and were included in the meta- analysis. The meta-analysis for behaviour change inter- ventions versus standard care for change in body mass demonstrated a significant effect in favour of the inter- vention (MD: -2.74; 95% CI: −4.42 to −1.07, p < 0.01, Fig. 4) [28, 32, 34, 35, 44, 52–54, 57]. Five studies provided data on changes in WC for the ex- perimental and control groups at the post-intervention follow-up and were included in the meta-analysis. The meta-analysis for behaviour change interventions versus standard care for change in WC demonstrated a signifi- cant effect in favour of the intervention (MD: -2.21; 95% CI: −4.01 to −0.42, p = 0.02, Fig. 6) [34, 35, 40, 50, 53]. Subgroup analyses indicated that behaviour change in- terventions resulted in a significant changes in body mass when follow-up measurement was 6 months and under (MD: -3.15; 95% CI: −5.96 to −0.34, p = 0.03, Fig. 4), and greater than 6 months (MD: -2.37; 95% CI: −4.40 to −0.35, p = 0.02, Fig. 4). The evidence is very uncertain about the effect of behaviour change interven- tions on changes in mass in ambulatory hospital patients. The behaviour change interventions demonstrated sig- nificant changes in WC when follow-up measurement was 6 months and under (3 studies, 194 participants, MD, −3.91, 95% CI, −5.96 to −1.85, p < 0.01, Fig. 6), but not when the follow-up was greater than 6 months (MD: -0.66; 95% CI: −2.88 to 0.95, p = 0.42, Fig. 6). The evidence is very uncertain about the effect of behaviour change interventions on changes in WC in ambulatory hospital patients. The one exception was the analysis for Table 2 Summary of findings table Table 2 Summary of findings table Behaviour change interventions for changes and maintenance in PA and anthropometrics in adults attending ambulatory hospital clinics Outcome Anticipated absolute effects* (95% CI) №of participants (studies) Certainty of the evidence (GRADE) Informative statements Physical activity SMD 0.96 higher [0.45 to 1.48] 1454 (13 RCTs) ⨁◯◯◯ VERY LOW a,b,c,d,e Behaviour change interventions may increase physical activity in ambulatory hospital patients but the evidence is very uncertain. Effects of behaviour change interventions on changes in BMI Effects of behaviour change interventions on changes in BMI Fifteen studies provided data on changes in BMI for the experimental and control groups at the post- intervention follow-up and were included in the meta- analysis. The meta-analysis for behaviour change inter- ventions versus standard care for change in BMI demon- strated a significant effect in favour of the intervention (MD: -0.99; 95% CI: −1.48 to −0.50, p < 0.01, Fig. 5) [28, 32–36, 39, 40, 50, 52–55, 57, 59]. The behaviour change interventions demonstrated sig- nificant changes in BMI when follow-up measurement was 6 months and under (MD: -1.55; 95% CI: −2.58 to −0.53, p < 0.01, Fig. 5), and greater than 6 months (MD: -0.75; 95% CI: −1.35 to −0.16, p = 0.01, Fig. 5). Behav- iour change interventions may decrease BMI in ambula- tory hospital patients but the evidence is very uncertain. significant effect in favour of the intervention (SMD: 0.43; 95% CI: −0.07 to 0.93, p = 0.09, Fig. 3) [28, 39, 41, 50, 58]. Behaviour change interventions may increase PA in ambulatory hospital patients but the evidence is very uncertain. Effects of behaviour change interventions on changes in physical activity Education; Goal setting; Discrepancy between current behaviour and goal 1 month 4 months Anthropometric: Objective measurement; Physical Activity: Self-reported using the Seven Day PA Recall survey Williams, 2018 [57] Australia Overweight patients with chronic LBP 159 (41%) 57 ± 13 10 x individual counselling sessions over 6 months, delivered via telephone SDT; Setting graded tasks; Setting specific behaviour goals; Barrier identification Prompting self- monitoring of behav- iour and outcomes 6 months 6 months Anthropometric: Objective measurement; Physical Activity: Self-reported using the AAS AAS Active Australia Survey, CBT Cognitive Behaviour Therapy, COPD Chronic Obstructive Pulmonary Disease, CVD Cardiovascular disease, HTN Hypertension, IGT Impaired Glucose Tolerance, IPAQ International Physical Activity Questionnaire, LMTPA Minnesota Leisure Time Physical Activity, MI Motivational Interviewing, NS Not stated, OA Osteoarthritis, PA Physical Activity, SDT Self-determination Theory, SQUASH Short QUestionnaire to ASsess Health enhancing physical activity, SR Self-reported, T2DM Type 2 Diabetes Mellitus. aas described by the authors of the studies Barrett et al. International Journal of Behavioral Nutrition and Physical Activity (2021) 18:7 Page 10 of 19 Page 10 of 19 Page 10 of 19 (2021) 18:7 significant effect in favour of the intervention (SMD: 0.43; 95% CI: −0.07 to 0.93, p = 0.09, Fig. 3) [28, 39, 41, 50, 58]. Behaviour change interventions may increase PA in ambulatory hospital patients but the evidence is very uncertain. Fig. 2 Risk of bias of included studies Fig. 2 Risk of bias of included studies Effects of behaviour change interventions on changes in waist circumference Mass (kg) MD −2.74 lower [−4.42 to −1.07] 872 (9 RCTs) ⨁◯◯◯ VERY LOW a,c,d,e,f The evidence is very uncertain about the effect of behaviour change interventions on changes in mass in ambulatory hospital patients. BMI (kg/m2) MD −0.99 lower [−1.48 to −0.50] 4728 (15 RCTs) ⨁◯◯◯ VERY LOW a,b,c,d,e Behaviour change interventions may decrease BMI in ambulatory hospital patients but the evidence is very uncertain. Waist Circumference (cm) MD −2.21 lower [−4.01, −0.42] 530 (5 RCTs) ⨁◯◯◯ VERY LOW a,c,d,f The evidence is very uncertain about the effect of behaviour change interventions on changes in waist circumference in ambulatory hospital patients. The risk in the intervention group (and its 95% confidence interval) is based on the assumed risk in the comparison group and the relative effect of the intervention (and its 95% CI) Explanations aLarge number of studies with high risk of bias bHigh heterogeneity cDifferences in population and outcome measures dWide confidence intervals eAsymmetry in the pattern of results fModerate heterogeneity ur change interventions for changes and maintenance in PA and anthropometrics in adults attending ambulatory hospital clinics e Anticipated absolute effects (95% CI) №of participants (studies) Certainty of the evidence (GRADE) Informative statements Barrett et al. International Journal of Behavioral Nutrition and Physical Activity (2021) 18:7 Page 11 of 19 Barrett et al. International Journal of Behavioral Nutrition and Physical Activity Barrett et al. International Journal of Behavioral Nutrition and Physical Activity (2021) 18:7 Page 11 of 19 Fig. 3 Meta-analysis investigating behavioural lifestyle interventions for changes in physical activity WC change when follow-up measurement was 6 months and under, in which case the evidence suggests that be- haviour change interventions results in a slight reduction in WC in ambulatory hospital patients. WC change when follow-up measurement was 6 months and under, in which case the evidence suggests that be- haviour change interventions results in a slight reduction in WC in ambulatory hospital patients. significant, and within the 95% confidence intervals at the imputed r of 0.8 (Table 3). Statistically significant changes remained for all outcomes at the inputed r of 1.0 and 0.5. In the low risk of bias analyses, behaviour change interventions exhibited significant beneficial ef- fects in PA, body mass and BMI. Subgroup analyses demonstrated significant changes in body mass and BMI for individuals with cardiovascular diseases, and Sensitivity and subgroup analyses International Journal of Behavioral Nutrition and Physical Activity Page 13 of 19 Mean change (95% confidence interval) p- value Heterogen 0.96 [0.45, 1.48] < 0.01 95% 1.04 [0.15, 1.92] 0.02 96% 1.52 [0.22, 2.81] 0.02 94% 0.74 [0.17, 1.30] 0.01 95% 0.72 [0.32, 1.13] < 0.01 92% 1.86 [0.96, 2.76] < 0.01 98% 2.08 [1.18, 2.97] < 0.01 86% 0.51 [0.00, 1.02] 0.05 94% 0.52 [−0.08, 1.12] 0.09 92% 1.31 [0.35, 2.28] < 0.01 96% 0.86 [−0.13, 1.85] 0.09 88% 1.00 [0.04, 2.04] 0.06 96% 0.19 [−0.07, 0.45] 0.15 61% -2.74 [−4.42, −1.07] < 0.01 45% −2.59 [−4.49, −0.68] < 0.01 2% −2.25 [−4.16, −0.34] 0.02 48% −4.75 [−7.69, −1.81] < 0.01 0% −2.43 [−4.18, −0.69] < 0.01 0% −2.21 [−3.57, −0.84] < 0.01 98% −3.56 [−5.91, −1.21] < 0.01 0% −2.57 [−4.57, −0.38] 0.04 54% −5.46 [−9.91, −1.01] 0.02 NA −2.14 [−3.80, −0.49] 0.01 0% −3.82 [−8.26, 0.63] 0.09 78% −2.85 [−6.27, 0.56] 0.10 68% −4.75 [−7.69, −1.81] < 0.01 0% −0.76 [−3.29, 1.77] 0.56 0% −0.99 [−1.48, −0.50] < 0.01 77% −0.57 [−1.20, 0.05] 0.07 37% −0.99 [−1.52, −0.47] < 0.01 55% −0.97 [−2.24, 0.29] 0.13 80% −0.88 [−1.43, −0.34] < 0.01 59% −1.38 [−1.88, −0.88] < 0.01 98% −1.39 [−2.37, −0.42] < 0.01 0% Table 3 Sensitivity and subgroup analyses Characteristics No. of studies No. Sensitivity and subgroup analyses of participants (intervention/ control) Mean change (95% confidence interval) p- value Heterogeneity Physical activity Full analysis 13 1454 (729/725) 0.96 [0.45, 1.48] < 0.01 95% Excluding high risk of bias overall 5 677 (340/337) 1.04 [0.15, 1.92] 0.02 96% Objective measurement 4 224 (128/96) 1.52 [0.22, 2.81] 0.02 94% Self-reported measurement 9 1230 (601/629) 0.74 [0.17, 1.30] 0.01 95% r = 0.5 13 1454 (729/725) 0.72 [0.32, 1.13] < 0.01 92% r = 1.0 13 1454 (729/725) 1.86 [0.96, 2.76] < 0.01 98% Short-term intervention duration 4 235 (126/101) 2.08 [1.18, 2.97] < 0.01 86% Long-term intervention duration 9 1227 (603/624) 0.51 [0.00, 1.02] 0.05 94% Low intensity intervention 4 654 (337/317) 0.52 [−0.08, 1.12] 0.09 92% Medium intensity intervention 6 642 (313/329) 1.31 [0.35, 2.28] < 0.01 96% High intensity intervention 3 158 (79/79) 0.86 [−0.13, 1.85] 0.09 88% Obese subgroup 4 619 (250/269) 1.00 [0.04, 2.04] 0.06 96% CVD subgroup 5 675 (339/346) 0.19 [−0.07, 0.45] 0.15 61% Body Mass (kg) Full analysis 9 872 (419/453) -2.74 [−4.42, −1.07] < 0.01 45% Excluding high risk of bias overall 3 253 (123/130) −2.59 [−4.49, −0.68] < 0.01 2% Objective measurement 7 656 (321/336) −2.25 [−4.16, −0.34] 0.02 48% Self-reported measurement 2 226 (98/117) −4.75 [−7.69, −1.81] < 0.01 0% r = 0.5 9 872 (419/453) −2.43 [−4.18, −0.69] < 0.01 0% r = 1.0 9 872 (419/453) −2.21 [−3.57, −0.84] < 0.01 98% Short-term intervention duration 2 134 (64/70) −3.56 [−5.91, −1.21] < 0.01 0% Long-term intervention duration 7 738 (355/383) −2.57 [−4.57, −0.38] 0.04 54% Low intensity intervention 1 135 (66/79) −5.46 [−9.91, −1.01] 0.02 NA Medium intensity intervention 5 411 (196/215) −2.14 [−3.80, −0.49] 0.01 0% High intensity intervention 3 316 (157/159) −3.82 [−8.26, 0.63] 0.09 78% Obese subgroup 4 520 (250/270) −2.85 [−6.27, 0.56] 0.10 68% CVD subgroup 2 215 (98/117) −4.75 [−7.69, −1.81] < 0.01 0% Diabetes/IGT subgroup 2 73 (39/34) −0.76 [−3.29, 1.77] 0.56 0% BMI (kg/m2) Full analysis 15 4728 (2385/2343) −0.99 [−1.48, −0.50] < 0.01 77% Excluding high risk of bias overall 4 531 (265/266) −0.57 [−1.20, 0.05] 0.07 37% Objective measurement 11 2198 (1126/1072) −0.99 [−1.52, −0.47] < 0.01 55% Self-reported measurement 4 2530 (1259/1271) −0.97 [−2.24, 0.29] 0.13 80% r = 0 5 15 4728 (2385/2343) −0 88 [−1 43 −0 34] < 0 01 59% Table 3 Sensitivity and subgroup analyses Table 3 Sensitivity and subgroup analyses Characteristics No. Sensitivity and subgroup analyses Sensitivity analyses of the imputed correlation coeffi- cients revealed that effect sizes remained statistically Fig. 4 Meta-analysis investigating behavioural lifestyle interventions for changes in body mass Barrett et al. International Journal of Behavioral Nutrition and Physical Activity (2021) 18:7 Page 12 of Barrett et al. International Journal of Behavioral Nutrition and Physical Activity Page 12 of 19 Fig. 5 Meta-analysis investigating behavioural lifestyle interventions for changes in BMI subgroup analysis could be conducted on changes in WC between objective and self-reported measures. significant changes in BMI for individuals with type 2 diabetes/impaired glucose tolerance (Table 3). Lar- ger effect sizes were observed for PA and BMI changes when objective measurement was used. Lar- ger effect sizes were observed for changes in body mass when self-reported measurement was used. No Interventions with a short-term duration demon- strated significant effects for changes in PA, body mass, BMI and WC (Table 3). Interventions with a longer- term duration demonstrated significant effects for Interventions with a short-term duration demon- strated significant effects for changes in PA, body mass, BMI and WC (Table 3). Interventions with a longer- term duration demonstrated significant effects for Fig. 6 Meta-analysis investigating behavioural lifestyle interventions for changes in waist circumference Barrett et al. International Journal of Behavioral Nutrition and Physical Activity (2021) 18:7 Page 13 of 19 Barrett et al. International Journal of Behavioral Nutrition and Physical Activity (2021) 18:7 Barrett et al. Sensitivity and subgroup analyses of studies No. of participants control) Physical activity Full analysis 13 1454 (729/725) Excluding high risk of bias overall 5 677 (340/337) Objective measurement 4 224 (128/96) Self-reported measurement 9 1230 (601/629) r = 0.5 13 1454 (729/725) r = 1.0 13 1454 (729/725) Short-term intervention duration 4 235 (126/101) Long-term intervention duration 9 1227 (603/624) Low intensity intervention 4 654 (337/317) Medium intensity intervention 6 642 (313/329) High intensity intervention 3 158 (79/79) Obese subgroup 4 619 (250/269) CVD subgroup 5 675 (339/346) Body Mass (kg) Full analysis 9 872 (419/453) Excluding high risk of bias overall 3 253 (123/130) Objective measurement 7 656 (321/336) Self-reported measurement 2 226 (98/117) r = 0.5 9 872 (419/453) r = 1.0 9 872 (419/453) Short-term intervention duration 2 134 (64/70) Long-term intervention duration 7 738 (355/383) Low intensity intervention 1 135 (66/79) Medium intensity intervention 5 411 (196/215) High intensity intervention 3 316 (157/159) Obese subgroup 4 520 (250/270) CVD subgroup 2 215 (98/117) Diabetes/IGT subgroup 2 73 (39/34) BMI (kg/m2) Full analysis 15 4728 (2385/2343) Excluding high risk of bias overall 4 531 (265/266) Objective measurement 11 2198 (1126/1072) Self-reported measurement 4 2530 (1259/1271) r = 0.5 15 4728 (2385/2343) r = 1.0 15 4728 (2385/2343) Short-term intervention duration 2 134 (64/70) Barrett et al. International Journal of Behavioral Nutrition and Physical Activity (2021) 18:7 Page 14 of 19 Barrett et al. International Journal of Behavioral Nutrition and Physical Activity Page 14 of 19 Table 3 Sensitivity and subgroup analyses (Continued) Characteristics No. of studies No. Sensitivity and subgroup analyses of participants (intervention/ control) Mean change (95% confidence interval) p- value Heterogeneity Long-term intervention duration 13 4594 (2321/2273) −0.93 [−1.47, −0.39] < 0.01 80% Low intensity intervention 4 698 (349/349) −1.12 [−2.38, 0.15] 0.02 71% Medium intensity intervention 7 2569 (1275/1269) −0.56 [−1.17, 0.04] 0.07 39% High intensity intervention 4 1461 (761/700) −1.63 [−2.85, −0.42] < 0.01 72% Obese subgroup 3 277 (129/148) −2.08 [−5.56, 1.40] 0.24 82% CVD subgroup 2 139 (68/71) −1.88 [−3.04, −0.72] < 0.01 0% Diabetes/IGT subgroup 6 1725 (898/827) −0.99 [−1.19, −0.79] < 0.01 0% Waist Circumference (cm) Full analysis 5 530 (265/265) −2.21 [−4.01, −0.42] 0.02 41% Excluding high risk of bias overall 4 472 (236/236) −2.34 [−4.49, −0.18] 0.03 45% Objective measurement 4 460 (233/227) −1.64 [−3.43, 0.15] 0.07 28% Self-reported measurement – – – – – r = 0.5 5 530 (265/265) −2.40 [−4.20, −0.59] 0.02 55% r = 1.0 5 530 (265/265) −2.61 [−4.23, −0.99] < 0.01 99% Short-term intervention duration 2 134 (64/70) −3.68 [−6.05, −1.30] < 0.01 0% Long-term intervention duration 3 396 (201/195) −1.40 [−3.68, 0.88] 0.23 41% Low intensity intervention 1 278 (142/136) −0.90 [−2.71, 0.91] 0.33 NA Medium intensity intervention 4 252 (123/129) −2.87 [−5.04, −0.70] 0.01 32% High intensity intervention – – – – – CVD subgroup 2 348 (174/174) −2.34 [−5.63, 0.96] 0.40 70% Diabetes/IGT subgroup 2 118 (59/59) −2.05 [−6.85, 2.75] 0.06 53% BMI Body Mass Index, CVD Cardiovascular Disease, IGT Impaired Glucose Tolerance, NA Not applicable Table 3 Sensitivity and subgroup analyses (Continued) small positive changes in PA and anthropometrics can deliver beneficial health benefits [60]. The moderate to large effect sizes demonstrated here are likely to deliver important health outcomes for ambulatory hospital pa- tients [60]. Patients attending secondary care hospital clinics are more likely than the general population to have preventable chronic disease due to risk factors such as insufficient PA or overweight and obesity [61]. Behav- iour change interventions aimed at changes in PA and anthropometrics can go towards addressing health risks in this population [62]. Nevertheless, the heterogeneity of results for all outcomes were moderate to high, and the GRADE assessment indicated that the evidence is very uncertain about the effect of behaviour change in- terventions on changes in PA and anthropometrics. changes in body mass and BMI only (Table 3). Sensitivity and subgroup analyses In terms of intervention dose, interventions categorised as low in- tensity demonstrated significant effects for changes in body mass and WC (Table 3). Interventions categorised as medium intensity demonstrated significant effects for changes in PA and body mass (Table 3). Interventions categorised as high intensity demonstrated significant ef- fects for changes in BMI (Table 3). No subgroup analysis could be conducted on changes in WC between dose of intervention. The moderate to high heterogeneity found in the primary meta-analyses was consistent across the majority of sensitivity and subgroup analyses. Discussion This systematic review and meta-analyses provides evi- dence to support the use of behaviour change interven- tions for changes in PA and anthropometrics, initiated in the ambulatory hospital setting. The effect sizes were large for PA and moderate for anthropometric out- comes. These positive results are important as even The meta-analysis of 13 randomised controlled trials for behaviour change interventions versus standard care for changes in PA demonstrated a significant large effect (d = 0.96) in favour of the intervention. The effect size is larger than those reported for PA interventions aiming Barrett et al. International Journal of Behavioral Nutrition and Physical Activity (2021) 18:7 Page 15 of 19 Page 15 of 19 Page 15 of 19 to increase PA in older adults (d = 0.26) [63], chronically ill adults (d = 0.45) [64], healthy inactive adults (d = 0.32) [65] and young and middle aged adults (d = 0.32) [66], but similar to that reported for behaviour change interventions targeting individuals at risk of cardiovascu- lar disease [10, 11]. The heterogeneity of both interven- tions and outcome measures, and the wide confidence intervals observed in the included studies contributed to the downgrading of the certainty about the results to very low. Despite the low level of certainty, it is encour- aging to see a significant positive intervention effect across the diverse clinical populations with the included measures of PA participation. are the central tenets of prevention programs needed to address overweight and obesity prevalence [72]. This re- view adds to the evidence base to support the use of be- haviour change interventions to influence anthropometric changes in the ambulatory hospital setting. The 2.74 kg (95% CI: −4.42 to −1.07) reduction in body mass found in this meta-analysis compares to simi- lar reductions of 3.77 kg (95% CI: −4.55 to −2.99) [73] and 2.12 kg (95% CI: −2.61 to −1.63) [74] found in be- haviour change interventions for people at high risk for diabetes, and in nutritional education programs with a specific focus on weight loss (−2.07 kg; 95% CI: −1.52 to −2.62). The mean reduction in BMI of 0.99 kg/m2 (95% CI: −1.48 to −0.50) found in this meta-analysis lies be- tween the results from studies in secondary prevention behaviour change interventions, being −0.16 kg/m2 (95% CI: −0.62 to 0.31) [10] and −1.80 kg/m2 (95% CI: −2.62 to −0.99) [11]. Discussion The significant decrease in body mass and BMI over the longer-term follow-up is noteworthy given the mean age of the individuals in the analyses was 57. High proportions of middle aged individuals con- tinue to gain weight each year [75]. The magnitude of improvements observed for changes in anthropometrics found in this review are likely to be clinically significant. Favourable changes in anthropometrics are associated with decreased risk for cardiovascular events [76], type 2 diabetes [76, 77] and some cancers [77]. measures of PA participation. When stratified by follow-up duration, the analyses of the effect of behaviour change interventions on changes in PA demonstrated a significant increase in PA when the follow-up lasted for 6 months sessions or less. Inter- ventions with a follow-up of greater than 6 months dem- onstrated a non-significant effect in favour of the intervention. Samdal et al., (2017) found that strategies such as motivational interviewing and goal setting are ef- fective for assisting individuals in initiating PA behaviour change [67]. Cognitive strategies such as problem solving and relapse prevention, on the other hand, promote changes in cognition, PA beliefs and influence behaviour change maintenance [68]. Some of the most common strategies used in the studies included in this review were motivational interviewing, goal setting and general counselling/health coaching. These strategies are all ac- knowledged as important theoretical constructs for suc- cessful behaviour change [69]; however, very few of the included studies clearly demarcated the use of strategies for PA maintenance, which could have impacted the ef- fect size over the longer term follow-up. Only a small number of the included studies aimed to engage partici- pants in existing community resources. Referrals to spe- cific community programs, such as walking groups, strength training, and exercise for adults, have shown to have a positive effect on longer-term PA behaviour [69]. The meta-analyses of behaviour change interventions versus standard care for changes in anthropometric out- comes demonstrated significant positive effects in body mass, BMI and WC. Significant reductions in body mass, BMI and WC were found when the follow-up lasted for 6 months or less. Significant favourable changes in body mass and BMI were found when the follow-up lasted for greater than 6 months. The increasing prevalence of overweight and obesity over recent decades have been a major public health concern [70]. Discussion Overweight and obes- ity not only have a direct impact on morbidity, but con- tribute significantly towards further metabolic conditions, including insulin resistance, and type 2 dia- betes [71]. Behaviour change interventions, predomin- antly focusing on changes in PA and anthropometrics, When stratified by follow-up duration, the analyses of the effect of behaviour change interventions on changes in PA demonstrated a significant increase in PA when the follow-up lasted for 6 months sessions or less. Inter- ventions with a follow-up of greater than 6 months dem- onstrated a non-significant effect in favour of the intervention. Samdal et al., (2017) found that strategies such as motivational interviewing and goal setting are ef- fective for assisting individuals in initiating PA behaviour change [67]. Cognitive strategies such as problem solving and relapse prevention, on the other hand, promote changes in cognition, PA beliefs and influence behaviour change maintenance [68]. Some of the most common strategies used in the studies included in this review were motivational interviewing, goal setting and general counselling/health coaching. These strategies are all ac- knowledged as important theoretical constructs for suc- cessful behaviour change [69]; however, very few of the included studies clearly demarcated the use of strategies for PA maintenance, which could have impacted the ef- fect size over the longer term follow-up. Only a small number of the included studies aimed to engage partici- pants in existing community resources. Referrals to spe- cific community programs, such as walking groups, strength training, and exercise for adults, have shown to have a positive effect on longer-term PA behaviour [69]. Implications for practice p p Previous research has shown that experiencing health events such as hospital appointments can be the catalyst for changes in behaviour [15, 78]. Ambulatory hospital patients represent an ideal population to intervene with to lessen the risk of developing serious health condi- tions. Incorporating the use of behaviour change inter- ventions to increase PA in adults attending ambulatory hospital clinics aligns with the 2020 World Health Organization guidelines on PA and sedentary behaviour, which indicate the importance of PA for individuals with chronic conditions [79]. The current analysis incorpo- rates a wide range of participant populations attending ambulatory hospital clinics, ranging from younger to older adults, as well as individuals with health risk fac- tors to individuals with diagnosed chronic conditions. Hospital patients have indicated that they would like the healthcare system to provide guidance on behaviour change and healthy lifestyles [80]. Patients and public health at large might benefit from hospitals shifting their focus from predominantly curative care to a position of more holistic health promotion [81, 82]. p g [ ] The meta-analyses of behaviour change interventions versus standard care for changes in anthropometric out- comes demonstrated significant positive effects in body mass, BMI and WC. Significant reductions in body mass, BMI and WC were found when the follow-up lasted for 6 months or less. Significant favourable changes in body mass and BMI were found when the follow-up lasted for greater than 6 months. The increasing prevalence of overweight and obesity over recent decades have been a major public health concern [70]. Overweight and obes- ity not only have a direct impact on morbidity, but con- tribute significantly towards further metabolic conditions, including insulin resistance, and type 2 dia- betes [71]. Behaviour change interventions, predomin- antly focusing on changes in PA and anthropometrics, Hospitals considering integrating behaviour change in- terventions into routine care may be encouraged that the delivery of short duration interventions results in Barrett et al. International Journal of Behavioral Nutrition and Physical Activity (2021) 18:7 Page 16 of 19 Barrett et al. International Journal of Behavioral Nutrition and Physical Activity Page 16 of 19 (2021) 18:7 statistically significant changes in PA, body mass, BMI and WC for hospital patients. The subgroup analyses provide some indication of the effect of intervention dose on PA and anthropometric changes, with signifi- cant changes observed for medium and high intensity in- terventions. Abbreviations BMI: Body Mass Index; CI: Confidence Interval; CVD: Cardiovascular disease; IGT: Impaired Glucose Tolerance; MD: Mean Difference; PA: Physical Activity; PRISMA: Preferred Reporting Items for Systematic Reviews and Meta- Analyses; SMD: Standardised Mean Difference; T2DM: Type 2 Diabetes; WC: Waist Circumference Implications for practice Behaviour change interventions providing a higher number of sessions have been demonstrated to increase self-management skills, which may result in the significant outcomes observed for medium and high in- tensity interventions [83, 84]. Another potential advan- tage highlighted in this review was the range of health professionals that were able to deliver the behaviour change intervention. The diversity in clinicians might be advantageous when applying the intervention across dif- fering sectors of the ambulatory hospital setting. result in moderate to high heterogeneity [87]. Finally, only 5 of the 29 included studies reported on interven- tion fidelity [33, 34, 36, 41, 44]. Without a clear meas- urement of fidelity, reports of the effectiveness of interventions must be interpreted cautiously, as the pos- sibility that the intervention was not delivered as intended cannot be ruled out [65]. Supplementary Information Supplementary Information h l l Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s12966-020-01076-6. Additional file 1. Additional file 2. Additional file 3. Additional file 4. Additional file 5. Additional file 1. Additional file 2. Additional file 3. Additional file 4. Additional file 5. y p g The meta-analyses included studies with small sample sizes, and differences in the duration of interventions. The review also included studies with heterogeneous intervention components including differences in the frequency and duration of the sessions, and differences in the professionals providing the intervention. The het- erogeneity also existed in the delivery format, including face-to-face, telephone calls and group counselling deliv- ery. This heterogeneity makes the independent contribu- tion of any of the intervention components, or a combination of these factors, difficult to establish, and partially explains the moderate to high heterogeneity of the meta-analyses. The moderate to high heterogeneity was reported in the majority of sub-group analyses, indi- cating a consistency of results across the examination of the different components of the interventions. Behaviour change interventions tend to exhibit both clinical and methodological diversity, often resulting in statistical heterogeneity within the meta-analyses [87]. Indeed, al- most one third of meta-analyses have been shown to Acknowledgements The authors would like to thank Mrs. Angela Mundy and Mrs. Angela Johns- Hayden for their help in building the final search database. We acknowledge the support of the Bendigo Tertiary Education Anniversary Foundation and Holsworth Research Initiative for Professor Kingsley’s research. Conclusion Thi i This review indicates that behaviour change interven- tions resulted in large improvements in PA, and moder- ate changes in anthropometric outcomes in adults presenting to ambulatory hospital clinics. The results in- dicate the value of behaviour change interventions for mitigating chronic disease risk factors, and supports the implementation of behaviour change interventions in ambulatory secondary care clinics. The heterogeneity in study populations, reported outcomes, and intervention components downgraded the certainty of the evidence, and prevents the drawing of firmer conclusions from the evidence provided. In order to improve the translation of these findings into clinical practice, future studies of behaviour change interventions should include clearly defined interventions and assessments of treatment fidelity. Limitations This review has a number of limitations. The wide range of PA measures used within the interventions suggest that caution should be applied when interpreting the translatability of these results. Additionally, only 4 of the studies in the PA meta-analysis used objective measure- ment [34, 38, 43, 58]. Social desirability bias can lead to over-reporting of PA levels in self-reported measures [85]. Although the majority of self-report questionnaires were based on valid and reliable measures, objective measurements have demonstrated a higher degree of re- producibility and validity for quantifying duration and intensity of PA [86]. The effect size calculated from studies that used objectively measured PA was higher than the overall effect size observed for PA change (Table 3), which improves confidence in the effective- ness of behaviour change interventions to increase PA in ambulatory hospital settings. Authors’ contributions 1 3 SB1, SB3 and MK conceived the study idea and design. SB1, JL, SB3 PO’H and MK screened the articles. SB1 and OH extracted the data and performed the risk of bias and quality assessments. SB1, SB3 and MK conducted the data analysis. SB1 wrote the draft manuscript. All authors contributed to the interpretation of the results and critically reviewed the manuscript. All authors read and approved the final manuscript. References ld 1. 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https://openalex.org/W4387455384	https://www.researchsquare.com/article/rs-3400744/latest.pdf	English	null	Time-penalized trees (TpT): a new tree-based data mining algorithm for time-varying covariates	Research Square (Research Square)	2,023	cc-by	22,046	Claude Bernard University Lyon 1 Claude Bernard University Lyon 1 Time-penalized trees (TpT): a new tree-based data mining algorithm for time-varying covariates Mathias Valla Abstract This article proposes a new decision tree algorithm that accounts for time-varying covari- ates in the decision-making process. Traditional decision tree algorithms assume that the covariates are static and do not change over time, which can lead to inaccurate predictions in dynamic environments. Other existing methods suggest workaround solutions such as the pseudo-subject approach, discussed in the article. The proposed algorithm utilizes a diﬀer- ent structure and a time-penalized splitting criterion that allow a recursive partitioning of both the covariates space and time. Relevant historical trends are then inherently involved in the construction of a tree, and are visible and interpretable once it is ﬁt. This approach allows for innovative and highly interpretable analysis in settings where the covariates are subject to change over time. The eﬀectiveness of the algorithm is demonstrated through real-world data analysis, highlighting its potential applications in various ﬁelds, including healthcare, ﬁnance, insurance, environmental monitoring, and data mining in general. Key words: decision tree, time-varying covariate, data mining, longitudinal study, algo- rithm ∗Email: mathias.valla@gmail.com , URL: https://mathias-valla.com Research Article Additional Declarations: Competing interest reported. Work(s) conducted within the Research Chair DIALog under the aegis of the Risk Foundation, an initiative by CNP Assurances. Version of Record: A version of this preprint was published at Annals of Mathematics and Arti¬cial Intelligence on August 22nd, 2024. See the published version at https://doi.org/10.1007/s10472-024- 09950-w. Time-penalized trees (TpT): a new tree-based data mining algorithm for time-varying covariates Mathias Valla∗1,2 ´e Claude Bernard Lyon 1, Institut de Science Financi`ere et d’Assurances (ISFA), Laboratoire SAF EA2429, F-69366, Lyon, France. 2Faculty of Economics and Business, KU Leuven, Belgium. 1Univ Lyon, Universit´e Claude Bernard Lyon 1, Institut de Science Financi`ere et d’Assurances (ISFA), Laboratoire SAF EA2429, F-69366, Lyon, France. 2Faculty of Economics and Business, KU Leuven, Belgium. 1 Introduction Decision trees are a popular machine learning tool for data mining as well as classiﬁcation and regression predictions. Growing such a tree is a data-driven process based on a set of input covariates and a target variable. The most famous decision tree algorithm is arguably Classiﬁcation and Regression Trees (CART), introduced by Breiman et al.[5]. CART constructs a binary tree by recursively partitioning the feature space into smaller and smaller subsets, based on a splitting criterion that maximizes the separation between the target variable’s values in each subset. However, traditional decision tree algorithms like CART assume that the input features or covariates are static and do not change over time. In many real-world settings, this assumption is unrealistic, and the time-dynamic nature of the covariates is highly informative and should be included in the tree construction. In such settings, not accounting for dynamic features results in information loss, hence a loss of accuracy and richness of analysis. Other data-driven approaches can already eﬃciently seize the time dimension of features in prediction and data-mining settings. One can think of neural networks (See [30] or [45] for instance). Yet conventional parametric statistical models or machine learning approaches such as logistic regression or most tree-based models cannot handle time-varying covariates straightforwardly. They assume that individual observations are independently and identically distributed. Because of the longitudinal structure of a time-varying dataset (see Section 2.2 for more details), this independence hypothesis cannot be met: diﬀerent observations of a single ∗Email: mathias.valla@gmail.com , URL: https://mathias-valla.com 1 Introduction 2 subject are naturally strongly correlated. To address this limitation, some existing tree-based methods suggest workarounds such as the pseudo-subject approach in survival trees ([17]), which create artiﬁcial left-truncated and right-censored subjects by pooling observations over time, or the inclusion of a mixed eﬀect model structure around a tree-based core ([20, 40, 21]). Such computationally intensive methods proved to yield competitive results in many prediction frameworks, yet we argue in the following sections that they are not entirely satisfying in terms of interpretability. In this article, we propose Time-penalized Tree (TpT), a new decision tree algorithm that accounts for time-varying covariates in the decision-making process. Our algorithm utilizes a diﬀerent structure and a time-penalized splitting criterion that allows for recursive partitioning of both the covariates space and time. 2.1 Classiﬁcation and regression trees In this section, we brieﬂy describe the mechanisms of a simple yet powerful data-mining and prediction model: decision trees, and more speciﬁcally, Classiﬁcation and Regression trees or CART [5]. Here, we assume that all covariates are time-independent. Let D = (x(i), y(i)) N i=1 be a dataset of N individuals with x(i) = x(i) 1 , . . . , x(i) p , the vector of p covariates and y(i) the target variable for the i-th subject. The covariates and target spaces are respectively denoted X and Y. Decision trees create a recursive partition of X based on binary decision rules. This partitioning can be visualized directly in the case where there are two covariates x1 and x2 (see Figure 1). In that case, individual observations are represented as dots that are eventually clustered into nL distinct, non-overlapping regions of X denoted (L1, . . . , LnL). Figure 1: Decision tree recurcive partitioning More generally it can be visualized as a tree (see Figure 2), with yes/no questions within each node and terminal nodes - or leaves - corresponding to the nL regions of the covariates space. Because the regions deﬁned by leaves are non-overlapping, every individual i belongs to a single leaf L(i), and a prediction is made for all individuals falling in the leaf. More generally, let g be a node, at g, we deﬁne D(g) = (x(i), y(i)) ∈D where (x(i), y(i)) ∈g, the set of observations in the node g. The quantity N(g) = \|D(g)\| is then the number of individuals in the node. Figure 1: Decision tree recurcive partitioning Figure 1: Decision tree recurcive partitioning More generally it can be visualized as a tree (see Figure 2), with yes/no questions within each node and terminal nodes - or leaves - corresponding to the nL regions of the covariates space. Because the regions deﬁned by leaves are non-overlapping, every individual i belongs to a single leaf L(i), and a prediction is made for all individuals falling in the leaf. More generally, let g be a node, at g, we deﬁne D(g) = (x(i), y(i)) ∈D where (x(i), y(i)) ∈g, the set of observations in the node g. The quantity N(g) = \|D(g)\| is then the number of individuals in the node. x1 ≤d1 ? x2 ≤d2 ? fT (L1) fT (L2) Yes x2 ≤d3 ? x1 ≤d4 ? 1 Introduction We detail the algorithm and show simulated real data-mining and visualization applications but do not discuss the theoretical convergence guarantees or the ways it can be used as a predictive tool. Such work is of primary interest to us and will constitute forthcoming studies; in that sense, any collaboration on an optimized implementation or on the study of the theoretical properties of TpT in regression and survival settings is more than welcomed. The rest of this paper is structured as follows. We recall the basics about classiﬁcation and regression trees as well as time-varying covariates analysis in Section 2, we also brieﬂy present existing approaches and frame their interpretability ﬂaws. Then we detail the speciﬁci- ties of TpT in Section 3 and explain its beneﬁts, which is the main contribution of this work. In Section 4, we show a concrete application of our framework on a real-world life-insurance dataset, with visuals and illustration work, demonstrating the interpretability properties of TpT. Eventually, Section 5 concludes this paper and details future works. 2 Preliminaries 3 Algorithm 1: Grow algorithm for CART A decision tree is therefore deﬁned by its splitting criterion (SplittingCriterion), its stopping rule(s) (StoppingRules), and its pruning process (Prune). 1or prediction, in such contexts 2 2The set of classes for categorical covariates Input : D, g Output: CART Consider the current node g, if no current node exists, create a new tree T with a single initial node g. if StoppingRules(g) then if StoppingRules(g) then Let g be a leaf with the prediction fT (g). else For all possible covariates and threshold ﬁnd the pair (xk, d) that obtain the best SplittingCriterion(D, xk, d). For all possible covariates and threshold ﬁnd the pair (xk, d) that obtain the best S li i C i i (D d) Split the node g along covariate xk at threshold d into two child nodes gr and gl plit the node g along covariate xk at threshold d into two child nodes gr and gl. row(D(gr) gr) Preliminaries In both cases, a decision tree yields a single constant label1 for any entire region: its mode or mean. The accuracy of a tree is then based on its ability to minimize the error it commits when assigning labels. Among all possible trees - thus, all possible partitions of X - the optimal one should be the one maximizing the label assignment accuracy. Such a tree theoretically exists but cannot generally be found in a computationally reasonable time. Therefore algorithms like CART use a top-down greedy approach: they start from an initial node - the root - containing all observations in D. Then they ﬁnd the covariate xj and the threshold d2 such that they optimize a splitting criterion. The root is then split into those two child nodes for which the same splitting process is repeated until a stopping criterion is triggered. Once grown, this tree is called maximal tree. Such a tree overﬁts the data, and predictions made on observations that were not used to grow the tree are usually inaccurate. That is why a last step is required: the maximal tree is pruned to a subtree that has better general- ization abilities. From an algorithmic perspective, growing a CART can be summarized as such: 2.1 Classiﬁcation and regression trees fT (L3) fT (L4) x1 ≤d5 ? ... ... fT (LnL−1) fT (LnL) No Figure 2: Decision tree example Yes x2 ≤d2 ? x1 ≤d4 ? Figure 2: Decision tree example In a classiﬁcation context, the label given by the tree T for subject i is given by In a classiﬁcation context, the label given by the tree T for subject i is given by fT (x(i)) = mode {y(j), ∀j \| x(j) ∈L(i)} = fT (L(i)). fT (x(i)) = mode {y(j), ∀j \| x(j) ∈L(i)} = fT (L(i)). In a regression context, the label given by the tree T for subject i in leaf Lj is given by fT (x(i)) = mean {y(j), ∀j \| x(j) ∈L(i)} = fT (L(i)). In a regression context, the label given by the tree T for subject i in leaf Lj is given by fT (x(i)) = mean {y(j), ∀j \| x(j) ∈L(i)} = fT (L(i)). 4 2 Preliminaries • φ(p1, . . . , pK) ≥0, 2 Preliminaries (2) (2) Then, the gain function to maximize when splitting the parent node gp into the two child nodes gl and gr is obviously Then, the gain function to maximize when splitting the parent node gp into the two child nodes gl and gr is obviously G(gp; gl, gr) = MSE(gp) − N(gl) N(gp)MSE(gl) + N(gr) N(gp)MSE(gr) . (3) (3) Even if technically, MSE is not an impurity function, we clearly see that Equation 3 is the perfect regression equivalent of Equation 1. Even if technically, MSE is not an impurity function, we clearly see that Equation 3 is the perfect regression equivalent of Equation 1. Thus in the following sections, we use the general notations of equation 1 with I(g) ≡MSE(g) when the target variable is numerical. 2 Preliminaries 5 5 • The minimum of φ is reached whenever any of the pk = 1, then φ(p1, . . . , pK) = 0, • The minimum of φ is reached whenever any of the pk = 1, then φ(p1, . . . , pK) = 0, • The maximum of φ is reached for φ( 1 K , . . . , 1 K ), • The maximum of φ is reached for φ( 1 K , . . . , 1 K ), • φ is symmetric with regard to its arguments. • φ is symmetric with regard to its arguments. For CART, usual classiﬁcation impurities are Gini (φ(p1, . . . , pK) = −P i pilog(pi)), the entropy (φ(p1, . . . , pK) = 1 2 P i pi(1 −pi)) or the twoing measure. For our purposes, no further speciﬁcities are needed and in all generality, the impurity - or heterogeneity - of node g is measured by I(g) = φ(p1(g), . . . pK(g)). At each node of a CART, the optimal split is chosen as the split that reduces the impurity the most. That is to say, the split that maximizes the following gain function by splitting the parent node gp into the two child nodes gl and gr is G(gp; gl, gr) = I(gp) − N(gl) N(gp)I(gl) + N(gr) N(gp)I(gr) . (1) (1) Of course, various other criteria and ideas for splitting exist. This paper does not aim to review all of them but we refer the astute reader to such comparisons of splitting methods (see [31], [8], [4], [41] or [13] for instance). The eﬃcacy of each splitting criterion has been discussed but no deﬁnitive consensus over which one is the ﬁnest exists. All measures prove desirable properties in particular scenarios while demonstrating drawbacks in others. Regression: In a regression context, the best split can be chosen with the target variable empirical variance or mean squared error, a natural choice of heterogeneity measure. We deﬁne MSE(g) the mean squared error at node g, as MSE(g) = ( MSE(g) = P i∈g (ˆyg −yi)2 ˆyg = 1 N(g) P i∈g yi. 2.1.1 Splitting Criterion Originally, CART produces, at every node, a split that minimizes the heterogeneity regarding the target variable within each child node. Equivalently, the optimal split is to maximize the loss of heterogeneity between the considered node and its child nodes: the so-called goodness- on-split. Therefore, measures of heterogeneity are needed when the target variable is categorical - for classiﬁcation tasks - and when it is numerical - for regression tasks -. Classiﬁcation: In classiﬁcation, let’s deﬁne pk, k ∈{1, . . . , P} as the proportion of observa- tions of class k in D. We extend this idea by deﬁning pk(g) as the proportion of observation of class k in D(g). An impurity function φ, is a function measuring the heterogeneity, deﬁned for pk, k ∈{1, . . . , P}, with pk ≥0 and P k pk = 1 such that: • φ(p1, . . . , pK) ≥0, • φ(p1, . . . , pK) ≥0, 2 Preliminaries 2.1.2 Stopping rules Stopping rules can be speciﬁed. In that case, the growing phase continues until one of them is met. First of all, a node will not split any further if all observations it contains have the same target variable value. Then, a minimum improvement in the splitting criterion (for statistical test-based splitting criteria for instance), a maximum depth of the tree (parameter: maxdepth), a minimum number of observations in a node (parameter: minsplit), or a minimum number of observations in the hypothetical child nodes that would result from a new split. 6 2 Preliminaries 6 2.1.3 Tree Pruning The stopping rules aﬀect the size of the maximal tree. No or weak stopping rules will generate a high-variance/low-bias over-ﬁtted tree whereas constraining ones will lead to smaller, more interpretable low-variance/high-bias under-ﬁtted trees. The idea of cost-complexity pruning developed by Breiman emerged from the need to ﬁnd a compromise between the two extremes. The main idea behind cost-complexity pruning is to consider sub-trees of the maximal tree and evaluate them with a cost function that increases as the error rate rises and decreases as the number of leaves drops. When a tree is pruned at a node, the weighted summed error of the leaves increases while the number of leaves reduces, thus a pruned sub-tree is selected only if the error gain is counter-balanced by the complexity loss. The cost of a tree T is given by: Cα(T ) = R(T ) + αψ(nL), α ≥0, (4) (4) where R(T ) is the sum of all errors or impurities of the leaves of T , weighted by the number of individuals they represent. The function ψ is an increasing function of nL, it is originally set to ψ(nL) = nL in Breiman’s work [5], but as demonstrated relevant properties when set to ψ(nL) = √nL in classiﬁcation settings (see Appendix A.1 for more details and references). The penalty α is the complexity parameter: the higher it is, the smaller the pruned tree. With a reasonable choice of ψ, the interest of α is that for a ﬁxed complexity parameter value, there exists a unique smallest subtree T of the maximal tree Tmax that minimizes Cα(T ). Thus by decreasing α, we can construct a sequence of pruned optimal subtrees [T1, T2, . . . , Tmax] of diﬀerent sizes. This tree sequence is such that T1 is the root node, T2 a sub-tree of T with more leaves and accuracy than T1 and so on until Tmax, the unpruned maximal tree. With Breiman’s notation, we have Tmax ⊇· · · ⊇T2 ⊇T1. The optimal complexity parameter value, hence the best tree in the sequence is usually selected using cross-validation. The optimal complexity parameter value, hence the best tree in the sequence is usually selected using cross-validation. 2.2 Longitudinal notations We build Dlong 7 2 Preliminaries 2 as the collection of all observations structured longitudinally : as the collection of all observations structured longitudinally : as the collection of all observations structured longitudinally : as the collection of all observations structured longitudinally : Dlong = i, n t(i) j , t(i) j+1, x(i) j , y(i) j on(i)−1 j=0 N i=1 Dlong = i, n t(i) j , t(i) j+1, x(i) j , y(i) j on(i)−1 j=0 N i=1 Or, if displayed in a table: Or, if displayed in a table: ID Time window Start Time window End Covariate 1 ... Covariate p Target variable 1 t(1) 0 t(1) 1 x(1) 0,1 ... x(1) 0,p y(1) 0 1 t(1) 1 t(1) 2 x(1) 1,1 ... x(1) 1,p y(1) 1 1 t(1) 2 t(1) 3 x(1) 2,1 ... x(1) 2,p y(1) 2 1 t(1) 3 t(1) 4 x(1) 3,1 ... x(1) 3,p y(1) 3 2 t(2) 0 t(2) 1 x(2) 0,1 ... x(2) 0,p y(2) 0 3 t(3) 0 t(3) 1 x(3) 0,1 ... x(3) 0,p y(3) 0 3 t(3) 1 t(3) 2 x(3) 1,1 ... x(3) 1,p y(3) 1 3 t(3) 2 t(3) 3 x(3) 2,1 ... x(3) 2,p y(3) 2 . . . . . . . . . . . . . . . . . . . . . Table 1: A longitudinally structured dataset Time window Start Time window End Covariate 1 ... Covariate p Target variable ID Time window Start Time window End Covariate 1 ... Covariate Table 1: A longitudinally structured dataset 3Note that the same reasoning can be applied to categorical time-varying covariates as well. 2.2 Longitudinal notations This paper aims to enrich the growing process of decision trees in the presence of time-varying covariates. To do so, let us introduce some notations borrowed from the existing longitudinal literature including works of Rizopoulos or Yao et al [36, 47]. Let us assume a very general setting where we want to build a dataset Dlong, encompassing the time-varying features of N subjects, which are repeatedly measured over time. In all generality, let us assume that among the p covariates, pTV of them are time-varying and pTI others are time-invariant. At time t, the set of covariates is given by X(t) = (X1, X2, . . . , XpT I, XpT I+1(t), . . . , Xp(t)). In order to simplify the notations, we consider all constant features as a special case of time-varying covariates, with X(t) = (X1(t), X2(t), . . . , Xp(t)) with Xk(t) = Xk, ∀t and ∀k ∈[1, . . . , pTI]. Let n(i) be the number of distinct times t(i) j , j = 0, 1, . . . , n(i) −1 at which subject i has been observed. At time t(i) j , subject i has a vector of covariates denoted x(i) j = x(i) j,1, . . . , x(i) j,p . Classical longitudinal setting: For a given subject i, covariates are stored in rows, one row per observation window [t(i) j , t(i) j+1). Each row contains the unique t(i) j , t(i) j+1, x(i) j , y(i) j elements, with y(i) j the target variable observed at time t(i) j . They are completed by the subject unique identiﬁer i. Each row represents what we will now call an observation. We build Dlong Classical longitudinal setting: For a given subject i, covariates are stored in rows, one row per observation window [t(i) j , t(i) j+1). Each row contains the unique t(i) j , t(i) j+1, x(i) j , y(i) j elements, with y(i) j the target variable observed at time t(i) j . They are completed by the subject unique identiﬁer i. Each row represents what we will now call an observation. 2.3 Existing longitudinal tree-based algorithms The problem when splitting time-varying covariates: Whether they are designed for survival analysis or not, longitudinal tree-based models exist and propose various methods to include time-varying covariates that cannot naturally ﬁt in the tree-growing algorithm described in Algorithm 1. As an illustrative example, let x1(t) be a numerical time-varying covariate. At each node, a splitting rule of the form “x1(t) ≤s”3 should be able to split subjects into two child nodes. A subject for which this rule is true at all observed times will go in one child node without any ambiguity. On the other hand, the general case where the rule is true for some periods but false for anywhere else is unclear and needs to be addressed. The “eventually not longitudinal” methods: The most na¨ıve model would be a regular CART, trained on all observations in the longitudinal dataset without taking the correlation between observations of the same subject into account. As stated by Segal [39], this would simply ignore the capital aspect of dealing with longitudinal data: “The covariation induced by making several observations of some continuous response on the same unit, as occurs with repeated measures designs, cluster designs, and longitudinal studies, poses data analytic problems. Analysis of such designs that ignore the covariance structure are known to produce incorrect variance estimate.”. Other na¨ıve attempts consist of summarising the longitudinal trajectories of time-varying covariates with a small number of parameters. For instance, one could think of only keeping the mean value of every trajectory, the median, its ﬁnal slope, the baseline value, or the most recent one, ignoring all the remaining information. This leads to a loss of precious information. A similar idea is to regress every longitudinal covariate against Preliminaries 2 8 time and possibly other features, within-subjects to include the parameters of the regression - intercept and slope - as baseline covariate. It can be argued that if the longitudinal covariates are all strongly linearly associated with time, which is rarely the case in practice, this kind of alternative solution can be relevant. Eo and Cho [14] proposed a model called mixed-eﬀects longitudinal tree (MELT) able to handle longitudinal response by ﬁtting a mixed-eﬀect model at each node of the tree. Subjects are then split based on the heterogeneity of their slopes. 2.3 Existing longitudinal tree-based algorithms Kundu [24] extended this idea of resuming information contained in the longitudinal covariates by a combination of splits on baseline covariates and implemented it in the R package LongCART. Other approaches (such as [35] and more recently [32]) designed longitudinal trees that use lagged response values as potential predictors, but still do not treat either the outcome or the covariates as inherently dynamic with time. Overall, in these methods, information is lost during the process, and the number of measurements per subject in real datasets can be too small to obtain satisfying regression parameters. The “CART-extended” methods: Segal and De’ath [39, 11] independently proposed the ﬁrst applications that clearly deﬁne an extension to the CART method and directly account for correlation in the response variable. They both suﬀered limitations as they were designed for a longitudinal response but time-ﬁxed covariates where all the subjects were measured at the same observation times, with the same interval between them. Segal’s regression tree consisted of imputing a covariance structure in the split procedure. This led to many theoretical questions about deﬁning that covariance structure as well as practical ones regarding the complexity of the computations. On the other hand, De’ath procedure simply modiﬁed the CART algorithm by allowing it to consider an entire matrix containing all the observations for one subject as a single observation. Allowing that was done by using the gain of MSE as a splitting criterion, and replacing the 1-dimensional mean in the MSE with a multi-dimensional mean modiﬁed with a covariance structure; the prediction given by the tree would then be the multi-dimensional mean of the observation in the terminal nodes. In both cases, those methods can be seen as ﬁtting a model to the longitudinal outcome at every node as part of the splitting criterion. Segal’s procedure has never been publically available but De’Ath’s algorithm of the tree was published as the R package mvpart (see [12]) which is no longer maintained and accessible today. More recent works by Larsen and Speckman [25] as well as Hsiao et al. [23] followed and improved the idea of De’ath, by redeﬁning the node impurity measure with the Mahalanobis distance and estimating the covariance matrix from the whole data set. 4Louis Capitaine also worked on a promising generalization of decision trees and forests that must be acknowl- edged. We refer to Appendix A.2 for further details. 2.3 Existing longitudinal tree-based algorithms It is worth mentioning that other articles extended the idea of Segal, to binary responses and classiﬁcation trees (see [48]), in a clustering context using deviance as a goodness-of-ﬁt criterion for partitioning (see [1]) and then to every type of longitudinal response - not only continuous or binary - using Generalized estimating equations (see the works of Lee [26, 28, 27]). Such models show advantages in terms of predictive ability and interpretability but do not handle time-varying covariates. The “state-of-the-art” methods: In the work of Hajjem et al. [19], Sela et al. [40] and their respective extensions (see [21, 10]4 and [16]), a general mixed-eﬀect model is assumed for the longitudinal outcome. The tree-based part only predicts ﬁxed eﬀects whereas individual estimated parameters account for all the time-varying eﬀects. Such approaches can estimate longitudinal outcomes but the inclusion of time-varying covariates is handled via the pseudo- subject workaround detailed in the next paragraph. It relies on the assumption that all the 9 2 Preliminaries 9 2 dependency between several observations of the same subject is captured by the random eﬀect of the mixed model. In a survival setting, Du et al. [17] and its extensions (see [46]) proposed a model based on those ideas: they allowed subjects to be divided into pseudo-subjects and used an adjusted log-rank test in the splitting procedure to accommodate for left truncation and ensure that the independence implicit assumption does not lead to biased results. We strongly refer the astute reader to the works mentioned in this paragraph as we consider they are the most advantageous approaches today. The algorithms corresponding to their respective work are the R packages REEMtree, LongituRF, LTRCtrees and LTRCforests, the R function REEMctree and the Python library MERF. Pseudo-subjects For LTRC and mixed-eﬀect tree-based models, the time dependency is dealt with by design and assumptions: every observation is considered independent regarding the splitting process of the tree. They consider the unmodiﬁed Dlong as an input and run through a CART-like growing process, ﬁnding optimal binary decision rules at each node of the tree. Whenever a split produces an ambiguity as described in paragraph 2.3, the periods h t(i) j , t(i) j+1 where their splitting rule “x1(t) ≤s” is true would go to the left node, and the other would go to the right node, thus dividing one subject into several pseudo-subjects. 2.3 Existing longitudinal tree-based algorithms It cleverly addresses the time-handling issue when the bias that comes with correlated right-censored and left-truncated (LTRC) observations is neutralized otherwise. In such models, any individual can be spread in many diﬀerent tree leaves - even if, at any ﬁxed time, any individual will have a single observation that will fall into a unique one. It is undoubtedly an unbiasing and desirable property in terms of prediction but it may be at the expense of interpretability. In our opinion, none of these procedures can inherently handle time-varying covariates in a satisfying way for data-mining and visualization purposes. Indeed, following CART’s example, a unique trajectory per subject would ensure a clear visualization of the data: the algorithm should be designed to separate individuals whose features are signiﬁcantly diverging regarding the target variable rather than pseudo-subjects. 3 Time penalized trees 10 3 Time penalized trees We present here the building blocks of a new way to think about decision trees in the presence of time-varying covariates: time-penalized trees or TpT. Let Dlong be a longitudinal dataset, and T = [0; max i;j (t(i) j )] be the continuous observation interval of time. We deﬁne D(t) as the dataset containing, for every subject i, her unique observation with the maximal observation time t(i) j such that t(i) j ≤t and T (i) ≥t, where x(i)(t) ∈X(t) is the vector of covariates and y(i)(t) ∈Y(t) the target variable at time t. Eventually, N(t) = \|D(t)\| is the total number of subjects under study at time t. Let g be a node, which is also identiﬁed with a subregion of X × ○T it represents. We thus deﬁne D(g), the set of observations in the node g and N(g) = \|D(g)\| the number of subjects it contains. The idea behind TpT is to build a tree that beneﬁts from all the longitudinal informa- tion available and where the concept of time is central: at each node, we chose to split along covariates and time. As stated in Section 2.1, a tree-growing algorithm is deﬁned by its splitting criterion, stopping rule(s), and pruning process. This applies to TpT and the algorithm we suggest can be described as: Input : Dlong, gp Output: TpT Input : Dlong, gp Output: TpT Consider the current parent node gp, if no current node exists, create a new tree T with a single initial node gp at time tp = 0. p p if StoppingRules(gp) then Let gp be a leaf. else For all possible covariates xk, threshold d and time point tc >= tp ﬁnd the triplet (xk, d, tc) that obtain the best SplittingCriterion(Dlong(tc), xk, d). Split the node gp: all subjects with T (i) < tc go to a “duration leaf” gt. All other subjects - with T (i) ≥tc - are split along covariate xk at threshold d into two child nodes gr and gl. Iteratively run Grow(Dlong(tc), gr). Iteratively run Grow(Dlong(tc), gl). return Prune(T ) Algorithm 2: Grow algorithm for TpT In the end, a ﬁnal Time-penalized Tree would look like that: p if StoppingRules(gp) then pp g gp Let gp be a leaf. Let gp be a leaf. 3 Time penalized trees se For all possible covariates xk, threshold d and time point tc >= tp ﬁnd the triplet (xk, d, tc) that obtain the best SplittingCriterion(Dlong(tc), xk, d). For all possible covariates xk, threshold d and time point tc >= tp ﬁnd the triplet (xk, d, tc) that obtain the best SplittingCriterion(Dlong(tc), xk, d). ( ) g g( ) Split the node gp: all subjects with T (i) < tc go to a “duration leaf” gt. All other subjects - with T (i) ≥tc - are split along covariate xk at threshold d into two child nodes gr and gl. Split the node gp: all subjects with T (i) < tc go to a “duration leaf” gt. All other subjects - with T (i) ≥tc - are split along covariate xk at threshold d into two child nodes gr and gl. Iteratively run Grow(Dlong(tc), gr). Iteratively run Grow(Dlong(tc), gr). Iteratively run Grow(Dlong(tc), gr). Iteratively run Grow(Dlong(tc), gl). t P (T ) Algorithm 2: Grow algorithm for TpT Algorithm 2: Grow algorithm for TpT In the end, a ﬁnal Time-penalized Tree would look like that: 11 3 Time penalized trees x1 ≤d1 at t = t1 ? x2 ≤d2 at t = t6 ? x1 ≤d8 at t = t7 ? ˆY (t7) ˆY (t7) x3 ≤d9 at t = t8 ? ... ... Yes x2 ≤d3 at t = t2 ? x3 ≤d4 at t = t3 ? ˆY (t3) ˆY (t3) x1 ≤d5 at t = t4 ? x3 ≤d6 at t = t7 ? ... ... x3 ≤d7 at t = t5 ? ... ... No Duration < t1 Duration < t2 Duration < t2 Duration < t3 Duration < t4 Duration < t7 Duration < t5 Duration < t7 Duration < t8 Time • • • • • • • • • t0 = 0 t1 t2 t3 t4 t5 t6 t7 t8 x1 ≤d1 at t = t1 ? x2 ≤d2 at t = t6 ? x1 ≤d8 at t = t7 ? ˆY (t7) ˆY (t7) x3 ≤d9 at t = t8 ? ... ... Yes x2 ≤d3 at t = t2 ? x3 ≤d4 at t = t3 ? ˆY (t3) ˆY (t3) x1 ≤d5 at t = t4 ? x3 ≤d6 at t = t7 ? ... ... x3 ≤d7 at t = t5 ? ... ... No Duration < t1 Duration < t2 Duration < t2 Duration < t3 Duration < t4 Duration < t7 Duration < t5 Duration < t7 Duration < t8 Time • • • • • • • • • t0 = 0 t1 t2 t3 t4 t5 t6 t7 t8 x1 ≤d5 at t = t4 ? With the root node appearing in blue, leaf nodes appearing in green (duration leaves displayed horizontally from every node and terminal leaves at the very end of every branch) and the timeline being depicted on the left side of the ﬁgure. Deﬁning TpT stopping rules is exactly similar to CART (see Section 2.1.2). Its splitting criterion and pruning process, meanwhile, are modiﬁed and discussed in the sections below. 3.1 TpT splitting criterion The split function for TpT is the critical contribution of the algorithm but it is rather straightforward. We want to select the split on a covariate, at a threshold and a time that will maximize a time-penalized split criterion. A single split of a node gp would look like this: 12 12 3 Time penalized trees gp - x1(t1) ≤d1 ? gl Yes gr No Duration leaf: gt - Duration < t1 Time • • t0 t1 gp - x1(t1) ≤d1 ? gl Yes gr No Duration leaf: gt - Duration < t1 Time • • t0 t1 To obtain such a split, we have to deﬁne a time-penalized split criterion, as Gγ(gp; gl, gr, gt) = I(gp) − N(gl) N(gp)I(gl) + N(gr) N(gp)I(gr) + N(gt) N(gp)I(gt) · e−γ·(tc−tp), With γ ∈R+. With γ ∈R+. With I(g) an impurity or MSE function as described in Section 2.1.1, tp and tc the re- spective times of the parent node and child nodes and γ the penalty parameter. We can immediately see that this is simply the classical CART splitting criterion with an additional exponential penalty term, depending on how distant in time the considered split is. The exponential penalty that we propose induces that the more time distance there is between a parent node and its potential child node, the more penalized the split. In other words, splits are chosen where covariates AND time points are informative about the target variable; we ﬁrst try to ﬁnd close splits if they can detect heterogeneity but distant splits will be considered if they are very informative. We can ﬁnd examples of this type of exponential consideration of time in time series analysis with exponential smoothing (see [7, 22]), where exponential functions are used to assign exponentially decreasing weights over time. We only referenced two rather distant instances of tree-based modiﬁed splitting criteria where exponential weights were introduced. First in Section 5.5 of Bremner’s thesis [6] that used localized splitting criteria that are based on local averaging in regression trees or local proportions in classiﬁcation trees, weighted by exponential weights. The weights have no link to time or a measure of distance from the previous node. Goldstein [18] also suggested using exponential weights in tree-based algorithms to promote splits on covariates that were already used in previous splits over others. 3.1 TpT splitting criterion The partitioning procedure of TpT can also be visualized similarly to Figure 1. Con- sider two time-varying covariates x1(t) and x2(t): at depth 0 and t = 0, the tree is only a root and D(0) is not partitionned. We can see that the ﬁrst split, at t = t0 creates a division of D(t1) such as 3 Time penalized trees 13 Figure 3: TpT 1-depth recursive partitioning Figure 3: TpT 1-depth recursive partitioning Figure 3: TpT 1-depth recursive partitioning At depth 2 and t = t1, all subjects that have been observed up to t = t2 within each partition, are once again split into two subgroups. This creates a division of D(t2) such as Figure 4: TpT 2-depth recursive partitioning Figure 4: TpT 2-depth recursive partitioning 3 Time penalized trees 14 Eventually, the complete partitioning procedure can be visualized as in Figure 5. It is the representation of a classical binary split procedure, with the inclusion of a time dimension. The routes of all subjects can be displayed in that representation: the red, blue and green paths in Figure 5 are examples of such individual trajectories. Figure 5: TpT recursive partitioning and individual trajectories Figure 5: TpT recursive partitioning and individual trajectories Remark 1 Several things need to be noted regarding the partitioning illustration depicted in Figure 5. First, duration leaves are not represented here: the red trajectory for instance, does not split after time t = t1 because the policy it represents has a seniority inferior to t2. Its course after time t1 being unknown, it stops and this region of D(t1) is a duration leaf for similar subjects. Secondly, this illustration shows that divisions of early steps transpose into continuous partitions in further steps: this is not true in general. The two groups formed by the partition of D(t1) may not be represented by a unique region of D(t2), split at a constant threshold over one covariate. For instance, the set of all individuals with a policy face amount (FA) ≤10, 000 at time t1 is 15 Time penalized trees 3 composed of individuals without any observation at time t2, of policyholders with an FA > 10, 000 and ones with an FA ≤10, 000. Eventually, it is also to be noted that not every disjoint region splits at every step of the partitioning. There are times when several splits occur, others where only one region is partitioned, and others where none. All those points are not depicted in Figure 5 for simplicity’s sake. We can already foresee that higher values of γ ensure that the next optimal split is more likely to be close in time to the previous node (a distant split is to be chosen only if it is very interesting). The produced TpT will be close to a CART with all longitudinal covariates values blocked at t = t0. And it can be easily proven that TpT(Dlong) −→ γ→+∞CART(Dlong(t0)). (5) (5) It allows a TpT to explore the covariates space but prevents it from exploring the time dimension. On the contrary, lower values of γ are more likely to produce distant splits and the constructed TpT will show similarities with a CART where all longitudinal covariates values rapidly approach their ﬁnal value. It allows a TpT to split along the time dimension quickly but prevents it from exploring the covariates space at any given time. It allows a TpT to explore the covariates space but prevents it from exploring the time dimension. 3.2 TpT Pruning process For a TpT, the penalty parameter γ aﬀects the tree’s dimensions (depth and number of leaves, see Section 4 for an analysis on the matter). An optimal γ that minimizes the impurity of the tree (the weighted sum of all leaves impurities) can be chosen but it is not a pruning process comparable to cost-complexity pruning. For a given γ, a maximal TpT can be grown and may overﬁt the data. It is obvious in a prediction context but also true in a data-mining framework. To control for bias and overﬁtting, various pruning strategies can be considered. First, Breiman’s cost-complexity pruning is still well-deﬁned under the TpT framework, for a given γ, and can be applied as long as all term nodes - denoted as gt in previous illustrations and algorithm - are considered as leaves. We suggest a slightly diﬀerent adaptation of this pruning strategy to select both α and γ simultaneously. It consists of selecting the pair (α, γ) that minimizes Cα(T ), the cost of the tree. Simpler pruning strategies such as Reduced Error Pruning (see [34]) can also be used. Their advantages and ﬂaws are notably discussed in Esposito’s article [15] as well as their tendency to over/under-prune. Remark 2 Because the impurities of the parent and child nodes can be computed at diﬀerent time points, it can happen that Gγ < 0. Such cases imply that a speciﬁc stopping rule must be enforced for TpT: Gγ must be positive for a node to split. Otherwise, it would allow ineﬀective splits. Remark 1 On the contrary, lower values of γ are more likely to produce distant splits and the constructed TpT will show similarities with a CART where all longitudinal covariates values rapidly approach their ﬁnal value. It allows a TpT to split along the time dimension quickly but prevents it from exploring the covariates space at any given time. 4 Applications We decided to test TpT on real datasets. Such a longitudinal data mining algorithm can prove useful in various ﬁelds (medicine, sports analytics, taxonomy, biology), here we applied it to a life insurance customer segmentation analysis. For that purpose, we use a real-world dataset of 983 policyholders (PH) and we investigate the link between the PH’s characteristics through time and the ﬁnal outcome of their policies. Throughout the lifetime of such insurance poli- cies, a series of events can occur. Firstly, one policyholder’s coverage can be increased with premium payments that are highly ﬂexible, both in terms of amount and frequency, and are adjusted according to the policyholder’s ﬁnancial circumstances and preferences. Additionally, policyholders may decide to reduce their coverage by withdrawing a portion of their policy. We refer to these events as partial lapses, enabling PHs to adjust their coverage to better align with their changing needs. Other ﬁnancial operations can occur, such as the payment of inter- est or proﬁt sharing from the insurer to the PH, and the payment of fees from the PH to the insurer. Insurance companies’ information systems are usually designed to keep track of those operations at the policy level, thus actuaries and life insurers often have access to the complete history of their policyholders as the information system is updated in real-time. Eventually, one’s insurance plan ends whenever the PH dies or decides to terminate it by lapsing. In the end, the timeline of such insurance policies can be illustrated below:5 Figure 6: Example of policyholders timelines Figure 6: Example of policyholders timelines At any given time a policy is either active, has been lapsed by the policyholder, or has ended because of her death. Among all insurance plans subscribed between 1998 and 2019, in our dataset, 57.4% are active, 22.8% ended with the death of the policyholders, and 19.8% lapsed. We only consider uncensored observations here, we thus have 55% of churned policies and 45% that ended with death. For this application, our data mining goal is to gain insights into the PH’s pathways that lead to these diﬀerent outcomes. We want to ﬁnd time-dependent clusters of individuals with similar timelines and outcomes at a given time. 5Illustration taken from [42] 3.3 Time-to-event Survival analysis can also be carried out directly under the TpT framework. Indeed, for data mining purposes, subjects are distributed in the ﬁnal tree considering their last observed time T (i). Censorship and event occurrences are visible in the term nodes gt. Adapting TpT for prediction tasks would require additional work to account for censorship (see [44]), but this speciﬁc topic is not in the scope of our paper. 4 Applications 16 4 Applications This is thus a time-dependent classiﬁcation problem, where the target variable is the ﬁnal outcome of the policies, the tree grows with the survival time and splits on potentially time-varying covariates such as age, rate, Customer Lifetime Value (CLV), face amount (FA) or gender. More detailed descriptions of the dataset used can be found in Valla et al. and in Valla’s works [43, 42] 4 Applications 17 4.1 Properties of TpT for the maximal tree 4.1 Properties of TpT for the maximal tree First of all, Table 2 below displays the results obtained by TpT with various choices for the time penalty parameter γ. It shows the dimensions of TpTs (depth and number of leaves), their global impurities and costs, the highest time point when a split occurred, and the average time at which any subject is split. Graphs of those results can be found in Figure 7. Here we considered unpruned trees using the time-penalized version of the Gini impurity measure as a splitting rule and without any stopping criterion. For this application, we computed the cost of the tree with a choice of α = q 3 log 2 2N , suggested by Scott [37]. The pruning process then only consist of selecting γ as the solution of argminCα(T ). Figure 7: Characteritics of unstopped and unpruned TpT depending on the time penalty More results and graphs obtained with this setting, but also with diﬀerent impurity measures Figure 7: Characteritics of unstopped and unpruned TpT depending on the time penalty More results and graphs obtained with this setting, but also with diﬀerent impurity measures are displayed in Appendix A.3. More results and graphs obtained with this setting, but also with diﬀerent impurity measures are displayed in Appendix A.3. Table 2: Characteritics of TpT depending on the time penalty 4.1 Properties of TpT for the maximal tree 18 Applications 4 Time penalty γ Runtime Depth # of terminal leaves # of duration leaves Total # of leaves Tree cost Max of split times Mean of split tim 0.0000 719.07 7 27 17 44 0.412 20.0 5.768 0.0025 733.95 7 27 16 43 0.411 20.0 5.276 0.0050 732.73 7 27 15 42 0.408 20.0 5.192 0.0100 725.95 9 30 15 45 0.410 20.0 5.429 0.0125 795.26 10 35 19 54 0.416 20.0 4.832 0.0175 862.99 11 35 21 56 0.416 20.0 3.81 0.0200 883.2 11 35 20 55 0.414 20.0 3.599 0.0300 868.98 11 35 20 55 0.415 20.0 3.601 0.0325 1007.65 11 55 33 88 0.413 20.0 4.315 0.0350 970.33 11 53 27 80 0.421 20.0 3.977 0.0450 1046.33 10 61 30 91 0.421 20.0 3.763 0.0500 1062.24 10 71 36 107 0.424 20.0 4.175 0.0625 1069.06 10 69 36 105 0.422 20.0 4.209 0.0650 1141.95 11 79 47 126 0.425 18.0 4.277 0.0700 1137.4 11 81 49 130 0.426 18.0 4.282 0.0775 1117.22 11 83 44 127 0.417 18.0 4.272 0.0800 1122.76 11 87 46 133 0.417 18.0 4.26 0.0825 1133.39 11 87 45 132 0.416 18.0 4.238 0.0850 1165.53 11 89 46 135 0.416 18.0 3.947 0.0900 1287.23 15 98 49 147 0.408 18.0 3.579 0.0950 1350.29 15 105 51 156 0.390 18.0 3.085 0.1025 1340.77 15 106 50 156 0.389 18.0 3.083 0.1050 1346.53 15 104 44 148 0.378 18.0 2.906 0.1075 1349.57 15 104 44 148 0.379 18.0 2.898 0.1125 1351.12 15 105 44 149 0.380 18.0 2.888 0.1200 1347.56 15 105 43 148 0.375 18.0 2.929 0.1300 1378.96 15 112 41 153 0.374 16.0 2.929 0.1400 1561.86 15 118 48 166 0.385 16.0 2.645 0.1425 1559.3 15 122 44 166 0.381 16.0 2.416 0.1475 1685.14 17 124 43 167 0.377 16.0 2.129 0.1500 1712.25 15 128 40 168 0.375 15.0 1.778 0.1525 1677.49 16 130 39 169 0.377 15.0 1.76 0.1550 1709.18 16 131 39 170 0.378 15.0 1.749 0.1575 1692.19 16 137 38 175 0.371 14.0 1.773 0.1700 1686.58 16 137 36 173 0.370 14.0 1.762 0.1775 1687.16 16 140 35 175 0.370 13.0 1.663 0.1850 1695.6 15 143 33 176 0.369 13.0 1.647 0.1925 1687.49 15 144 33 177 0.370 13.0 1.64 0.1950 1704.68 15 146 33 179 0.367 13.0 1.608 0.1975 1704.85 15 146 33 179 0.363 13.0 1.618 0.2075 1679.89 15 149 33 182 0.364 12.0 1.617 0.2100 1721.42 15 150 32 182 0.366 12.0 1.521 0.2125 1741.25 15 152 31 183 0.366 12.0 1.519 0.2150 1740.3 15 155 29 184 0.367 12.0 1.502 0.2175 1746.67 15 157 29 186 0.368 12.0 1.492 0.2275 1730.43 15 158 28 186 0.365 12.0 1.465 0.2350 1717.29 15 160 28 188 0.366 12.0 1.457 0.2375 1724.14 15 161 27 188 0.366 12.0 1.455 0.2500 1796.99 15 165 25 190 0.359 14.0 1.184 0.2525 1809.4 15 168 23 191 0.359 13.0 1.158 0.2550 1810.04 15 169 22 191 0.359 13.0 1.143 0.2575 1832.64 15 169 21 190 0.357 13.0 1.142 0.2675 1854.5 19 169 23 192 0.360 11.0 1.198 0.2725 1850.47 17 173 17 190 0.356 10.0 0.981 0.2775 1857.1 17 174 16 190 0.356 10.0 0.965 0.2875 2025.16 17 173 11 184 0.358 10.0 0.645 0.2900 2045.07 17 174 10 184 0.358 10.0 0.648 0.3050 1976.4 17 175 10 185 0.359 10.0 0.639 0.3125 1969.5 17 176 9 185 0.361 10.0 0.635 0.3250 1960.95 17 176 10 186 0.362 10.0 0.64 0.3300 1949.79 17 176 10 186 0.360 10.0 0.606 0.3325 1976.88 17 177 9 186 0.359 10.0 0.604 0.3375 1965.69 17 176 9 185 0.357 11.0 0.534 0.3425 1953.18 17 179 5 184 0.358 6.0 0.267 0.3475 1947.54 18 181 4 185 0.358 5.0 0.265 0.3650 1962.15 18 181 4 185 0.359 5.0 0.236 0.3900 1918.64 18 183 4 187 0.360 5.0 0.226 0.4325 1809.19 18 183 4 187 0.361 5.0 0.223 0.4625 1683.22 18 185 3 188 0.362 5.0 0.216 0.4725 1651.89 18 187 3 190 0.364 5.0 0.213 0.4950 1620.57 18 188 3 191 0.363 5.0 0.199 0.5000 1626.91 18 189 2 191 0.365 5.0 0.111 0.5500 1636.21 18 193 1 194 0.374 5.0 0.057 0.6000 1629.73 18 193 1 194 0.375 5.0 0.055 0.7000 1572.03 18 194 1 195 0.377 5.0 0.021 0.8000 1566.33 18 194 0 194 0.378 5.0 0.016 0.9000 1571.42 18 197 0 197 0.382 5.0 0.007 1.0000 1575.33 18 198 0 198 0.383 5.0 0.006 1.1000 1549.67 18 199 0 199 0.384 5.0 0.005 1.3000 1509.43 18 200 0 200 0.384 5.0 0.004 1.4000 1511.22 18 201 0 201 0.385 5.0 0.003 4 Applications 4 Applications 19 We can observe that the depth and number of leaves grow with γ. 4.1 Properties of TpT for the maximal tree This was to be ex- pected, as a TpT that does not penalize time-distant splits will quickly ﬁnd high impurity-gain splits at distant times thus preventing the exploration of less distant time periods. Conversely, the same phenomenon explains that the average time when splits occur is a decreasing function of γ. As the penalty parameters get high values, any future split is heavily penalized and can not compete with splits at time t0, regardless of their potential unpenalized gain. Eventually and very interestingly, we observe that the unpenalized TpT, as well as the heavily penalized one, are not optimal in terms of global impurity. There exists an optimal choice of γ that generates a TpT minimizing the sum of its leaves impurities. This tree has a penalty parameter of 0.2725, a depth of 17 and a number of leaves of 190 - 173 terminal leaves and 17 duration leaves - and is displayed in Appendix A.3. Such trees, without stopping criterion and post-pruning are useful to discuss the proper- ties of TpTs but do not yield immediate insights on our dataset. Nevertheless, there is one statistic that proves to be insightful: the distribution of times when splits occur. Obviously, with an exponentially penalized splitting criterion, the more distant from its parent time tp a split time tc is, the more penalized it is and the less likely it is to be selected. The a priori probability for time to be selected as a split time is ∝e−γ·(tp−tc) Thus, by weighting this distribution with an exponential factor, we balance this bias and retrieve the importance of the time periods. In the optimal unpruned and unstopped tree, the splitting time points are distributed as such: Figure 8: Split times distribution for the optimal unstopped and unpruned TpT Figure 8: Split times distribution for the optimal unstopped and unpruned TpT In the weighted histogram, we can clearly see that some periods seem to be critical split points that diﬀerentiate active policies from lapsers or policies that are likely to end with the policyholder’s death. Interestingly, we see that t = 0 and t = 8 are particularly important in terms of diﬀerentiation between policies’ outcomes. 4.1 Properties of TpT for the maximal tree However, every split at t = 3 is either a split on CLV or the FA of the policy, thus we can argue that the ﬁnal outcome of a policy seems dependent on its FA 3 years after subscription. Similarly, the unpenalized suboptimal TpT with γ = 0, depicted in Figure 11 only splits at times t = 0, t = 3 or t ≥8, with respectively 1, 1 and 24 splits. This application has also been tried on a larger longitudinal dataset, containing the 119,431 observations of 9,873 PHs. Characteritics of TpTs grown with various γ are discribed in Figure 9 It gives the split times distribution in Figure 10. Due to the heavy computation time, all other analysis are carried out on the smaller dataset. This application has also been tried on a larger longitudinal dataset, containing the 119,431 observations of 9,873 PHs. Characteritics of TpTs grown with various γ are discribed in Figure 9 It gives the split times distribution in Figure 10. Due to the heavy computation time, all other analysis are carried out on the smaller dataset. Figure 9: Characteritics of unstopped and un- pruned TpT, trained on 9,873 PHs with various time penalties Figure 10: Split times distribution for the optimal unstopped and unpruned TpT, trained on 9,873 PHs Figure 9: Characteritics of unstopped and un- pruned TpT, trained on 9,873 PHs with various time penalties Figure 10: Split times distribution for the optimal unstopped and unpruned TpT, trained on 9,873 PHs In all following vizualisations, all leaves or regions that contain a majority of policies that ended with the PH’s death are labelled “D”, and all those that contain a majority of policies that ended with lapse (or churn) are labelled “C”. In terms of colors, the proportion of each class is represented by a nuance between (for a 100% proportion of “D”) and (for a 100% proportion of “C”). For example, a leaf or region with 50% of churners is represented by the color . In all following vizualisations, all leaves or regions that contain a majority of policies that ended with the PH’s death are labelled “D”, and all those that contain a majority of policies that ended with lapse (or churn) are labelled “C”. 4.1 Properties of TpT for the maximal tree For t = 0, the insight is clear: most of the information that separates the churners from policies that end with the PH’s death can be retrieved from the baseline covariates: for instance, the age at subscription seems to be very informative - older PHs are more exposed to the mortality risk - and thus is selected at baseline. Regarding the important splits at t = 8, they correspond to splits on age, CLV, or 20 4 Applications 4 FA. CLV is highly dependent on both age and FA, thus we could argue that age and FA are the most informative covariates at t = 8. By law, French life insurance plans ensure that when a given policy is at least eight years old, the policyholder can lapse without any surrender penalty. This is a clear incentive not to churn before one’s policy reaches 8 years of seniority. It seems consistent to observe that this threshold is pointed out in our analysis. The third year of seniority comes right after t = 0 and t = 8 in terms of temporal importance, which does not have any obvious business justiﬁcation. However, every split at t = 3 is either a split on CLV or the FA of the policy, thus we can argue that the ﬁnal outcome of a policy seems dependent on its FA 3 years after subscription. Similarly, the unpenalized suboptimal TpT with γ = 0, depicted in Figure 11 only splits at times t = 0, t = 3 or t ≥8, with respectively 1, 1 and 24 splits. FA. CLV is highly dependent on both age and FA, thus we could argue that age and FA are the most informative covariates at t = 8. By law, French life insurance plans ensure that when a given policy is at least eight years old, the policyholder can lapse without any surrender penalty. This is a clear incentive not to churn before one’s policy reaches 8 years of seniority. It seems consistent to observe that this threshold is pointed out in our analysis. The third year of seniority comes right after t = 0 and t = 8 in terms of temporal importance, which does not have any obvious business justiﬁcation. 4.1 Properties of TpT for the maximal tree In terms of colors, the proportion of each class is represented by a nuance between (for a 100% proportion of “D”) and (for a 100% proportion of “C”). For example, a leaf or region with 50% of churners is represented by the color . 21 4 Applications Figure 11: Overﬁtted unpenalized, unstopped and unpruned TpT Figure 11: Overﬁtted unpenalized, unstopped and unpruned TpT 4.2 Use-case with minsplit A clear strength of decision trees is their interpretability. Obviously, trees with hundreds of leaves each containing a handful of subjects can not be interpreted. Here we decided to inves- 22 Applications 4 tigate the results obtained by TpTs with various γ, using the time-penalized version of the Gini impurity measure as a splitting rule and including a stopping criterion: any leaf must contain at least 50 individuals otherwise it does not split. This choice of stopping rule is not really close to the default value for the minsplit parameter in most CART implementations, but it will generate shorter, less overﬁtted TpTs, better suited for direct interpretability and data analysis. Here are the results for TpT on our longitudinal dataset, with minsplit= 50. Graphs of those results can be found in Figure 12. Time penalty γ Runtime Depth # of terminal leaves # of duration leaves Total # of leaves Tree cost Max of split times Mean of split times 0.0000 587.03 4 9 7 16 0.319 15.0 4.309 0.0025 736.31 6 11 6 17 0.306 15.0 2.134 0.0075 730.35 6 11 5 16 0.306 15.0 2.102 0.0200 726.84 6 11 4 15 0.306 15.0 1.97 0.0275 789.2 6 13 6 19 0.300 8.0 2.203 0.0325 784.67 6 13 5 18 0.300 8.0 2.131 0.0350 817.64 6 14 4 18 0.296 9.0 1.762 0.0650 840.38 7 15 3 18 0.297 9.0 1.129 0.0925 841.11 7 15 2 17 0.299 8.0 0.88 0.1100 850.24 7 15 1 16 0.300 8.0 0.564 0.1250 873.15 7 16 1 17 0.298 5.0 0.423 0.1375 873.64 6 15 2 17 0.297 5.0 0.303 0.1900 899.41 6 15 1 16 0.297 3.0 0.157 0.2050 886.07 6 15 1 16 0.298 3.0 0.125 0.7000 752.84 6 15 0 15 0.299 1.0 0.032 0.8000 743.89 6 15 0 15 0.300 0.0 0.0 Table 3: Characteritics of TpT (minsplit: 50) depending on the time penalty Table 3: Characteritics of TpT (minsplit: 50) depending on the time penalty Table 3: Characteritics of TpT (minsplit: 50) depending on the time penalty Figure 12: Characteritics of TpT (minsplit: 50) depending on the time penalty Figure 12: Characteritics of TpT (minsplit: 50) depending on the time penalty In this table are a few remarkable TpT. First of all, we also see here that the trees with γ = 0 and γ →∞are not the best in terms of global cost. 4.2 Use-case with minsplit An important temporal dependence that can be learned from the tree is the fact that there exists an incentive not to lapse before eight years of seniority. It is clearly depicted in the optimal TpT - γ = 0.035 - as the duration leaves generated by splits occurring at times ≥8 contain a majority of policyholders that did not lapse. It means that regardless of their age, subjects with a seniority ≤8 years do not lapse. The TpT with no time penalty - γ = 0 - can capture the same temporal dependence for splits that occur immediately after 8 years for older PH but fails to do so for younger ones. This is explained by the fact that for the latter, the unpenalized TpT quickly ﬁnds an excellent split at time t = 15, which prevents splits around 8 years from being found. This is a compelling argument in favor of a time penalty. Furthermore, the TpT with a very high time penalty produces a tree that only splits at time t = 0, thus no temporal insights can be found with it. If we were to conclude from such a tree, we could say that Age is the most important covariate, and allow for a good partitionning of D but we cannot have any temporal analysis. This is an argument in favor of TpT and the suggested γ selection process. 4.2 Use-case with minsplit This is a critical result: γ = 0 is the case where the last observed observation points are quickly considered whereas early periods are not really considered, and high γ represents the case where a tree is grown only one the baseline values of all time-varying covariates. Thus, TpT shows that considering time in the splitting process improves the global purity of the tree, it better diﬀerentiates between individuals with diﬀerent outcomes and trajectories. In terms of interpretability, Figure 13 shows that the optimal TpT is a compromise between small TpTs with time-distant splits and a large baseline tree without any temporal information. Whole-page versions of those trees can be found in Appendix A.3.3. Applications 23 Figure 13: TpTs with γ = 0, γ = 0.035 and γ →∞, respectively Figure 13: TpTs with γ = 0, γ = 0.035 and γ →∞, respectively An important temporal dependence that can be learned from the tree is the fact that there exists an incentive not to lapse before eight years of seniority. It is clearly depicted in the optimal TpT - γ = 0.035 - as the duration leaves generated by splits occurring at times ≥8 contain a majority of policyholders that did not lapse. It means that regardless of their age, subjects with a seniority ≤8 years do not lapse. The TpT with no time penalty - γ = 0 - can capture the same temporal dependence for splits that occur immediately after 8 years for older PH but fails to do so for younger ones. This is explained by the fact that for the latter, the unpenalized TpT quickly ﬁnds an excellent split at time t = 15, which prevents splits around 8 years from being found. This is a compelling argument in favor of a time penalty. Furthermore, the TpT with a very high time penalty produces a tree that only splits at time t = 0, thus no temporal insights can be found with it. If we were to conclude from such a tree, we could say that Age is the most important covariate, and allow for a good partitionning of D but we cannot have any temporal analysis. This is an argument in favor of TpT and the suggested γ selection process. 5.2 Limitations and future work For data-mining purposes and with the algorithm as it is deﬁned, many improvement paths can be considered. First of all, the introduction of a penalized splitting criterion, and thus a penalty pa- rameter could be discussed more thoroughly. The current multiplicative exponential form of penalization has been duly justiﬁed but one could explore the eﬀects of diﬀerent approaches. Other distributions of the future time cut-oﬀpenalties such as Gamma (with parameters α < 1, β ≥1 or α = 1, β > 2), Pareto, Weibull (with parameter k < 1) or Log-logistic (β ≤1) could be justiﬁed on concrete examples. Moreover, in the algorithm as it stands, every point in time can be considered for a potential cut-oﬀ; some time-horizon limit where distant splits would simply be ignored would have an impact on the shape of the ﬁnal tree. Eventually, an additive time penalty similar to a Lasso or Ridge regression penalty term is yet to be explored. In all those scenarios, the penalty parameter aﬀects the width and length of the ﬁnal tree and can even be interpreted as a pre-pruning parameter. The properties of that pre-pruning as well as the choice of an optimal γ are yet to be discussed. On a ﬁnal note, we do not know if any technical properties (see [4, 8]) of the penalized splitting criterion still hold. That knowl- edge will certainly not aﬀect the concrete applications of TpT but is more of a theoretical interest. Secondly, we showed in illustrative applications that time-outliers can be easily miscate- gorized as the TpT can send them early in one direction of the tree from which they will not escape. In addition to that, those observations are likely to end up being isolated in a leaf if the stopping rules allow it. First of all, it is not clear if this constitutes a ﬂaw of TpT. On the one hand, it forces observation into an early path that may not be consistent with later observations. On the other hand, this behavior is linked to an abrupt change of the covariates and target variable trajectories in time, which is a discriminating feature that can justify that such subjects end up in a speciﬁc leaf. We see two ways to handle this speciﬁc property: • A ﬁrst idea would be to modify the TpT algorithm to make it less greedy. 4.3 Pathways vizualizations In terms of data mining and clustering, let us focus on the optimal TpT obtained in the previous section and depicted in Figure 14. In the same way a decision tree is a representation of all observations in a cross-sectional dataset, a TpT is a complete representation of a longitudinal one and we can highlight the pathway of any given policyholder in the tree. Unlike any other longitudinal tree-based model, any individual has a unique continuous trajectory in the tree. The pathways of ﬁve policyholders selected at random from our dataset are represented in the following TpT. 24 24 4 Applications Figure 14: Individual longitudinal trajectories in the optimal TpT (minsplit = 50) Figure 14: Individual longitudinal trajectories in the optimal TpT (minsplit = 50) 25 4 Applications Thus, the longitudinal dataset and all individual timelines can be easily represented as a parti- tionning. All PH are represented on the y-axis and the region of the covariate space where they belong changes as a function of time, on the x-axis. In this example, the 18 leaves of Figure 14 correspond to the 18 ﬁnal regions of Figure 15, as t →∞. Figure 15: Global timeline and individual longitudinal trajectories Figure 15: Global timeline and individual longitudinal trajectories Figure 15: Global timeline and individual longitudinal trajectories The numbers in each regions, as well as their hights is the number of PH they contained, and the ﬁve individual trajectories represented as pathways in the tree correspond to the ﬁve horizontal lines within the global timeline. This type of visualization allow to better interpret the periods where changes in the outcome can occur. 5 Conclusion, limitations and future work 26 5.1 Conclusion This paper exhibits TpT, a new tree-based data-mining algorithm that accounts for time-varying covariates through time-penalized splitting criteria. As it is the ﬁrst article on the subject, it is meant to be a mere introduction of the algorithm with few applications on real data. Our methods handle time-varying covariates as well as a longitudinal target variable inherently. Con- trary to existing approaches, it does not need workaround strategies such as the pseudo-subject method and provides a tree that separates ”complete individuals”, as each subject covariates trajectory corresponds to a single unique trajectory in the ﬁnal tree. Pruning strategies were proposed and tested with real datasets and illustrative examples. The algorithm proves to have appealing data-mining and visualization potential in various ﬁelds that could be explored more deeply in the future. 5.2 Limitations and future work Instead of choosing the best split at each node, we could consider ﬁnding the best sequence of several consecutive splits. This N-step ahead strategy would ensure that abrupt changes in covariates in the future are anticipated in early splits. In cases where the penalty parameter is low, this approach also ensures that the TpT does not grow too fast with time. 5 Conclusion, limitations and future work 27 • Another innovative solution is to introduce the possibility for an outlier in a node to teleport into another one, at a similar depth/split time in the tree. For instance, if it so happens that a subject trajectory suddenly becomes signiﬁcantly diﬀerent from the other ones in the same node, it can be clever to acknowledge that it is no longer consistent to keep it in the said node. This solution has drawbacks: it requires testing for outliers in every node, at every step. If one is found, it can only be teleported if another node within which the subject would not be an outlier is found. Moreover, it is a straightforward solution for data mining but other adaptations are necessary if TpT is to be used for predictive tasks. Despite this, it would still ensure individual trajectories for every subject in the tree and it would consolidate the global within-node homogeneity. Then, our last point raises another capital question: the applications shown in Section 4 only exhibit the potential of TpT in a data mining setting; can it be adapted to prediction tasks? In our context, a prediction is an estimation of a subject’s target variable y(i) at a time t, given its covariate history up to t. An obvious research path in that direction is to mimic the example of CART. For a subject in node g, predicting the mean of the target variable at time t of all subjects emerging from node g is to be tried. If the target variable is a time-to-event and the observations are censored, it could be weighted by the inverse probabilities of censoring weights (IPCW). It perfectly translates in terms of interpretability: the prediction of an outcome at time t for individual i is the mean of the outcomes at time t of all subjects taking the same trajectory in the tree. There are several obvious drawbacks to this approach: there needs to exist observations of other subjects at time t. 5.2 Limitations and future work And even if some exist, the variance of the prediction is directly linked to the number of such subjects. Exploring the properties and pre- dictive performance of this approach is left as future work and other methods such as ﬁtting a longitudinal model6 at every node, not for splitting but for prediction purposes are also studied. Eventually, if prediction is made possible in the future, exploring the performance of en- semble methods for TpT looks like a reasonable next step. Such approaches are in contradiction with the research of interpretability that motivated TpT, but competitive predictive performance could justify them. 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In this article, they illustrate the prediction ability of Fr´echet forests on longitudinal data and the robustness of their method to missing data and time shifts. Several limitations can be pointed out: ﬁrstly the mathematical assumption of the existence of the Fr´echet mean in the output space must be veriﬁed and that mean must be approximated as precisely as possible. Another limitation is the interpretability, as it is always the case with bagging techniques, but here it is also true for individual Fr´echet trees: if covariates’ importance can be analyzed, relevant threshold and time points can not be easily observed. Eventually, the computational burden of this algorithm is also important. This method has been implemented in the R package FrechForest. A.1 About cost-complexity pruning Even thougt the original cost function of the CART algorithm described by [5] is penalized proportionnaly to its number of leaves nL, several works on the matter suggest other types of penalty. [2] shows that applying risk bounds to CART imply a penalty with ψ(nL) = √nL. In later works, [29], [33], then [38] also showed that risk bounds with a penalty using ψ(nL) = √nL can be derived for classiﬁcation trees whereas penalties proportionnal to nL can only be derived in speciﬁc cases discusse by [3]. In summary, square-root penalties appear to have a much stronger theoretical foundation than nL proportionnal ones in various context, notably for classiﬁcation tasks. The maximum depth achieved is 17 The number of leaves is 190 173 terminal leaves and 17 duration leaves The tree impurity is : 0.06270710057403012 The penalized tree impurity is : 0.3556124586066797 The maximum time where a split occured is 10.0 The average split time is 0.9805922147055561 The tree is : depth = 0 if Age <= 65.5 at t = 0.0, samples: 983 and no duration leaf then depth = 1 if Age <= 42.5 at t = 0.0, samples: 463 and no duration leaf then depth = 2 if GENDER <= 1.5 at t = 0.0, samples: 110 and no duration leaf then depth = 3 if Age <= 30.5 at t = 0.0, samples: 58 and no duration leaf then depth = 4{value: CHURNED, samples: 29} else depth = 4 if CLV <= 9.16 at t = 0.0, samples: 29 A.3.1 Results without stopping criterion The maximal unpruned and unstopped TpT, obtained with the time-penalized Gini split- ting criterion and an optimal time penalty achieves a depth of 17, has 190 leaves - 173 terminal leaves, 17 duration leaves - and is too large to be fully displayed as a tree here. However, we can still represent it as a list of decisions describing how the dataset is partitionned: 32 32 A Appendix A Appendix then depth = 5{value: CHURNED, samples: 9} else depth = 5 if FACE AMOUNT <= 7197.19 at t = 2.0, samples: 20 and no duration leaf then depth = 6 if FACE AMOUNT <= 3654.22 at t = 2.0, samples: 9 and no duration leaf then depth = 7 if Age <= 39.5 at t = 2.0, samples: 5 and no duration leaf then depth = 8{value: CHURNED, samples: 3} else depth = 8{value: CHURNED 0.5, samples: 2} else depth = 7{value: DEATH, samples: 4} else depth = 6{value: CHURNED, samples: 11} else depth = 3{value: CHURNED, samples: 52} else depth = 2 if GENDER <= 1.5 at t = 0.0, samples: 353 and no duration leaf then depth = 3 if Age <= 53.5 at t = 0.0, samples: 154 and no duration leaf then depth = 4 if Age <= 52.5 at t = 0.0, samples: 53 and no duration leaf then depth = 5 if CLV <= 13.11 at t = 0.0, samples: 47 and no duration leaf then depth = 6 if FACE AMOUNT <= 3196.44 at t = 3.0, samples: 10 and duration leaf has 1 samples. A Appendix Label is : CHURNED 1.0 then depth = 7{value: CHURNED, samples: 7} else depth = 7{value: CHURNED 0.5, samples: 2} else depth = 6 if CLV <= 88.4 at t = 0.0, samples: 37 and no duration leaf then depth = 7 if Nb Contrats <= 1.5 at t = 0.0, samples: 14 and no duration leaf then depth = 8 if CLV <= 40.05 at t = 0.0, samples: 12 and no duration leaf then depth = 9 if Age <= 45.5 at t = 0.0, samples: 8 and no duration leaf then depth = 10{value: DEATH, samples: 3} else depth = 10 if CLV <= 17.03 at t = 0.0, samples: 5 and no duration leaf then depth = 11{value: DEATH, samples: 2} else depth = 11{value: CHURNED, samples: 3} else depth = 9{value: DEATH, samples: 4} else depth = 8{value: CHURNED, samples: 2} else depth = 7 if CLV <= 591.46 at t = 0.0, samples: 23 and no duration leaf then depth = 8 if CLV <= 352.28 at t = 0.0, samples: 18 and no duration leaf then depth = 9 if CLV <= 155.3 at t = 0.0, samples: 16 and no duration leaf then depth = 10 if CLV <= 190.4 at t = 1.0, samples: 9 and no duration leaf then depth = 11{value: CHURNED 0.5, samples: 2} else depth = 11{value: CHURNED, samples: 7} else depth = 10 if CLV <= 178.0 at t = 0.0, samples: 7 and no duration leaf then depth = 11{value: DEATH, samples: 2} else depth = 11 if CLV <= 259.66 at t = 0.0, samples: 5 and no duration leaf then depth = 12{value: CHURNED, samples: 3} else depth = 12{value: CHURNED 0.5, samples: 2} else depth = 9{value: DEATH, samples: 2} else depth = 8{value: CHURNED, samples: 5} else depth = 5{value: CHURNED, samples: 6} else depth = 4 if CDI NOM PRODUIT <= 1.5 at t = 0.0, samples: 101 and no duration leaf then depth = 5 if FACE AMOUNT <= 10325.88 at t = 4.0, samples: 83 and duration leaf has 3 samples. Label is : DEATH 1.0 then depth = 6 if CLV <= 16.28 at t = 4.0, samples: 41 and no duration leaf then depth = 7 if Age <= 60.5 at t = 6.0, samples: 8 and duration leaf has 1 samples. A Appendix Label is : CHURNED 1.0 then depth = 8{value: CHURNED 0.5, samples: 2} else depth = 8{value: DEATH, samples: 5} else depth = 7 if CLV <= 89.04 at t = 4.0, samples: 33 and no duration leaf then depth = 8{value: CHURNED, samples: 8} else depth = 8 if CLV <= 100.4 at t = 4.0, samples: 25 and no duration leaf then depth = 9{value: DEATH, samples: 2} else depth = 9 if CLV <= 181.96 at t = 4.0, samples: 23 and no duration leaf then depth = 10{value: CHURNED, samples: 5} else depth = 10 if CLV <= 310.27 at t = 5.0, samples: 18 and no duration leaf then depth = 11{value: DEATH, samples: 3} else depth = 11 if Age <= 68.5 at t = 5.0, samples: 15 and no duration leaf then depth = 12 if FACE AMOUNT <= 3972.54 at t = 5.0, samples: 11 and no duration leaf then depth = 13{value: DEATH, samples: 3} else depth = 13 if CLV <= 748.3 at t = 6.0, samples: 8 and no duration leaf then depth = 14{value: CHURNED, samples: 4} else depth = 14 if CLV <= 917.37 at t = 6.0, samples: 4 and no duration leaf then depth = 15{value: DEATH, samples: 2} else depth = 15{value: CHURNED, samples: 2} else depth = 12{value: CHURNED, samples: 4} else depth = 12{value: CHURNED 0.5, samples: 2} { else depth = 9{value: DEATH, samples: 2} else depth = 8{value: CHURNED, sample { h = 5{value: CHURNED, samples: 6} else depth = 5{value: CHURNED, samples: 6} then depth = 5 if FACE AMOUNT <= 10325.88 at t = 4.0, samples: 83 then depth = 8{value: CHURNED 0.5, samples: 2} { } else depth = 8{value: DEATH, samples: 5} then depth = 8{value: CHURNED, samples: 8} { } e depth = 11 if Age <= 68.5 at t = 5.0, samples: 15 then depth = 13{value: DEATH, samples: 3} then depth = 14{value: CHURNED, samples: 4} { } else depth = 14 if CLV <= 917.37 at t = 6.0, samples: 4 and no duration leaf then depth = 15{value: DEATH, samples: 2} else depth = 15{value: CHURNED, samples: 2} { else depth = 12{value: CHURNED, samples: 4} 33 A Appendix then depth = 10{value: DEATH, samples: 3} p { , p } else depth = 11{value: CHURNED, samples: 2} { else depth = 10{value: DEATH, samples: and no duration leaf then depth = 8{value: CHURNED 0.5, samples: 4} else depth = 8 if CLV <= 2068.21 at t = 0.0, samples: 6 and no duration leaf and no duration leaf then depth = 9{value: CHURNED, samples: 4} else depth = 9{value: CHURNED 0.5, samples: 2} then depth = 4 if Age <= 60.5 at t = 0.0, samples: 167 then depth = 7{value: DEATH, samples: 2} { } else depth = 7{value: CHURNED 0.5, samples: 2} { } else depth = 6 if CLV <= 2145.92 at t = 3.0, samples: 11 then depth = 10 if FACE AMOUNT <= 12512.22 at t = 6.0, samp and no duration leaf then depth = 11 if CLV <= 217.81 at t = 8.0, samples: 65 and duration leaf has 4 samples. Label is : CHURNED 0.5 p then depth = 12 if Age <= 66.5 at t = 8.0, samples: 24 g and no duration leaf p and no duration leaf then depth = 14 if Age <= 55.5 at t = 10.0, samples: 7 and duration leaf has 1 samples. Label is : CHURNED 1.0 p then depth = 15{value: CHURNED, samples: 3} { } else depth = 15{value: DEATH, samples: 3} then depth = 15{value: CHURNED, samples: 10} { } else depth = 15{value: CHURNED 0.67, samples: 3} then depth = 14{value: DEATH, samples: 2} { } else depth = 14{value: CHURNED 0.5, samples: 2} { else depth = 11 if Age <= 60.5 at t = 7.0, samples: 13 ation leaf has 1 samples. and no duration leaf Label is : DEATH 1.0 depth = 12 if FACE AMOUNT <= 16037.28 at t = 8.0, samples: o duration leaf then depth = 13{value: DEATH, samples: 3} then depth = 13{value: DEATH, samples: 3} else depth = 13 if Age <= 57.5 at t = 8.0, sampl { } else depth = 13 if Age <= 57.5 at t = 8.0, samples: 5 34 A Appendix and no duration leaf then depth = 14{value: DEATH 0.67, samples: 3} else depth = 14{value: CHURNED, samples: 2} else depth = 12{value: CHURNED, samples: 4} else depth = 10{value: DEATH, samples: 2} else depth = 9{value: CHURNED, samples: 12} else depth = 8{value: DEATH, samples: 2} else depth = 7{value: CHURNED, samples: 16} else depth = 5 if Age <= 64.5 at t = 0.0, samples: 52 and no duration leaf then depth = 6 if CLV <= 251.48 at t = 0.0, samples: 43 and no duration leaf then depth = 7 if FACE AMOUNT <= 73.34 at t = 3.0, samples: 29 and no duration leaf then depth = 8{value: DEATH, samples: 3} else depth = 8 if CLV <= 119.45 at t = 3.0, samples: 26 and no duration leaf then depth = 9 if CLV <= 54.46 at t = 4.0, samples: 11 and no duration leaf then depth = 10{value: CHURNED 0.67, samples: 3} else depth = 10{value: CHURNED, samples: 8} else depth = 9 if FACE AMOUNT <= 3331.54 at t = 3.0, samples: 15 and no duration leaf then depth = 10{value: DEATH, samples: 2} else depth = 10 if CLV <= 445.55 at t = 3.0, samples: 13 and no duration leaf then depth = 11{value: CHURNED, samples: 3} else depth = 11 if CLV <= 709.34 at t = 3.0, samples: 10 and no duration leaf then depth = 12 if CLV <= 622.04 at t = 3.0, samples: 4 and no duration leaf then depth = 13{value: CHURNED 0.5, samples: 2} else depth = 13{value: DEATH, samples: 2} else depth = 12 if FACE AMOUNT <= 16863.76 at t = 3.0, samples: 6 and no duration leaf then depth = 13{value: CHURNED, samples: 3} else depth = 13{value: DEATH 0.67, samples: 3} else depth = 7 if Nb Contrats <= 1.5 at t = 0.0, samples: 14 and no duration leaf then depth = 8 if FACE AMOUNT <= 22229.94 at t = 1.0, samples: 11 and no duration leaf then depth = 9{value: DEATH 0.67, samples: 3} else depth = 9{value: DEATH, samples: 8} else depth = 8{value: CHURNED, samples: 3} else depth = 6 if CLV <= 633.18 at t = 0.0, samples: 9 and no duration leaf then depth = 7{value: CHURNED, samples: 7} else depth = 7{value: CHURNED 0.5, samples: 2} else depth = 4 if CLV <= 2081.39 at t = 0.0, samples: 32 and no duration leaf then depth = 5 if Age <= 63.5 at t = 0.0, samples: 30 and no duration leaf then depth = 6 if FACE AMOUNT <= 37433.94 at t = 3.0, samples: 26 and duration leaf has 2 samples. and no duration leaf Label is : CHURNED 1.0 then depth = 7{value: CHURNED, samples: 21} else depth = 7{value: CHURNED 0.67, samples: 3} else depth = 6 if Age <= 64.5 at t = 0.0, samples: 4 and no duration leaf then depth = 7{value: CHURNED 0.5, samples: 2} else depth = 7{value: CHURNED, samples: 2} else depth = 5{value: CHURNED 0.5, samples: 2} else depth = 1 if Age <= 72.5 at t = 0.0, samples: 520 and no duration leaf then depth = 2 if CLV <= 2.73 at t = 0.0, samples: 180 and no duration leaf then depth = 3 if CLV <= 99.7 at t = 2.0, samples: 13 and no duration leaf then depth = 4 if CLV <= 11.97 at t = 3.0, samples: 11 and no duration leaf then depth = 5{value: CHURNED, samples: 9} else depth = 5{value: CHURNED 0.5, samples: 2} else depth = 4{value: DEATH, samples: 2} else depth = 3 if CLV <= 2982.24 at t = 0.0, samples: 167 and no duration leaf then depth = 4 if Nb Contrats <= 2.5 at t = 0.0, samples: 165 and no duration leaf then depth = 5 if CDI NOM PRODUIT <= 1.5 at t = 0.0, samples: 159 and no duration leaf then depth = 6 if CLV <= 153.51 at t = 0.0, samples: 146 and no duration leaf then depth = 7 if Nb Contrats <= 1.5 at t = 1.0, samples: 61 and no duration leaf then depth = 8 if Age <= 70.5 at t = 1.0, samples: 58 and no duration leaf then depth = 9 if GENDER <= 1.5 at t = 1.0, samples: 30 and no duration leaf then depth = 10 if CLV <= 152.49 at t = 1.0, samples: 17 and no duration leaf then depth = 11{value: DEATH, samples: 10} else depth = 11 if CLV <= 212.33 at t = 1.0, samples: 7 and no duration leaf then depth = 12{value: CHURNED 0.5, samples: 2} else depth = 12{value: DEATH, samples: 5} else depth = 10 if FACE AMOUNT <= 3475.12 at t = 1.0, samples: 13 and no duration leaf else depth = 13{value: DEATH 0.67, sam then depth = 9{value: DEATH 0.67, samples: 3} p { , p } else depth = 7{value: CHURNED 0.5, samples: 2} then depth = 7{value: CHURNED, samples: 21} then depth = 7{value: CHURNED 0.5, samples: 2} else depth = 7{value: CHURNED, samples: 2 { else depth = 5{value: CHURNED 0.5, sampl { } else depth = 5{value: CHURNED 0.5, samples: 2} { else depth = 4{value: DEATH, samples: 2} then depth = 4 if Nb Contrats <= 2.5 at t = 0.0, samples: 165 nd no duration leaf then depth = 7 if Nb Contrats <= 1.5 at t = 1.0, samples: 61 d d ti l f and no duration leaf then depth = 9 if GENDER <= 1.5 at t = 1.0, samples: 30 and no duration leaf then depth = 10 if CLV <= 152.49 at t = 1.0, samples: 17 then depth = 11{value: DEATH, samples: 10} { } else depth = 11 if CLV <= 212.33 at t = 1.0, samples: 7 and no duration leaf then depth = 12{value: CHURNED 0.5, samples: 2} then depth = 12{value: CHURNED 0.5, samples: else depth = 12{value: DEATH, samples: 5} else depth = 12{value: DEATH, samples: 5} else depth = 10 if FACE AMOUNT <= 3475.12 at t = 35 A Appendix then depth = 11 if FACE AMOUNT <= 1499.92 at t = 1.0, samples: 7 and no duration leaf then depth = 12{value: DEATH, samples: 2} else depth = 12 if Age <= 69.5 at t = 1.0, samples: 5 and no duration leaf then depth = 13{value: CHURNED, samples: 3} else depth = 13{value: CHURNED 0.5, samples: 2} else depth = 11 if CLV <= 195.51 at t = 1.0, samples: 6 and no duration leaf then depth = 12{value: DEATH, samples: 4} else depth = 12{value: CHURNED 0.5, samples: 2} else depth = 9 if FACE AMOUNT <= 2307.93 at t = 1.0, samples: 28 and no duration leaf then depth = 10 if CLV <= 35.73 at t = 1.0, samples: 6 and no duration leaf then depth = 11{value: DEATH, samples: 4} else depth = 11{value: CHURNED 0.5, samples: 2} else depth = 10{value: DEATH, samples: 22} else depth = 8{value: CHURNED 0.67, samples: 3} else depth = 7 if CLV <= 161.62 at t = 0.0, samples: 85 and no duration leaf then depth = 8{value: CHURNED, samples: 2} else depth = 8 if CLV <= 1185.78 at t = 0.0, samples: 83 and no duration leaf then depth = 9 if CLV <= 1072.6 at t = 0.0, samples: 69 and no duration leaf then depth = 10 if Nb Contrats <= 1.5 at t = 0.0, samples: 67 and no duration leaf then depth = 11 if CLV <= 296.66 at t = 0.0, samples: 61 and no duration leaf then depth = 12 if CLV <= 240.56 at t = 0.0, samples: 22 and no duration leaf then depth = 13 if CLV <= 396.72 at t = 1.0, samples: 14 and no duration leaf then depth = 14 if CLV <= 342.37 at t = 1.0, samples: 7 and no duration leaf then depth = 15{value: CHURNED 0.5, samples: 2} else depth = 15{value: DEATH, samples: 5} else depth = 14 if CLV <= 428.3 at t = 1.0, samples: 7 and no duration leaf then depth = 15{value: CHURNED, samples: 3} else depth = 15 if GENDER <= 1.5 at t = 1.0, samples: 4 and no duration leaf then depth = 16{value: DEATH, samples: 2} else depth = 16{value: CHURNED 0.5, samples: 2} else depth = 13{value: DEATH, samples: 8} else depth = 12 if CLV <= 522.24 at t = 0.0, samples: 39 and no duration leaf then depth = 13 if CLV <= 388.65 at t = 0.0, samples: 24 and no duration leaf then depth = 14 if Age <= 70.5 at t = 0.0, samples: 12 and no duration leaf then depth = 15 if CLV <= 308.23 at t = 0.0, samples: 8 and no duration leaf then depth = 16{value: CHURNED 0.5, samples: 2} else depth = 16{value: DEATH, samples: 6} else depth = 15 if CLV <= 320.33 at t = 0.0, samples: 4 and no duration leaf then depth = 16{value: CHURNED, samples: 2} else depth = 16{value: CHURNED 0.5, samples: 2} else depth = 14 if CLV <= 427.18 at t = 0.0, samples: 12 and no duration leaf then depth = 15{value: CHURNED, samples: 4} else depth = 15 if CLV <= 507.19 at t = 0.0, samples: 8 and no duration leaf then depth = 16 if GENDER <= 1.5 at t = 0.0, samples: 6 and no duration leaf then depth = 17{value: DEATH, samples: 3} else depth = 17{value: CHURNED 0.67, samples: 3} else depth = 16{value: CHURNED, samples: 2} else depth = 13 if CLV <= 735.13 at t = 0.0, samples: 15 and no duration leaf then depth = 14{value: DEATH, samples: 8} else depth = 14 if CLV <= 767.92 at t = 0.0, samples: 7 and no duration leaf then depth = 15{value: CHURNED, samples: 2} else depth = 15 if FACE AMOUNT <= 47527.88 at t = 1.0, samples: 5 and no duration leaf then depth = 16{value: DEATH, samples: 3} else depth = 16{value: CHURNED 0.5, samples: 2} else depth = 11{value: DEATH, samples: 6} else depth = 10{value: CHURNED, samples: 2} else depth = 9 if Age <= 71.0 at t = 0.0, samples: 14 and no duration leaf then depth = 10{value: DEATH, samples: 11} else depth = 10{value: DEATH 0.67, samples: 3} else depth = 6 if FACE AMOUNT <= 7905.44 at t = 2.0, samples: 13 and no duration leaf then depth = 7 if FACE AMOUNT <= 1385.41 at t = 3.0, samples: 8 and duration leaf has 2 samples. and no duration leaf Label is : CHURNED 1.0 then depth = 8{value: DEATH, samples: 2} else depth = 8{value: CHURNED, samples: 4} else depth = 7{value: DEATH, samples: 5} else depth = 5 if Nb Contrats <= 4.5 at t = 1.0, samples: 6 and no duration leaf then depth = 11 if FACE AMOUNT <= 1499.92 at t = 1.0, samples: 7 and no duration leaf then depth = 12{value: DEATH, samples: 2} else depth = 12 if Age <= 69.5 at t = 1.0, samples: 5 and no duration leaf then depth = 13{value: CHURNED, samples: 3} else depth = 13{value: CHURNED 0.5, samples: 2} else depth = 11 if CLV <= 195.51 at t = 1.0, samples: 6 and no duration leaf then depth = 12{value: DEATH, samples: 4} else depth = 12{value: CHURNED 0.5, samples: 2} else depth = 9 if FACE AMOUNT <= 2307.93 at t = 1.0, samples: 28 and no duration leaf then depth = 10 if CLV <= 35.73 at t = 1.0, samples: 6 and no duration leaf then depth = 11{value: DEATH, samples: 4} else depth = 11{value: CHURNED 0.5, samples: 2} else depth = 10{value: DEATH, samples: 22} else depth = 8{value: CHURNED 0.67, samples: 3} else depth = 7 if CLV <= 161.62 at t = 0.0, samples: 85 and no duration leaf then depth = 8{value: CHURNED, samples: 2} else depth = 8 if CLV <= 1185.78 at t = 0.0, samples: 83 and no duration leaf then depth = 9 if CLV <= 1072.6 at t = 0.0, samples: 69 and no duration leaf then depth = 10 if Nb Contrats <= 1.5 at t = 0.0, samples: 67 and no duration leaf then depth = 11 if CLV <= 296.66 at t = 0.0, samples: 61 and no duration leaf then depth = 12 if CLV <= 240.56 at t = 0.0, samples: 22 and no duration leaf then depth = 13 if CLV <= 396.72 at t = 1.0, samples: 14 and no duration leaf then depth = 14 if CLV <= 342.37 at t = 1.0, samples: 7 and no duration leaf then depth = 15{value: CHURNED 0.5, samples: 2} else depth = 15{value: DEATH, samples: 5} else depth = 14 if CLV <= 428.3 at t = 1.0, samples: 7 and no duration leaf then depth = 15{value: CHURNED, samples: 3} else depth = 15 if GENDER <= 1.5 at t = 1.0, samples: 4 and no duration leaf then depth = 16{value: DEATH, samples: 2} else depth = 16{value: CHURNED 0.5, samples: 2} else depth = 13{value: DEATH, samples: 8} else depth = 12 if CLV <= 522.24 at t = 0.0, samples: 39 and no duration leaf then depth = 13 if CLV <= 388.65 at t = 0.0, samples: 24 and no duration leaf then depth = 14 if Age <= 70.5 at t = 0.0, samples: 12 and no duration leaf then depth = 15 if CLV <= 308.23 at t = 0.0, samples: 8 and no duration leaf then depth = 16{value: CHURNED 0.5, samples: 2} else depth = 16{value: DEATH, samples: 6} else depth = 15 if CLV <= 320.33 at t = 0.0, samples: 4 and no duration leaf then depth = 16{value: CHURNED, samples: 2} else depth = 16{value: CHURNED 0.5, samples: 2} else depth = 14 if CLV <= 427.18 at t = 0.0, samples: 12 and no duration leaf then depth = 15{value: CHURNED, samples: 4} else depth = 15 if CLV <= 507.19 at t = 0.0, samples: 8 and no duration leaf then depth = 16 if GENDER <= 1.5 at t = 0.0, samples: 6 and no duration leaf then depth = 17{value: DEATH, samples: 3} else depth = 17{value: CHURNED 0.67, samples: 3} else depth = 16{value: CHURNED, samples: 2} else depth = 13 if CLV <= 735.13 at t = 0.0, samples: 15 and no duration leaf then depth = 14{value: DEATH, samples: 8} else depth = 14 if CLV <= 767.92 at t = 0.0, samples: 7 and no duration leaf then depth = 15{value: CHURNED, samples: 2} else depth = 15 if FACE AMOUNT <= 47527.88 at t = 1.0, samples: 5 and no duration leaf then depth = 16{value: DEATH, samples: 3} else depth = 16{value: CHURNED 0.5, samples: 2} else depth = 11{value: DEATH, samples: 6} else depth = 10{value: CHURNED, samples: 2} else depth = 9 if Age <= 71.0 at t = 0.0, samples: 14 and no duration leaf then depth = 10{value: DEATH, samples: 11} else depth = 10{value: DEATH 0.67, samples: 3} else depth = 6 if FACE AMOUNT <= 7905.44 at t = 2.0, samples: 13 and no duration leaf then depth = 7 if FACE AMOUNT <= 1385.41 at t = 3.0, samples: 8 and duration leaf has 2 samples. and no duration leaf Label is : CHURNED 1.0 then depth = 8{value: DEATH, samples: 2} l d th 8{ l CHURNED l 4} then depth = 12{value: DEATH, samples: 2} then depth = 17{value: DEATH, samples: 3} { } else depth = 17{value: CHURNED 0.67, samples: 3} { } else depth = 16{value: CHURNED, samples: 2} then depth = 14{value: DEATH, samples: 8} { } else depth = 14 if CLV <= 767.92 at t = 0.0, samples: 7 then depth = 16{value: DEATH, samples: 3} p { , p } else depth = 16{value: CHURNED 0.5, samples: 2} { else depth = 16{value: CH { else depth = 16{value: CHURNED 0 p { else depth = 11{value: DEATH, samples: 6} p { , p } else depth = 10{value: CHURNED, samples: 2} depth = 9 if Age <= 71.0 at t = 0.0, samples: 14 no duration leaf then depth = 10{value: DEATH, samples: 11} { } else depth = 10{value: DEATH 0.67, samples: 3} else depth = 6 if FACE AMOUNT <= 7905.44 at t = 2.0, samples: 13 and no duration leaf then depth = 7 if FACE AMOUNT <= 1385.41 at t = 3.0, samples: 8 then depth = 8{value: DEATH, samples: 2} 36 A Appendix 36 then depth = 6{value: CHURNED, samples: 4} else depth = 6{value: DEATH, samples: 2} else depth = 4{value: CHURNED, samples: 2} else depth = 2 if CLV <= 24.19 at t = 0.0, samples: 340 and no duration leaf then depth = 3 if CLV <= 23.77 at t = 0.0, samples: 70 and no duration leaf then depth = 4 if Age <= 81.5 at t = 0.0, samples: 68 and no duration leaf then depth = 5 if Age <= 76.5 at t = 0.0, samples: 53 and no duration leaf then depth = 6 if CDI NOM PRODUIT <= 1.5 at t = 0.0, samples: 32 and no duration leaf then depth = 7 if CLV <= 1.72 at t = 0.0, samples: 24 and no duration leaf then depth = 8 if CLV <= 2.86 at t = 1.0, samples: 7 and no duration leaf then depth = 9{value: DEATH, samples: 3} else depth = 9{value: CHURNED 0.5, samples: 4} else depth = 8{value: DEATH, samples: 17} else depth = 7 if GENDER <= 1.5 at t = 4.0, samples: 8 and duration leaf has 2 samples. and no duration leaf Label is : CHURNED 0.5 then depth = 8{value: CHURNED, samples: 2} else depth = 8 if CLV <= 56.71 at t = 4.0, samples: 4 and no duration leaf then depth = 9{value: CHURNED 0.5, samples: 2} else depth = 9{value: DEATH, samples: 2} else depth = 6 if CLV <= 1.5 at t = 0.0, samples: 21 and no duration leaf then depth = 7{value: CHURNED, samples: 3} else depth = 7 if CLV <= 101.49 at t = 3.0, samples: 18 and duration leaf has 1 samples. and no duration leaf Label is : DEATH 1.0 then depth = 8 if Age <= 79.5 at t = 3.0, samples: 13 and no duration leaf then depth = 9{value: CHURNED, samples: 2} else depth = 9 if GENDER <= 1.5 at t = 3.0, samples: 11 and no duration leaf then depth = 10 if CLV <= 24.93 at t = 3.0, samples: 4 and no duration leaf then depth = 11{value: DEATH, samples: 2} else depth = 11{value: CHURNED, samples: 2} else depth = 10 if Age <= 80.5 at t = 3.0, samples: 7 and no duration leaf then depth = 11{value: DEATH 0.67, samples: 3} else depth = 11{value: DEATH, samples: 4} else depth = 8{value: CHURNED, samples: 4} else depth = 5{value: DEATH, samples: 15} else depth = 4{value: CHURNED, samples: 2} else depth = 3 if CDI NOM PRODUIT <= 1.5 at t = 0.0, samples: 270 and no duration leaf then depth = 4 if Age <= 74.5 at t = 0.0, samples: 240 and no duration leaf then depth = 5 if CLV <= 303.99 at t = 0.0, samples: 41 and no duration leaf then depth = 6 if Age <= 73.5 at t = 0.0, samples: 23 and no duration leaf then depth = 7 if CLV <= 391.43 at t = 2.0, samples: 6 and no duration leaf then depth = 8{value: CHURNED 0.5, samples: 2} else depth = 8{value: DEATH, samples: 4} else depth = 7{value: DEATH, samples: 17} else depth = 6 if CLV <= 334.97 at t = 0.0, samples: 18 and no duration leaf then depth = 7{value: CHURNED, samples: 3} else depth = 7 if CLV <= 1380.47 at t = 0.0, samples: 15 and no duration leaf then depth = 8{value: DEATH, samples: 13} else depth = 8{value: CHURNED 0.5, samples: 2} else depth = 5 if Age <= 89.5 at t = 0.0, samples: 199 and no duration leaf then depth = 6 if FACE AMOUNT <= 65229.84 at t = 3.0, samples: 192 and duration leaf has 2 samples. Label is : DEATH 1.0 then depth = 7 if FACE AMOUNT <= 5858.16 at t = 3.0, samples: 160 and no duration leaf then depth = 8 if FACE AMOUNT <= 5693.4 at t = 4.0, samples: 27 and duration leaf has 3 samples. and no duration leaf Label is : DEATH 1.0 then depth = 9{value: DEATH, samples: 22} else depth = 9{value: CHURNED, samples: 2} else depth = 8 if Age <= 78.5 at t = 3.0, samples: 133 and no duration leaf then depth = 9 if FACE AMOUNT <= 14620.96 at t = 3.0, samples: 13 and no duration leaf then depth = 10 if CLV <= 743.21 at t = 3.0, samples: 5 and no duration leaf then depth = 11{value: DEATH, samples: 3} else depth = 11{value: CHURNED 0.5, samples: 2} else depth = 10{value: DEATH, samples: 8} else depth = 9 if Age <= 81.5 at t = 3.0, samples: 120 and no duration leaf then depth = 10 if Age <= 80.5 at t = 3.0, samples: 49 and no duration leaf then depth = 11{value: DEATH, samples: 33} else depth = 11 if CLV <= 1337.19 at t = 3.0, samples: 16 and no duration leaf then depth = 12 if CLV <= 1236.12 at t = 3.0, samples: 7 then depth = 6{value: CHURNED, samples: 4} else depth = 6{value: DEATH, samples: 2} else depth = 4{value: CHURNED, samples: 2} else depth = 2 if CLV <= 24.19 at t = 0.0, samples: 340 and no duration leaf then depth = 3 if CLV <= 23.77 at t = 0.0, samples: 70 and no duration leaf then depth = 4 if Age <= 81.5 at t = 0.0, samples: 68 and no duration leaf then depth = 5 if Age <= 76.5 at t = 0.0, samples: 53 and no duration leaf then depth = 6 if CDI NOM PRODUIT <= 1.5 at t = 0.0, samples: 32 and no duration leaf then depth = 7 if CLV <= 1.72 at t = 0.0, samples: 24 and no duration leaf then depth = 8 if CLV <= 2.86 at t = 1.0, samples: 7 and no duration leaf then depth = 9{value: DEATH, samples: 3} else depth = 9{value: CHURNED 0.5, samples: 4} else depth = 8{value: DEATH, samples: 17} else depth = 7 if GENDER <= 1.5 at t = 4.0, samples: 8 and duration leaf has 2 samples. and no duration leaf Label is : CHURNED 0.5 then depth = 8{value: CHURNED, samples: 2} else depth = 8 if CLV <= 56.71 at t = 4.0, samples: 4 and no duration leaf then depth = 9{value: CHURNED 0.5, samples: 2} else depth = 9{value: DEATH, samples: 2} else depth = 6 if CLV <= 1.5 at t = 0.0, samples: 21 and no duration leaf then depth = 7{value: CHURNED, samples: 3} else depth = 7 if CLV <= 101.49 at t = 3.0, samples: 18 and duration leaf has 1 samples. and no duration leaf Label is : DEATH 1.0 then depth = 8 if Age <= 79.5 at t = 3.0, samples: 13 and no duration leaf then depth = 9{value: CHURNED, samples: 2} else depth = 9 if GENDER <= 1.5 at t = 3.0, samples: 11 and no duration leaf then depth = 10 if CLV <= 24.93 at t = 3.0, samples: 4 and no duration leaf then depth = 11{value: DEATH, samples: 2} else depth = 11{value: CHURNED, samples: 2} else depth = 10 if Age <= 80.5 at t = 3.0, samples: 7 and no duration leaf then depth = 11{value: DEATH 0.67, samples: 3} else depth = 11{value: DEATH, samples: 4} else depth = 8{value: CHURNED, samples: 4} else depth = 5{value: DEATH, samples: 15} else depth = 4{value: CHURNED, samples: 2} else depth = 3 if CDI NOM PRODUIT <= 1.5 at t = 0.0, samples: 270 and no duration leaf then depth = 4 if Age <= 74.5 at t = 0.0, samples: 240 and no duration leaf then depth = 5 if CLV <= 303.99 at t = 0.0, samples: 41 and no duration leaf then depth = 6 if Age <= 73.5 at t = 0.0, samples: 23 and no duration leaf then depth = 7 if CLV <= 391.43 at t = 2.0, samples: 6 and no duration leaf then depth = 8{value: CHURNED 0.5, samples: 2} else depth = 8{value: DEATH, samples: 4} else depth = 7{value: DEATH, samples: 17} else depth = 6 if CLV <= 334.97 at t = 0.0, samples: 18 and no duration leaf then depth = 7{value: CHURNED, samples: 3} else depth = 7 if CLV <= 1380.47 at t = 0.0, samples: 15 and no duration leaf then depth = 8{value: DEATH, samples: 13} else depth = 8{value: CHURNED 0.5, samples: 2} else depth = 5 if Age <= 89.5 at t = 0.0, samples: 199 and no duration leaf then depth = 6 if FACE AMOUNT <= 65229.84 at t = 3.0, samples: 192 and duration leaf has 2 samples. Label is : DEATH 1.0 then depth = 7 if FACE AMOUNT <= 5858.16 at t = 3.0, samples: 160 and no duration leaf then depth = 8 if FACE AMOUNT <= 5693.4 at t = 4.0, samples: 27 and duration leaf has 3 samples. and no duration leaf Label is : DEATH 1.0 then depth = 9{value: DEATH, samples: 22} else depth = 9{value: CHURNED, samples: 2} else depth = 8 if Age <= 78.5 at t = 3.0, samples: 133 and no duration leaf then depth = 9 if FACE AMOUNT <= 14620.96 at t = 3.0, samples: 13 and no duration leaf then depth = 10 if CLV <= 743.21 at t = 3.0, samples: 5 and no duration leaf then depth = 11{value: DEATH, samples: 3} else depth = 11{value: CHURNED 0.5, samples: 2} else depth = 10{value: DEATH, samples: 8} else depth = 9 if Age <= 81.5 at t = 3.0, samples: 120 and no duration leaf then depth = 10 if Age <= 80.5 at t = 3.0, samples: 49 and no duration leaf then depth = 11{value: DEATH, samples: 33} else depth = 11 if CLV <= 1337.19 at t = 3.0, samples: 16 and no duration leaf then depth = 12 if CLV <= 1236.12 at t = 3.0, samples: 7 then depth = 11{value: DEATH 0.67, samples: 3} p { , p else depth = 11{value: DEATH, samples: 4} p { , else depth = 8{value: CHURNED, samples: 4} { else depth = 5{value: DEATH, samples: 15} else depth = 4{value: CHURNED, samples: 2} p { , p } else depth = 3 if CDI NOM PRODUIT <= 1.5 at t = then depth = 8{value: CHURNED 0.5, samples: 2} else depth = 8{value: DEATH, samples: 4} p { , p } else depth = 7 if CLV <= 1380.47 at t = 0.0, samples: 15 then depth = 8{value: DEATH, samples: 13} { } else depth = 8{value: CHURNED 0.5, samples: 2} then depth = 8 if FACE AMOUNT <= 5693.4 at t = 4.0, samples: 27 then depth = 9{value: DEATH, samples: 22} else depth = 9{value: CHURNED, samples: 2} { else depth = 10{value: DEATH, samples: 8} then depth = 11{value: DEATH, samples: 33} { } else depth = 11 if CLV <= 1337.19 at t = 3.0, samples: 16 then depth = 12 if CLV <= 1236.12 at t = 3.0, samples: 7 37 A Appendix and no duration leaf then depth = 13{value: DEATH, samples: 5} else depth = 13{value: CHURNED 0.5, samples: 2} else depth = 12{value: DEATH, samples: 9} else depth = 10{value: DEATH, samples: 71} else depth = 7 if Age <= 79.5 at t = 3.0, samples: 30 and no duration leaf then depth = 8 if CLV <= 6469.44 at t = 3.0, samples: 6 and no duration leaf then depth = 9{value: CHURNED, samples: 2} else depth = 9{value: DEATH, samples: 4} else depth = 8 if CLV <= 4697.78 at t = 4.0, samples: 24 and duration leaf has 2 samples. and no duration leaf Label is : DEATH 1.0 then depth = 9{value: CHURNED 0.5, samples: 2} else depth = 9{value: DEATH, samples: 20} else depth = 6 if GENDER <= 1.5 at t = 0.0, samples: 7 and no duration leaf then depth = 7{value: CHURNED 0.5, samples: 2} else depth = 7{value: DEATH, samples: 5} else depth = 4 if Age <= 80.5 at t = 0.0, samples: 30 and no duration leaf then depth = 5 if GENDER <= 1.5 at t = 0.0, samples: 11 and no duration leaf then depth = 6 if Age <= 75.5 at t = 0.0, samples: 4 and no duration leaf then depth = 7{value: CHURNED 0.5, samples: 2} else depth = 7{value: DEATH, samples: 2} else depth = 6 if Age <= 79.5 at t = 0.0, samples: 7 and no duration leaf then depth = 7{value: CHURNED, samples: 4} else depth = 7{value: CHURNED 0.67, samples: 3} else depth = 5{value: DEATH, samples: 19} and no duration leaf then depth = 13{value: DEATH, samples: 5} else depth = 13{value: CHURNED 0.5, samples: 2} else depth = 12{value: DEATH, samples: 9} else depth = 10{value: DEATH, samples: 71} else depth = 7 if Age <= 79.5 at t = 3.0, samples: 30 and no duration leaf then depth = 8 if CLV <= 6469.44 at t = 3.0, samples: 6 and no duration leaf then depth = 9{value: CHURNED, samples: 2} else depth = 9{value: DEATH, samples: 4} else depth = 8 if CLV <= 4697.78 at t = 4.0, samples: 24 and duration leaf has 2 samples. and no duration leaf Label is : DEATH 1.0 then depth = 9{value: CHURNED 0.5, samples: 2} else depth = 9{value: DEATH, samples: 20} else depth = 6 if GENDER <= 1.5 at t = 0.0, samples: 7 and no duration leaf then depth = 7{value: CHURNED 0.5, samples: 2} else depth = 7{value: DEATH, samples: 5} else depth = 4 if Age <= 80.5 at t = 0.0, samples: 30 and no duration leaf then depth = 5 if GENDER <= 1.5 at t = 0.0, samples: 11 and no duration leaf then depth = 6 if Age <= 75.5 at t = 0.0, samples: 4 and no duration leaf then depth = 7{value: CHURNED 0.5, samples: 2} else depth = 7{value: DEATH, samples: 2} else depth = 6 if Age <= 79.5 at t = 0.0, samples: 7 and no duration leaf then depth = 7{value: CHURNED, samples: 4} else depth = 7{value: CHURNED 0.67, samples: 3} else depth = 5{value: DEATH, samples: 19} The unstopped and unpruned TpTs, obtained with the time-penalized entropy splitting criterion, and various time penalties yields the following results: The unstopped and unpruned TpTs, obtained with the time-penalized entropy splitting criterion, and various time penalties yields the following results: The unstopped and unpruned TpTs, obtained with the time-penalized entropy splitting criterion, and various time penalties yields the following results: The unstopped and unpruned TpTs, obtained with the time-penalized entropy splitting criterion, and various time penalties yields the following results: A.3.2 Results with minsplit = 25 The maximal unpruned and unstopped TpTs, obtained with the time-penalized entropy splitting criterion, minsplit= 25, and various time penalties yields the following results: A.3.2 Results with minsplit = 25 Table 4: Characteritics of unstopped and unpruned entropy TpTs depending on the time penal pp p py p p g p A.3.2 Results with minsplit = 25 The maximal unpruned and unstopped TpTs, obtained with the time-penalized entropy splitting criterion, minsplit= 25, and various time penalties yields the following results: The maximal unpruned and unstopped TpTs, obtained with the time-penalized entropy splitting criterion, minsplit= 25, and various time penalties yields the following results: The maximal unpruned and unstopped TpTs, obtained with the time-penalized entropy splitting criterion, minsplit= 25, and various time penalties yields the following results: 38 A Appendix Time penalty γ Runtime Depth # of terminal leaves # of duration leaves Total # of leaves Tree cost Max of split times Mean of split time 0.0000 671.79 7 26 16 42 0.743 20.0 5.689 0.0025 703.08 7 27 15 42 0.740 20.0 5.306 0.0050 796.89 8 30 17 47 0.721 20.0 4.366 0.0100 939.1 10 40 23 63 0.704 20.0 3.835 0.0125 937.04 10 40 24 64 0.704 20.0 3.837 0.0250 940.39 10 41 24 65 0.702 20.0 3.933 0.0275 944.95 10 43 25 68 0.692 20.0 4.277 0.0300 1034.06 10 53 31 84 0.680 20.0 3.933 0.0425 1054.6 10 54 32 86 0.677 20.0 3.875 0.0450 1058.25 10 55 32 87 0.675 20.0 3.845 0.0475 1056.82 10 59 34 93 0.674 20.0 3.84 0.0500 1246.9 14 73 45 118 0.620 19.0 3.613 0.0525 1354.92 14 69 40 109 0.620 20.0 2.419 0.0575 1383.59 14 77 41 118 0.598 20.0 2.484 0.0600 1389.81 14 82 44 126 0.587 19.0 2.666 0.0650 1383.68 14 82 43 125 0.583 19.0 2.639 0.0700 1379.63 14 83 43 126 0.574 19.0 2.605 0.0750 1427.5 15 86 44 130 0.569 19.0 2.581 0.0775 1427.62 15 87 42 129 0.566 19.0 2.606 0.0950 1421.46 15 91 42 133 0.561 19.0 2.591 0.0975 1421.71 15 92 42 134 0.560 19.0 2.606 0.1000 1420.78 15 93 42 135 0.557 19.0 2.549 0.1075 1447.24 15 95 41 136 0.551 19.0 2.712 0.1150 1438.93 15 97 40 137 0.545 19.0 2.688 0.1175 1447.51 15 97 37 134 0.549 19.0 2.657 0.1200 1458.82 15 98 38 136 0.547 19.0 2.674 0.1250 1502.87 15 114 45 159 0.506 18.0 2.643 0.1300 1504.6 15 115 45 160 0.506 18.0 2.628 0.1375 1504.56 17 119 45 164 0.494 15.0 2.647 0.1400 1647.49 17 121 46 167 0.488 15.0 2.672 0.1425 1724.77 17 124 43 167 0.468 15.0 2.119 0.1475 1750.56 17 125 44 169 0.467 17.0 1.787 0.1500 1756.44 17 132 44 176 0.464 17.0 1.78 0.1525 1766.5 17 135 44 179 0.455 15.0 1.63 0.1550 1791.99 18 135 37 172 0.452 15.0 1.633 0.1575 1798.21 18 136 35 171 0.449 15.0 1.638 0.1600 1800.5 18 136 34 170 0.450 15.0 1.636 0.1625 1844.87 18 143 28 171 0.438 15.0 1.415 0.1650 1825.93 18 143 29 172 0.436 15.0 1.416 0.1675 1884.66 18 154 23 177 0.412 11.0 1.187 0.1750 1889.17 18 155 22 177 0.407 11.0 1.158 0.1775 1910.45 19 156 18 174 0.394 11.0 1.003 0.1825 1901.92 19 157 17 174 0.395 11.0 1.003 0.1875 1940.97 19 164 13 177 0.390 11.0 0.865 0.1950 1938.27 19 166 13 179 0.398 11.0 0.846 0.1975 1909.02 19 166 13 179 0.401 11.0 0.843 0.2050 1931.62 19 165 12 177 0.398 11.0 0.809 0.2100 1921.77 19 165 11 176 0.399 11.0 0.807 0.2175 1938.66 21 165 9 174 0.395 12.0 0.844 0.2200 1940.89 21 166 9 175 0.401 12.0 0.828 0.2250 1946.0 21 166 8 174 0.400 12.0 0.818 0.2375 1941.67 21 167 9 176 0.402 12.0 0.819 0.2450 1939.31 21 160 16 176 0.428 14.0 1.059 0.2475 1917.16 21 161 16 177 0.430 14.0 1.05 0.2575 1920.18 19 162 18 180 0.435 14.0 1.013 0.2600 1916.36 19 163 17 180 0.437 14.0 1.007 0.2625 1968.98 19 172 10 182 0.408 11.0 0.771 0.2650 1965.17 20 172 17 189 0.422 13.0 0.849 0.2675 1969.52 21 173 16 189 0.423 13.0 0.85 0.2700 1960.23 21 174 14 188 0.421 13.0 0.85 0.2725 1997.83 21 175 14 189 0.424 13.0 0.817 0.2775 1977.08 21 176 13 189 0.420 13.0 0.816 0.2800 2194.84 24 182 11 193 0.419 13.0 0.791 0.2850 2167.05 24 179 6 185 0.410 7.0 0.649 0.2925 2138.61 24 179 6 185 0.411 7.0 0.647 0.3125 2115.89 24 179 5 184 0.411 7.0 0.641 0.3225 2088.42 24 178 5 183 0.410 7.0 0.626 0.3300 2076.5 24 178 5 183 0.413 7.0 0.612 0.3650 2027.6 24 180 4 184 0.412 7.0 0.596 0.3750 2051.34 24 180 4 184 0.413 7.0 0.584 0.4000 2079.19 24 181 4 185 0.413 7.0 0.56 0.4025 2015.83 24 186 5 191 0.419 7.0 0.444 0.4325 1864.67 24 187 5 192 0.420 7.0 0.431 0.4475 1757.62 19 182 5 187 0.420 7.0 0.095 0.4575 1731.83 19 183 3 186 0.420 4.0 0.092 0.4800 1577.62 19 187 3 190 0.420 4.0 0.057 0.5500 1577.43 19 188 3 191 0.420 4.0 0.053 0.6500 1577.71 19 189 3 192 0.425 4.0 0.048 0.7000 1573.9 19 190 2 192 0.427 4.0 0.045 0.8000 1578.4 19 193 1 194 0.433 4.0 0.04 0.9000 1589.0 19 192 0 192 0.447 3.0 0.032 1.0000 1592.38 19 195 0 195 0.445 3.0 0.022 1.1000 1576.86 19 195 0 195 0.447 2.0 0.02 1.3000 1576.14 19 196 0 196 0.446 2.0 0.015 Table 4: Characteritics of unstopped and unpruned entropy TpTs depending on the time pen 39 A Appendix Time penalty γ Runtime Depth # of terminal leaves # of duration leaves Total # of leaves Tree cost Max of split times Mean of split times 0.0000 668.38 5 14 8 22 0.664 15.0 5.722 0.0025 690.38 5 14 7 21 0.664 15.0 5.172 0.0050 782.22 6 17 8 25 0.638 15.0 4.346 0.0100 840.15 7 16 8 24 0.646 15.0 3.552 0.0150 914.61 8 17 8 25 0.637 15.0 3.301 0.0250 912.15 8 18 9 27 0.636 15.0 3.307 0.0275 917.44 8 18 7 25 0.640 15.0 3.033 0.0300 978.72 8 18 8 26 0.637 15.0 2.347 0.0400 1085.14 8 25 10 35 0.618 15.0 1.953 0.0475 1102.22 8 27 12 39 0.613 15.0 1.959 0.0500 1216.25 8 31 14 45 0.586 9.0 1.578 0.0575 1213.44 8 31 15 46 0.584 9.0 1.568 0.0675 1213.17 8 31 13 44 0.585 9.0 1.5 0.0850 1205.48 9 31 15 46 0.581 10.0 1.52 0.1000 1196.86 9 32 14 46 0.571 10.0 1.434 0.1125 1194.82 9 32 13 45 0.569 10.0 1.418 0.1175 1195.85 8 30 11 41 0.581 11.0 1.244 0.1200 1253.33 11 33 9 42 0.562 9.0 0.823 0.1350 1242.11 11 33 9 42 0.561 9.0 0.807 0.1625 1345.51 11 34 9 43 0.562 9.0 0.714 0.1700 1321.99 11 34 8 42 0.562 9.0 0.708 0.1925 1288.5 11 34 9 43 0.563 9.0 0.689 0.1950 1279.26 11 34 8 42 0.567 9.0 0.651 0.2000 1304.58 11 34 5 39 0.565 9.0 0.38 0.2050 1323.32 11 34 4 38 0.563 8.0 0.316 0.2400 1333.81 11 34 3 37 0.567 6.0 0.275 0.2525 1340.74 11 33 3 36 0.568 6.0 0.242 0.3150 1358.63 11 32 1 33 0.570 3.0 0.173 0.3725 1310.12 11 32 0 32 0.574 2.0 0.045 0.3750 1350.37 11 32 0 32 0.575 2.0 0.014 Table 5: Characteritics of Entropy TpTs (minsplit=25) depending on the time penalty Table 5: Characteritics of Entropy TpTs (minsplit=25) depending on the time penalty Table 5: Characteritics of Entropy TpTs (minsplit=25) depending on the time penalty Figure 16: Characteritics of Entropy TpTs (minsplit=25) depending on the time penalty Figure 16: Characteritics of Entropy TpTs (minsplit=25) depending on the time penalty 40 40 A Appendix A.3.3 Results with minsplit = 50 A.3.3 Results with minsplit = 50 A.3.3 Results with minsplit = 50 Figure 17: Gini TpT (minsplit=50) with γ = 0 Figure 17: Gini TpT (minsplit=50) with γ = 0 41 41 A Appendix Figure 18: Gini TpT (minsplit=50) with the optimal time penalty Figure 18: Gini TpT (minsplit=50) with the optimal time penalty Figure 18: Gini TpT (minsplit=50) with the optimal time penalty A Appendix 42 Figure 19: Gini TpT (minsplit=50) with γ →∞ A Appendix 42 Figure 19: Gini TpT (minsplit=50) with γ →∞
https://openalex.org/W1965041779	https://ccsenet.org/journal/index.php/ibr/article/download/27531/16655	English	null	Entrepreneurial Game Theoretic Approach to Planning Flexibility and Environmental Scanning	International business research	2,013	cc-by	8,810	Entrepreneurial Game Theoretic Approach to Planning Flexibility and Environmental Scanning Owolabi L. Kuye1, Bankole Abiola2 & Ben E. A. Oghojafor1 1 Department of Business Administration, University of Lagos, Lagos State, Nigeria 2 Department of Actuarial Science and Insurance, University of Lagos, Lagos State, Nigeria Correspondence: Owolabi L. Kuye, Department of Business Administration, University of Lagos, Lagos State, Nigeria. E-mail: labikuye@yahoo.com Accepted: April 26, 2013 Online Pu URL: http://dx.doi.org/10.5539/ibr.v6n6p95 Received: March 12, 2013 doi:10.5539/ibr.v6n6p95 Received: March 12, 2013 doi:10.5539/ibr.v6n6p95 Abstract This study focuses on planning flexibility and environmental scanning from the standpoint of corporate entrepreneurship, as represented by Top management. Game theory was applied in this study. In doing this, the problem was viewed from a “worst case design concept” which can be summarised as follows: The intrapreneur (the entrepreneur within a firm or corporate entrepreneurs) wishes to minimise the cost of operation while the environment tries to maximize the cost of operation. Hence, we introduce a cost functional, which is the cost of risk faced by the intrapreneur. We assume that the entrepreneur can apply flexibility in planning, hence denote this as control, while the environment that is needed to be scanned is denoted as uncertainty. The model of game is applied to allow for saddle point (a point where there is no ‘gain’ no ‘loss’) solution. The implication of this is that for a saddle point to exist in a “worst case” situation, the intrapreneur is likely to breaking-even. Thus, a proper mathematical treatment is given for such a game problem. Keywords: entrepreneurial, intrapreneur, corporate entrepreneurship, planning flexibility, environmental scanning, game theory, saddle point 1. Introduction Practitioners and scholar have recognised the challenges of pursuing entrepreneurship within an organisation (Mair & Rata, 2004) in the face of environmental uncertainty. Many entrepreneurship literature are of the view that it is necessary to promote entrepreneurial attitude and activities in firms (Lumpkin & Dess, 1996; Covin & Slevin, 1991; Zahra, 1993). For firms to become entrepreneurial and enhance their performance in the light of changing business environment, it is important that they employ planning flexibility as a strategy (Li et. al., 2006). Top management also needs to consider environmental scanning to be able to adopt planning flexibility. Environmental scanning can encourage entrepreneurial behaviour of firms in terms of taking risk, being proactive and innovative. The ability of firms to cope with uncertainty and behave entrepreneurially is a function of the intensity of their scanning efforts (Oghojafor et al., 2009). International Business Research; Vol. 6, No. 6; 2013 ISSN 1913-9004 E-ISSN 1913-9012 Published by Canadian Center of Science and Education International Business Research; Vol. 6, No. 6; 2013 ISSN 1913-9004 E-ISSN 1913-9012 Published by Canadian Center of Science and Education 2.1.1 The Need for Planning Flexibility Kukalis (1989) in Kemelgor (2002) suggests the need for firms in a rapidly changing business environment to adopt flexible planning. He also opined that firms in this situation will enhance their performance, if only they can employ planning flexibility approach. According to Alpkan et al. (2007), flexible planning involves the capacity of a firm’s strategic plans to become adaptive and responsive to change when necessary. Bhalla et al. (2006) opine that without flexibility in planning, rigidity in planning may be counterproductive in the long-run. Planning is critical to the systematic growth and adaptation of a firm in a changing environment (Heimann & Lusk, 1976). The authors believed that this is true of firms in all sectors. Inherently in the planning process is the consideration of uncertainty concerning future events. That uncertainty may include not only the occurrences of future states of nature but also the availabilities of future decision alternatives. Recognising the problem of uncertainty in the planning process, managers in actual practice and researchers in the development of decision models have mentioned the need for considering the flexibility of a plan, as a criterion for selecting a course of action in a planning process (Heimann & Lusk, 1976). According to Ancona et al. (2005), the need for flexibility is driven by intensifying competition so that capabilities for tailoring products and services to a range of customer needs are increasingly a source of competitive advantage. 2.1.2 Decision on Flexibility According to Ancona et al. (2005), the need for flexibility is driven by intensifying competition so that capabilities for tailoring products and services to a range of customer needs are increasingly a source of competitive advantage. 2.1.2 Decision on Flexibility As part of its strategic thinking, an organisation should consider whether it is imperative to increase its flexibility and if so, which approach is the most appropriate and effective (Aaker & Mascarenhas, 1984). The authors identified a series of four steps that can help address these dimensions: 1) Identifying environmental changes: This first step involves a careful external focusing on the potential environmental changes confronting the firm. They may include competitor threats, technological breakthroughs, political events, changes in cultural values and behaviours, or economic in development. They could also be highly specific like the development of a particular type and size of cell- phone memory. 2.1 Planning Flexibility Many firms are finding it difficult to rely on rigid rules, routines, and structures that have the key characteristics of bureaucracy and formal organisation. In recent times, firms are increasingly required to respond flexibly to diverse needs of various stakeholders (employees, customers, creditors, suppliers, distributors, shareholders and communities) in a manner that does not cause injustice and unfairness (Ancona et al., 2005). Firms operate in environments where the rate of change is increasing. These environments place demand on the ability of the organisation to change. Because of the rapidity of change that takes place in the environments, firms should be flexible in their approach to planning to enhance their productivity. Planning flexibility results from the capability of a firm’s strategic plan to adjust as environmental opportunities and threats emerge (Li et al., 2006). Flexible planning brings about proactiveness, creativity and innovativeness. Accordingly, it promotes a high degree of corporate entrepreneurship (Barringer & Bluedorn, 1999). 95 Vol. 6, No. 6; 2013 International Business Research www.ccsenet.org/ibr 2.1.1 The Need for Planning Flexibility 2) Appraising likely environmental changes: That is, consideration of the size, likelihood, and nature of the effect of each environmental change upon the firm. There will always be a large number of potential environmental changes. It is necessary to reduce them to a manageable number or at least to prioritise them. 3) The flexibility option: This step considers the flexibility option for each identified and screened environmental change. Flexibility needs to be considered in the context of a particular potential environmental change. The problem is that one approach might increase flexibility with respect to one potential environmental change but decrease it with respect to another. For instance, inventory might be used to buffer production uncertainties and fluctuations in sales and supplies. However, a technological change could cause the product to become obsolete leading to inventory not easily liquidated. 4) Other methods for coping with environmental changes: Flexibility is expected to be the most cost-effective technique for coping with environmental change and uncertainty. Other techniques too can be considered in addition to or in conjunction with flexibility: contingency planning, insurance, control, avoidance, and prediction. 2 2 l S 2.2 Environmental Scanning 2.2 Environmental Scanning 2.2 Environmental Scanning Creating and managing a successful business or venture requires research and planning. According to Sawyer (1993), research has revealed several reasons for the differences between planning in developed economies and less developed economies. Such reasons are attributable to dearth of the required technology required to scan and collect necessary data from the environment, and lack of information sources and other infrastructures for performing scanning activities (Sawyer, 1993). Consequently, organisations, profit and non-profit, are faced with challenges for survival and economic success. However, it should be noted that success results only when a firm strategically understands the external factors so as to be able to respond appropriately in a manner that will guarantee the firm’s survival and sustainable success. Environmental scanning is a means of gaining this understanding (Albright, 2004). For instance, business opportunities are daily springing up in all the sectors of the Nigerian economy as the nation’s population increases and new communities are established (Awaiko, 2004). To discover these business opportunities, environmental scanning is necessary. Scanning has to do with the gathering and utilisation of information concerning trends and events occurring in the external environment of a firm. It refers to awareness creation and communication of issues in the external environment that may likely affect the way a firm makes decisions (Albright, 2004). According to Choo (2001), environmental scanning could range from an informal conversation with someone to a formal business research 96 International Business Research www.ccsenet.org/ibr Vol. 6, No. 6; 2013 activity. Environmental scanning includes the actions taken to seek information in a firm’s external environment relating to trends and events, the awareness of which would assist senior management concerning the futurity of the firm’s current decisions (Aguilar, 1967 in Kourteli, 2005). Scanning intensity is the degree of comprehensiveness of the environmental scanning process and the effort towards the activities involved in scanning the environment (Barringer & Bluedorn, 1999). activity. Environmental scanning includes the actions taken to seek information in a firm’s external environment relating to trends and events, the awareness of which would assist senior management concerning the futurity of the firm’s current decisions (Aguilar, 1967 in Kourteli, 2005). Scanning intensity is the degree of comprehensiveness of the environmental scanning process and the effort towards the activities involved in scanning the environment (Barringer & Bluedorn, 1999). 2.2 Environmental Scanning Owualah (1999) asserts that the trends that need to be scanned are rooted in five macro environments: economic, social, technological, competitive and legal environments. Elenkov (1997) contests that the environments that need to be scanned can be classified into two layers. The first, which is closest to the organisation, is referred to as the task environment. Its elements include investors, employees, customers, suppliers, creditors, distributors, competitors and regulatory agencies. The second is the general environment. It refers to those factors that influence firms indirectly. The general environment includes socio-cultural, economic, and legal-political factors. The information gathered during scanning is provided to key managers within the firm and is used to guide management in future plans. It is also a means of assessing a firm’s strengths and weaknesses in response to external threats and opportunities. Thus, environmental scanning is a tool for identifying, collecting, and translating information about external influences into useful plans and decisions (Albright, 2004). 2.2.1 Why the Business Environment Should Be Scanned Firms need to do environmental scanning for the followings reasons: Environmental scanning is done so that firms are aware of the forces affecting change in the external environment in order to develop the strategies to sustain or enhance their competitiveness in the future (Choo, 2001). As a result of changes and uncertainties in the global environment and recently new business practices, firms can easily lag behind by not keeping up with new technology, current regulations, and various growing trends. Environmental scanning leads to greater anticipatory management (Albright, 2004) as it provides an awareness of the changing environment. According to Albright (2004), environmental scanning makes it possible for a firm to deal with external environmental issues that may, otherwise, have been difficult to identify. It is not just about information gathering; rather, its purpose is to focus on future impacts and potential influences on the organisation and how it can respond strategically (Albright, 2004). Environmental scanning enables a firm to highly focus on information from the external environment, as this will assist top management to become accessible to various sources that will assist them in their strategic planning and problem-solving situations (Popoola, 2001). Environmental scanning should be constantly done in order to be anticipatory and adaptive to changes in the environment. 2.2 Environmental Scanning Through constant monitoring of the external environment, it is easier for top management to respond to environmental change by making necessary adjustments in the light of the change. Hence, suggesting the difference between success and failure (Albright, 2004). 3. Methodology We let ) (t x represent the state of corporate entrepreneurship at any time ,t and let ) (t u represent planning flexibility which is the control exerted by the top management in case there is a deviation from the original plan occasioned by environmental scanning. Also we let ) (t v represent environmental scanning at any time .t For mathematical tractability; we model the problem on real Hilbert Space, as defined below: Mathematical Fundamentals: Consider a differential game modeled on Hilbert Spaces. Specifically, let 3,2,1 ,  i H i be real Hilbert spaces. Consider a differential game defined by: g y ) ( ) ( ) ( ) ( t Cv t Bu t Ax t x     (1) 1 0 ) 0 ( H x x   (2) ) ( ) ( ) ( ) ( t Cv t Bu t Ax t x     (1) 1 0 ) 0 ( H x x   (2) (1) (2) We associate a cost functional to equations (1) and (2), and define it as             T dt t Wv t v t Ru t u t Qx t x v u J ) ( ) ( ) ( ) ( ) ( ) ( ) ( (3) We associate a cost functional to equations (1) and (2), and define it as We associate a cost functional to equations (1) and (2), and define it as T We associate a cost functional to equations (1) and (2), and define it as             T dt t Wv t v t Ru t u t Qx t x v u J 0 ) ( ), ( ) ( ), ( ) ( ), ( ) , ( (3)             T dt t Wv t v t Ru t u t Qx t x v u J 0 ) ( ), ( ) ( ), ( ) ( ), ( ) , ( (3) Where we let  . and . 3. Methodology denote inner product and norm respectively on ,3,2,1 ,  i H i ) (t x represent the state vector in 1 H , ) (t u is the control strategy for player 1, with values in 2 H and ) (t v is the control strategy for player 2, with values in 3 H . 2.3 Corporate Entrepreneurship Entrepreneurship can be referred to as the process by which individuals, spurred by the desire for personal satisfaction or some rewards, act differently by adding value to an already existing venture or create an entirely new one not minding the risk involved (Kuye, 2008). Entrepreneurship includes the unique ability and determination to identify and exploit opportunity to create something new in the world (Martin & Osberg, 2007). Corporate entrepreneurship is entrepreneurship activities within an existing organisation which has been recognised as that which fosters economic prosperity, organisational performance, and wealth creation (Antoncic & Hisrich, 2004; Antoncic & Zorn, 2004). Corporate entrepreneurship is concerned with the development and sustainability of new ventures within an established firm (Garvin & Lavesque, 2006). 2.3.1 Entrepreneurial Postures 2.3.1 Entrepreneurial Postures There are three postures or dimensions (risk-taking, proactiveness and innovativeness) of entrepreneurial orientation as conceptualised by Kreiser et al. (2002); Moris et al. (2006); Chow (2006); Wiklund and Shepherd (2005); Covin and Sleving (1989). Innovation: Innovation is the process of adding value to a total firm, its employees, suppliers and customers via the creation of new techniques, procedures, services, products and marketing strategies (Shaw, O’Loughlin & McFadzean, 2005). ccording to Dess & Lumpkin (2005), risk-taking is the ability of a firm’s willingness to get involved Risk-taking: According to Dess & Lumpkin (2005), risk-taking is the ability of a firm’s willingness to get involved Risk-taking: According to Dess & Lumpkin (2005), risk-taking is the ability of a firm’s willingness to get involved 97 International Business Research Vol. 6, No. 6; 2013 www.ccsenet.org/ibr n a venture and act courageously without regards to the likelihood of the venture’s success or failure. in a venture and act courageously without regards to the likelihood of the venture’s success or failure. Proactiveness: Proactiveness is the process by which a firm continuously and actively searches rather than passively wait for market opportunities in view of the changing environment (Venkatraman, 1989). Proactiveness: Proactiveness is the process by which a firm continuously and actively searches rather than passively wait for market opportunities in view of the changing environment (Venkatraman, 1989). Even though, entrepreneurs are critical to economic development, developing countries especially in the Sub-Sahara Africa are yet to develop the wherewithal to exploit this resource (Bawuah, Buame & Hinson, 2006). 3. Methodology Remark (1): (i) It is in order, at this point, to explain that player 1 represents the intrapreneur who uses all efforts including factors of production to carryout production processes employing environmental scanning strategy u(t) at any time t to counteract measures that would make him deviate from the original plan. (ii) The environment in which the intrapreneur operates also employs strategy ) (t v to indirectly work against the efforts of player1 (intrapreneur). Hence in this paper we regard the environment as the player 11. (iii) The cost functional ) , ( v u J defined in (3) is the cost of the risk faced by the intrapreneur which must be minimized, but may be maximized by the environmental uncertainties if the intrapreneur fails to apply correct environmental scanning. A is a linear operator in 1 H with A D (domain of A) dense in 1 H . A is a closed operator. The resolvent: ) /( 1 ) , ( A I A R     exist if 0 Re   and     1 / ) , ( k A R . The operators: B: 1 2 H H  and 1 3 : H H C  are linear and uniformly bounded on ] ,. 0 [ T . The operator Q on 1 H is self-adjoint, symmetric and non-negative definite. The operators: B: 1 2 H H  and 1 3 : H H C  are linear and uniformly bounded on ] ,. 0 [ T . The operator Q on 1 H is self-adjoint, symmetric and non-negative definite. R and 1  R on 2 H are self adjoint symmetric and positive definite , while W and 1  W are self adjoint and negative definite. Player 1 apply strategy ) (t u at time t to minimize ) , ( v u J and player 2 apply strategy ) (t v at time t to maximize ) , ( v u J . Remark (2): The above explanations are necessary to ensure that the game proposed guaranteed existence of solution and mathematically solvable. Remark (2): The above explanations are necessary to ensure that the game proposed guaranteed existence of solution and mathematically solvable. Assumptions: A1: The pay-off functional can be expressed as follows: There exist 2 1,Q Q such that ] , [ 2 1 Q Q Q  and, A2: The controls ) (t u and ) (t v are separated on the right hand side of (1) i.e there exist 2 1, A A such that 2 1, [ A A A  ] 2 1, [ A A A  ] A3: ) , , , (   v u x H x    A4: 0 ) (  T  A5: 0 ) , , , (   v u x H u A6:  , , , ( v u x H v ) =0 A3: ) , , , (   v u x H x    A4: 0 ) (  T  A5: 0 ) , , , (   v u x H u A6:  , , , ( v u x H v ) =0 Remark (1): Given that ) , ( v u J is the optimal solution of ) , ( v u J , then the main problem is to show that: ) ,v u is the optimal solution of ) , ( v u J , then the main problem is to show that: Given that ) , ( v u J is the optimal solution of ) , ( v u J , then the main problem is to show that: ) , ( ) , ( ) , ( v u J v u J v u J   (4) Given that ) , ( v u J is the optimal solution of ) , ( v u J , then the main problem is to show that: ) , ( ) , ( ) , ( v u J v u J v u J   (4) ) , ( ) , ( ) , ( v u J v u J v u J   (4) ) , ( ) , ( ) , ( v u J v u J v u J   (4) (4) ) , ( ) , ( ) , ( v u J v u J v u J   We start achieving this aim by defining the Hamiltonian of the equation (1), (2) and (3) as follows:  ) , , , (  v u x H T t Wv t v t Qx t x        ) ( ), ( ) ( ), ( )] ( ) ( ) ( [ t Cv t Bu t Ax   (5) We start achieving this aim by defining the Hamiltonian of the equation (1), (2) and (3) as follows:  ) , , , (  v u x H T t Wv t v t Qx t x        ) ( ), ( ) ( ), ( )] ( ) ( ) ( [ t Cv t Bu t Ax   (5) (5) 98 Vol. 6, No. 6; 2013 International Business Research www.ccsenet.org/ibr The Multiplier ,  v u x such that problem 2 satisfies ). , , , ( ) , , , ( ) , , , (    v u x J v u x J v u x J   (8) Result 1: There exists a saddle point solution ( ) , . ,  v u x such that problem 2 satisfies ). , , , ( ) , , , ( ) , , , (    v u x J v u x J v u x J   Result 1: There exists a saddle point solution ( ) , . ,  v u x such that problem 2 satisfies ). , , , ( ) , , , ( ) , , , (    v u x J v u x J v u x J   (8) (8) Our aim is to establish the existence of the saddle point defined in (8). We shall do this in steps. Our aim is to establish the existence of the saddle point defined in (8). We shall do this in steps. Our aim is to establish the existence of the saddle point defined in (8). We shall do this in steps. er the control problem defined by: ) ( ) ( ) ( ) ( 1 t Bu t x t A t x    (9) Step1. Consider the control problem defined by: ) ( ) ( ) ( ) ( 1 t Bu t x t A t x    (9) With the pay off functional defined by: Step1. Consider the control problem defined by: Step1. Consider the control problem defined by: Step1. The Multiplier We now set          (6)          (6) (.)  is the multiplier, while (.)  and (.)  will be specified later. We transform (1)-(3) into unconstrained problem by introducing a penalty constant (.)  to get: Problem 1: Minimax (.)  is the multiplier, while (.)  and (.)  will be specified later. (.)  is the multiplier, while (.)  and (.)  will be specified later. The Multiplier We transform (1)-(3) into unconstrained problem by introducing a penalty constant (.)  to get: Problem 1: Minimax We transform (1)-(3) into unconstrained problem by introducing a penalty constant (.)  to get: Problem 1: Minimax We transform (1)-(3) into unconstrained problem by introducing a penalty constant (.)  to get: Problem 1: Minimax dt t Cv t x A t x dt t Bu t x A t x v J u J v u x J T T             0 2 2 0 2 1 ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , , , , (     + dt t Cv t x A t x dt t Bu t x A t x T T              ) ( ) ( ) ( , ) ( ) ( ) ( , 0 0 2 1   For computational convenience, we can update  and  as follows: For computational convenience, we can update  and  as follows: 1 0, ,....., 2,1,0 ,0 )], ( ) ( ) ( [ 2 ) ( ) ( 0 1 1                where k t Bu t x A t x t t k k k k k 0 1, ,..., 2,1,0 ,0 )], ( ) ( ) ( [ 2 ) ( ) ( 0 2 1                where k t Cv t x A t x t t k k k k (7) (7) 0 1, ,..., 2,1,0 ,0 )], ( ) ( ) ( [ 2 ) ( ) ( 0 2 1          where k t Cv t x A t x t t k k k k (7) Suppose 0    in problem 1, we have Suppose 0    in problem 1, we have Problem2: Min max Suppose 0    in problem 1, we have Problem2: Min max dt t Cv t x A t x dt t Bu t x A t x v J u J v u x j T T             0 2 2 2 0 1 ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , , , (    The following result is a consequence of the assumption A1and A2 The following result is a consequence of the assumption A1and A2 e exists a saddle point solution ( ) , . The Multiplier Consider the control problem defined by: ) ( ) ( ) ( ) ( 1 t Bu t x t A t x    (9) ) ( ) ( ) ( ) ( 1 t Bu t x t A t x    (9) ) ( ) ( ) ( ) ( 1 ( ) With the pay off functional defined by: With the pay off functional defined by:  dt t Ru t u t x Q t x u x J T        0 1 ) ( ), ( ) ( ), ( ) , ( (10) (10) Control (.) u is selected to minimise the pay off defined in (10) h i d i l f bl (9) d (10) i i Control (.) u is selected to minimise the pay off defined in (10) Control (.) u is selected to minimise the pay off defined in (10) Control (.) u is selected to minimise the pay off defined in (10) The unconstrained equivalence of problem (9) and (10) is given as: (11)  dt t Bu t x A t x t Ru t u t x Q t x u x J T           0 2 1 1 ) ( ) ( ) ( ) ( ), ( ) ( ), ( ) , , (   (11)  dt t Bu t x A t x t Ru t u t x Q t x u x J T           0 2 1 1 ) ( ) ( ) ( ) ( ), ( ) ( ), ( ) , , (   (11 99 International Business Research Vol. 6, No. 6; 2013 International Business Research Vol. 6, No. 6; 2013 International Business Research Vol. 6, No. The Multiplier 6, No. 6; 2013 International Business Research Vol. 6, No. The Multiplier 6; 2013 www.ccsenet.org/ibr Now, it is easy to note that the mild solution of (19) can be represented as Now, it is easy to note that the mild solution of (19) can be represented as     t d Cv t T x t T t x 0 0 ) ( ) ( ) ( ) (    (22) (22) Define operator P as follows: Define operator P as follows: Define operator P as follows: ] , [ , ) ( ) ( ) )( ( 3 0 2 H L v d Cv t T t Pv t        (23) ] , [ , ) ( ) ( ) )( ( 3 0 2 H L v d Cv t T t Pv t        (23) (23) Let ] , [ ) ( ), ( ) ( 1 2 0 H L t r t r x t T    , then the corresponding solution to (22) is denoted by: ) )( ( ) ( ) ( t Pv t r t x   (24) Let ] , [ ) ( ), ( ) ( 1 2 0 H L t r t r x t T    , then the corresponding solution to (22) is denoted by: ) )( ( ) ( ) ( t Pv t r t x   (24) (24) If we set: If we set:                K C A G C P A F C C W A A Q T T T T 2 2 2 2 2   (25)                K C A G C P A F C C W A A Q T T T T 2 2 2 2 2   (25) (25)         ) ( ), ( 2 1 ) ( ), ( 2 ) , ( 0 t v t v t Lv t r F v J  (26)         ) ( ), ( 2 1 ) ( ), ( 2 ) , ( 0 t v t v t Lv t r F v J  (26) Wh (26) Where ) ( 2 2 2 2 K A A C A P L T T T         (27)  P K P A G G G P P F T T T T    2 2 2       (28) (27) (28) 2 ) ( ) ( ), ( 2 2 0      t r A A t r F T  (29) (29) Since (.) v is a maximizer of (26) then the operator  must be negative definite, then at nth level of maximization process, Since (.) v is a maximizer of (26) then the operator  must be negative definite, then at nth level of maximization process, ) , ( 1 1 2 ) , ( 0 0 2   v J M m M m v J n n              (30) (30) M and m denote respectively the least upper and greatest lower bound of the spectrum of the operator  defined in (28). The Multiplier From (30) three cases are possible namely: M and m denote respectively the least upper and greatest lower bound of the spectrum of the operator  defined in (28). From (30) three cases are possible namely: M and m denote respectively the least upper and greatest lower bound of the spectrum of the operator  defined in (28). The Multiplier 6; 2013 www.ccsenet.org/ibr = dt t Bu A t x t x A t x t Bu t x t x t x t Bu B t u t x A A t x t Ru t u t x Q t x T T T T                                   0 1 1 1 1 ) ( ), ( 2 ) ( ), ( 2 ) ( ), ( 2 ) ( ), ( ) ( ), ( ) ( ), ( ) ( ), ( ) ( ), (       (12) = dt t Bu A t x t x A t x t Bu t x t x t x t Bu B t u t x A A t x t Ru t u t x Q t x T T T T                                   0 1 1 1 1 ) ( ), ( 2 ) ( ), ( 2 ) ( ), ( 2 ) ( ), ( ) ( ), ( ) ( ), ( ) ( ), ( ) ( ), (       (12) We can put (12) in a quadratic form as defined below: We can put (12) in a quadratic form as defined below: dt u x x B B R B B A B I A B A A A A Q u x x u x J T T T T                                        1 1 1 1 1 1 ) ( ) , , ( (13) (13) dt u x B B R B B A B I A u x x u x J T T                               1 1 ) ( ) , , ( (13) In a compact form, we can write (13) as:       1 In a compact form, we can write (13) as: In a compact form, we can write (13) as:  dt w w T    ) ( ) ( (14) (14)  dt w w T    ) ( ) ( (14) Where ] ,0 [ T   and                   B B R B B A B I A B A A A A Q T T T T          1 1 1 1 1 1 1 , Where ] ,0 [ T   and                   B B R B B A B I A B A A A A Q T T T T          1 1 1 1 1 1 1 ,   u x x wT , ,     u x x wT , ,   (15) (15) (Ibiejugba & Onumanyi 1984), had earlier established that if m and M are the greatest lower bound and the least upper bound of the spectrum of operator  then at nth level of iteration, the rate of convergence is given as: (Ibiejugba & Onumanyi 1984), had earlier established that if m and M are the greatest lower bound and the least upper bound of the spectrum of operator  then at nth level of iteration, the rate of convergence is given as: ) , , ( 1 1 ) , , ( 0 0 2   u x J M m M m u x J n n n              (16) ) , , ( 1 1 ) , , ( 0 0 2   u x J M m M m u x J n n n              (16) (16) Step 2: We will now establish that for (.) v a maximizer, at nth level of iteration, Step 2: We will now establish that for (.) v a maximizer, at nth level of iteration, Step 2: We will now establish that for (.) v a maximizer, at nth level of iteration, ) , , ( 1 1 ) , , ( 0 0 2   v x J M m M m v x J n n n              (17) ) , , ( 1 1 ) , , ( 0 0 2   v x J M m M m v x J n n n              (17) (17) To see this, Let ] ,0 [ T   be a fixed interval, denote by i H L , [ 2  ] the space of Strongly measurable function i H t z  ) ( such that: To see this, Let ] ,0 [ T   be a fixed interval, denote by i H L , [ 2  ] the space of Strongly measurable function i H t z  ) ( such that:    dt t z 2 ) ( Then ] , [ 2 i H L  is a real Hilbert space with an inner product defined by: Then ] , [ 2 i H L  is a real Hilbert space with an inner product defined by:      dt t z t w z w ) ( ), ( , (18)      dt t z t w z w ) ( ), ( , (18) (18) ) ( ) ( ) ( 2 t Cv t x A t x    (19) group generated by 2 A ) ( ) ( ) ( 2 t Cv t x A t x    (19) nuous semi- group generated by 2 A ) ( ) ( ) ( 2 t Cv t x A t x    (19) ) ( ) ( ) ( 2 t Cv t x A t x    (19) strongly continuous semi- group generated by 2 A Let ) (t T be strongly continuous semi- group generated by 2 A Let ) (t T be strongly continuous semi- group generated by 2 A Let ) (t T be strongly continuous semi- group generated by 2 A Consider the functional defined by:  dt t v t Wv t x t x Q v x J T        0 2 ) ( ), ( ) ( ), ( ) , ( (20) (20) W is negative definite, ) (t v tries to maximize ) , ( v x J .Transforming (19) and (20) to unconstraint problem we get:  dt t Cv t x A t x t Wv t v t x Q t x u x J T            0 2 2 2 ) ( ) ( ) ( ) ( ), ( ) ( ), ( ) , , (   (21 (21) 100 International Business Research Vol. 4. Conclusion Top management of a firm will pursue an entrepreneurial strategy if it hopes that entrepreneurship can make a notable difference in a firm’s ability to compete and achieve sustainable performance (Ramachandran, Devarajan & Ray, 2006). Then, top management is faced with a lot of challenges, majorly uncertainties, in the business environment that need to be dealt with. Top management, through its corporate entrepreneurs, wishes to minimise the costs of its operation in the face of uncertainties. It uses the factors of production to provide goods and services which constitute the costs of operation. The environment, on the other hand, tries to maximise these costs via various environmental constraints against the efforts of the top management. For a saddle point to be attained, an effective environmental scanning must be used to plan flexibly to break even. Top management of an entrepreneurial firm needs to carry out its environmental scanning activities and plan flexibly in a problem-solving manner. This, especially in relation to how innovation activity thrives in the firm and the value of specialised knowledge created is recognised and integrated to create wealth (Ramachandran, Devarajan & Ray, 2006). In all of this, planning flexibility and environmental scanning are considered effective strategies that can assist top management to cope with uncertainties in an entrepreneurial environment. Hence, a saddle point is ensured in a “worst case” situation. The Multiplier 6; 2013 www.ccsenet.org/ibr Case III: n n M m M m M m M m 2 2 1 1 1 1                          In this case, one can always choose (.) v such that In this case, one can always choose (.) v such that ) , ( 1 1 ) , ( 0 0 2   v J M m M m v J n              We can infer from all the three cases that: ) , ( ) , , ( ) , (    v J v u J u J   (31) ) , ( ) , , ( ) , (    v J v u J u J   (31) Remark (3) (i) The implication of our result is that for entrepreneurial firms, top management through its intrapreneurs requires minimal amount of resources (to do environmental scanning and planning flexibility) in dealing with the risk of entrepreneurship, in the presence of environmental uncertainty. (ii) The result in (31) represents a worst case situation, that is to say that the intrapreneur will brake-even no matter the situation as long as there is a saddle point as represented in (31). (iii) It should be very clear that the concept of “uncertainty” is artificial. We just assume that the system behaves according to some law; and model it accordingly. We seek the “best” under a “worst situation” (with respect to the uncertainty) in order to guarantee the desire performance over the entire range of the uncertainty. Alpkan, L., Yilmaz, C., & Kaya, N. (2007). Market Orientation and Planning Flexibility in SMEs: Performance Implications and an Empirical Investigation. International Small Business Journal, 25(2), 152-172. http://dx.doi.org/10.1177/0266242607074518 The Multiplier From (30) three cases are possible namely: Case I: n n M m M m M m M m 2 2 1 1 1 1                          Case I: n n M m M m M m M m 2 2 1 1 1 1                          Then, Then, Then, ) , ( ) , , ( ) , (    u J u v J v J   Case II: n n M m M m M m M m 2 2 1 1 1 1                          Then, ) , ( ) , , ( } , (    u J v u J v J   Then, ) , ( ) , , ( ) , (    u J u v J v J   Case II: n n M m M m M m M m 2 2 1 1 1 1                          Then, ) , ( ) , , ( ) , (    u J u v J v J   Case II: n n M m M m M m M m 2 2 1 1 1 1                          Then, ) , ( ) , , ( } , (    u J v u J v J   ) , ( ) , , ( } , (    u J v u J v J   101 101 International Business Research Vol. 6, No. Albright, K. 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https://openalex.org/W2620987933	https://www.biorxiv.org/content/biorxiv/early/2017/08/22/144691.full.pdf	English	null	Receptor uptake arrays for vitamin B<sub>12</sub>, siderophores and glycans shape bacterial communities	bioRxiv (Cold Spring Harbor Laboratory)	2,017	cc-by	22,628	Steven A. Frank∗ Molecular variants of vitamin B12, siderophores and glycans occur. To take up variant forms, bacteria may express an array of receptors. The gut microbe Bacteroides thetaiotaomicron has three diﬀerent receptors to take up variants of vitamin B12 and 88 receptors to take up various glycans. The design of receptor arrays reﬂects key processes that shape cellular evolution. Competition may focus each species on a subset of the available nutrient diversity. Some gut bacteria can take up only a narrow range of carbohydrates, whereas species such as B. thetaiotaomicron can digest many diﬀerent complex glycans. Comparison of diﬀerent nutrients, habitats, and genomes provide opportunity to test hypotheses about the breadth of receptor arrays. Another important process concerns ﬂuctuations in nutrient availability. Such ﬂuctuations enhance the value of cellular sensors, which gain information about environmental availability and adjust receptor deployment. Bacteria often adjust receptor expression in response to ﬂuctuations of particular carbohydrate food sources. Some species may adjust expression of uptake receptors for speciﬁc siderophores. How do cells use sensor information to control the response to ﬂuctuations? That question about regulatory wiring relates to problems that arise in control theory and artiﬁcial intelligence. Control theory clariﬁes how to analyze environmental ﬂuctuations in relation to the design of sensors and response systems. Recent advances in deep learning studies of artiﬁcial intelligence focus on the architecture of regulatory wiring and the ways in which complex control networks represent and classify environmental states. I emphasize the similar design problems that arise in cellular evolution, control theory, and artiﬁcial intelligence. I connect those broad conceptual aspects to many testable hypotheses for bacterial uptake of vitamin B12, siderophores and glycans. Keywords: Corrinoids, public goods, microbiome, systems biology, deep learning, control theory, phe- notypic plasticity Introduction 2 Vitamin B12 variants 3 Background . . . . . . . . . . . . . . . . . . . . 3 Corrinoids, growth and competition . . . . . . . 3 Receptor diversity and uptake arrays . . . . . . . 3 Design of corrinoid receptor arrays . . . . . . . . 4 Siderophores 7 Speciﬁcity of uptake receptors . . . . . . . . . . 8 Fluctuation and the timescales of acquisition and use . . . . . . . . . . . . . . . . . . . . . . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted August 22, 2017. ; https://doi.org/10.1101/144691 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted August 22, 2017. ; https://doi.org/10.1101/144691 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted August 22, 2017. ; https://doi.org/10.1101/144691 doi: bioRxiv preprint * Department of Ecology and Evolutionary Biology, University of California, Irvine, CA 92697–2525, USA web: https://stevefrank.org Receptor uptake arrays for vitamin B12, siderophores and glycans shape bacterial communities Steven A. Frank∗ Introduction iments demonstrate that particular receptors lead to competitive dominance in the presence of particular B12 variants, whereas other receptors cause domi- nance in the presence of other B12 variants. Recep- tors apparently diﬀer in their uptake eﬃcacies for a variety of B12 analogs. Degnan et al. 1 estimate that the human microbiome contains more than 30 recep- tor families for uptake of B12-like corrinoid variants. Genomic analyses predict that a signiﬁcant fraction of all bacteria and archaea require a corrinoid vari- ant for growth, yet only a minority can produce corri- noids2,3. Competition over corrinoid variants shapes the receptor arrays of individual species and the dy- namics of bacterial communities. Molecular variants of vitamin B12 occur. A bacterial cell may take up diﬀerent B12 forms by expressing multiple receptors. Bacterial cells also express multi- ple receptors to take up polymorphic iron-scavenging siderophores and energy containing glycans. I em- phasize broad questions about the characteristics of receptor uptake arrays for nutrient acquisition. How does natural selection set the number of re- ceptor variants? How does selection tune the bind- ing aﬃnities of diﬀerent receptor variants in an ar- ray? How does the design of receptor arrays shape the competitive and cooperative processes that deﬁne bacterial communities? Siderophores are secreted molecules that bind free iron. Bacteria use siderophore receptors to take up the siderophore-iron complexes. Iron often sets a limiting resource for microbial growth. Thus, com- petitive and cooperative processes over siderophore uptake shape bacterial community dynamics4–6. In- dividual bacteria may secrete more than one type of siderophore, or none at all. Bacteria typically have uptake receptors tuned for their own secreted types. In addition, bacteria often express an array of sider- ophore uptake receptors for types produced by other species7. Bacteria, yeast, and other fungi may take up each other’s secreted siderophores. The ubiqui- tous battle for free iron sets the design of siderophore uptake receptor arrays. Vitamin and metabolite receptor arrays provide an exceptional model to study conditional response to the environment. Nutrient availability ﬂuctuates. When cells can produce many diﬀerent receptors, it may pay to alter receptor expression levels according to the availability of matching nutrients. Plastic response requires sensor arrays to perceive availability. Environmental perception must then be transduced through a regulatory network that inte- grates information and alters deployment of the re- ceptor array. Steven A. Frank∗ It is made available under The copyright holder for this preprint (which was not this version posted August 22, 2017. ; https://doi.org/10.1101/144691 doi: bioRxiv preprint Steven A. Frank∗ . 8 Conditional versus continuous expression of re- ceptors . . . . . . . . . . . . . . . . . . . . 9 Competition and inverse public goods . . . . . . 10 Challenges for uptake receptor array design 10 Glycans 11 Receptor diversity and speciﬁcity . . . . . . . . . 11 Receptors for partially digested components . . . 12 Sensors and conditional response . . . . . . . . . 13 Hierarchy of preferred types . . . . . . . . . . . 13 Perception and response . . . . . . . . . . . . . . 14 Deep learning and control theory 15 Deep learning . . . . . . . . . . . . . . . . . . . 15 Control theory and the frequency domain . . . . 17 Discussion 18 Diversity of available nutrients and matching re- ceptors . . . . . . . . . . . . . . . . . . . . 18 Number and speciﬁcity of receptor variants per cell 19 Species interactions . . . . . . . . . . . . . . . . 20 Conditional response, ﬂuctuations and timescales 21 Regulatory overwiring . . . . . . . . . . . . . . . 22 Conclusions 23 * Department of Ecology and Evolutionary Biology, University of California, Irvine, CA 92697–2525, USA web: https://stevefrank.org Introduction git • master @ arXiv-2.0-0::8ec16e7-2017-08-21 (2017-08-22 01:10Z) • safrank . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted August 22, 2017. ; https://doi.org/10.1101/144691 doi: ioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted August 22, 2017. ; https://doi.org/10.1101/144691 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. Receptor diversity and uptake arrays The diversity of corrinoid forms has been known for many years11. Recent studies provide ﬁve lines of ev- idence about the diﬀerent functional characteristics of various corrinoids16. Genomic studies estimate that 76% of bacterial species require a B12 variant. Only one-half of those species that require a B12 variant can synthesize their own. The other species must take up the vitamin ex- ternally. Nearly all species that require a B12 variant, including those that can synthesize their own, have genes that encode uptake3. First, particular bacterial species appear to re- quire speciﬁc corrinoids for their corrinoid-dependent enzymatic reactions17–19. I deﬁne a ‘native’ corrinoid as a form required by a particular species. Second, individual species can remodel nonnative corrinoids into their required native form17,20. Allen and Stabler 21 detected eight distinct corrinoids in hu- man fecal samples from 20 individuals. Two individ- uals ingested high doses of cobalamin, the canonical B12 corrinoid. Those two individuals excreted broadly elevated amounts of the other detectable corrinoid forms. When those two individuals discontinued high supplementation, they reverted to a corrinoid distri- bution similar to the other individuals in the sample. Degnan et al. 16 suggest that the transient increase of diverse corrinoid forms in supplemented individuals implies that various bacterial gut species remodel the ingested B12 form into the diﬀerent native corrinoids particular for each species. Corrinoid synthesis is complex and energetically costly, whereas uptake is relatively inexpensive11. The cost diﬀerence between synthesis and uptake probably explains why uptake is so common, even among those species that can make their own. Only prokaryotes can make corrinoids. Free corri- noids most likely originate from release of intracellu- lar components following prokaryotic cell death. No active secretion is known. Corrinoids may also cycle among various prokaryotic and eukaryotic consumers and the environment. Background B12 variants comprise a family of corrinoid molecules11,12. The cobalt-containing corrin ring deﬁnes the group and plays a key role in corrinoid coenzyme activity. I use ‘B12 variant’ and ‘corrinoid’ interchangeably. Introduction It is made available under The copyright holder for this preprint (which was not this version posted August 22, 2017. ; https://doi.org/10.1101/144691 doi: bioRxiv preprint Vitamin B12 variants by a cell may be low and the half life of the molecules relatively long. Thus, low levels of free corrinoids do not necessarily mean that corrinoids are strongly lim- iting for growth. I begin with the variety of B12 molecules. How are dif- ferent receptors tuned to compete for taking up the diversity of B12 variants? Direct competition experiments provide the most compelling evidence for the importance of corrinoids. I describe a competition experiment at the end of the following subsection. Introduction How do aspects of environmental ﬂuc- tuation shape the evolutionary design of sensory per- ception and the regulatory control to achieve a con- ditional response? I discuss how perception, classiﬁcation, and re- sponse through a network relate to recent break- throughs in artiﬁcial intelligence, neural networks, and deep learning. Bacterial systems provide great opportunity to link conceptual aspects of evolution- ary design and deep learning to hypotheses that can be tested by comparative genomics and by experi- mental laboratory studies. Glycans are complex carbohydrates with diverse molecular structures. Bacteria use speciﬁc receptors and digestive enzymes to catabolize particular gly- cans. In habitats with diverse glycan sources, such as the mammalian gut, bacteria often express broad glycan receptor arrays. For example, many species of Bacteroidetes have diverse Polysaccharide Utilization Loci (PUL) gene clusters8–10. Each cluster typically encodes cell surface glycan-binding proteins that cap- ture speciﬁc glycans to initiate catabolism. Among B. thetaiotaomicron, B. ovatus and B. cellulosilyticus WH2, each has approximately 100 PULs. Species pairs vary in their number of shared PULs, proba- bly reﬂecting the diﬀerent habitats and the diﬀerent competitive tunings of their receptor arrays. I synthesize aspects of receptor arrays for vita- min B12 analogs, for siderophores, and for glycans. By considering these diﬀerent cases together, deeper principles of receptor variety and speciﬁcity emerge. Here, I use the word ‘receptor’ to include the various binding and transport processes that inﬂuence speci- ﬁcity and rate of uptake. For uptake of vitamin B12 analogs, Bacteroides thetaiotaomicron expresses three homologous vari- ants of the associated receptor1. Competition exper- 2 git • master @ arXiv-2.0-0::8ec16e7-2017-08-21 (2017-08-22 01:10Z) • safrank . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted August 22, 2017. ; https://doi.org/10.1101/144691 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted August 22, 2017. ; https://doi.org/10.1101/144691 doi: ioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. Why are there diﬀerent types of corrinoid? sequencing would add another 43 variants, for a total of 103 among the 313 species. The speciﬁc numbers are imprecise. However, the conclusion that there are a lot of transporter variants is likely to hold. Escape from attack. Colicins and phage use corrinoid receptors as a point of attack22–24. Colicins are tox- ins secreted by bacteria that can kill other bacteria. Typically, a colicin binds to a receptor that provides an important uptake function for the target cell. The receptor function makes it diﬃcult to hide or modify the receptor, providing a point for attack. Similarly, phage are viruses that initiate infection by binding to important cellular uptake receptors. A rare variant corrinoid and matching receptor can escape from at- tack by common colicins and phages. The advantage to rare variants promotes diversity25. Fourth, individual species may have multiple dis- tinct corrinoid uptake receptors. Degnan et al. 1 es- timated an average of 1.9 distinct uptake receptor genes among 57 human gut Bacteroidetes species, with a range of 1–4 variant receptors per species. Fifth, competition experiments demonstrated that particular receptors provide a strong growth ad- vantage when matched to particular corrinoids1. B. thetaiotaomicron expresses three distinct receptors, labeled btuB1, btuB2, and btuB3. An in vitro experi- ment competed a mutant that expressed only btuB1 against a mutant that expressed only btuB3. Each assay provided one of six distinct corrinoids, includ- ing cobalamin, the canonical B12 form. The btuB1 strain won in the presence of two corrinoid variants, whereas btuB3 won in the presence of the other four variants. Prevent uptake by competitors. Production and uptake of a novel variant prevents competition for uptake by other strains1,6. Continual selection for novel private variants could diversify corrinoids. Ini- tially, a strain may produce and take up its pri- vate form. When cell death releases sequestered molecules, nearby genetically related types can take up the novel form. As an initially private type gains an advantage and becomes more common, other strains may evolve uptake receptors tuned for the novel form. An in vivo experiment colonized germ-free mice with a wild-type strain that expressed all three re- ceptors and a mutant strain with btuB2 knocked out. Mice were fed a diet that included cobalamin. The wild-type strongly outcompeted the mutant. Rein- troduction of btuB2 to the mutant strain recovered nearly all of the lost competitive success. Corrinoids, growth and competition Many studies implicate corrinoids as a limiting growth factor for prokaryotes and marine eukaryotic plankton3,13. Most examples are based on three lines of evidence: absence of corrinoid-producing genes, presence of essential corrinoid-requiring genes with- out alternative corrinoid-independent pathways, and very low levels of free corrinoids. Additionally, sup- plementation of phytoplankton communities with B12 variants sometimes stimulates growth or signiﬁcantly alters community composition13,14. Third, many distinct corrinoid uptake receptors occur. Among 313 human gut species, Degnan et al. 1 inferred a minimum of 27 distinct corrinoid trans- porter families. They used a conservative 50% amino acid homology cutoﬀto deﬁne distinct transporter families. In B. thetaiotaomicron, a 50% homology cutoﬀdid not resolve two transporter genes that had distinct in vitro and in vivo functional uptake proper- ties. Using a 75% homology cutoﬀ, suﬃcient to dis- tinguish the functionally diﬀerent B. thetaiotaomicron transporters, raised the estimated number of distinct corrinoid transporter families to 60. Degnan et al. 1 further estimate that a two-fold increase in genomic However, it can be diﬃcult to interpret the im- portance of B12 variants from indirect lines of evi- dence15. For example, the amount of B12 required 3 3 git • master @ arXiv-2.0-0::8ec16e7-2017-08-21 (2017-08-22 01:10Z) • safrank . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted August 22, 2017. ; https://doi.org/10.1101/144691 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted August 22, 2017. ; https://doi.org/10.1101/144691 doi: bioRxiv preprint Why are there diﬀerent types of corrinoid? Thus, btuB2 provides a strong in vivo competitive advantage for the uptake of cobalamin1. Alternatively, a private variant may arise in a cross-feeding relationship. One strain may produce a novel variant that can be taken up by another strain. If the receiving strain gains a growth beneﬁt from up- take, and also has some positive feedback eﬀect on the initial producer strain, then a synergistic mutu- alism may drive the initial spread of a private vari- ant26,27. Community structure may be inﬂuenced by a network of private exchange channels. Any particularly successful and abundant private line of exchange will become subject to hijacking by other species, altering the community network. How are the properties of receptors tuned to maxi- mize the uptake beneﬁts of an array? I ﬁrst summarize what is known. I then turn to vari- ous conceptual issues that frame how we may under- stand receptor arrays. Degnan et al. 1 inferred that many species of hu- man gut Bacteroidetes express multiple corrinoid re- ceptors. However, the binding and uptake proper- ties of such receptor arrays for diﬀerent corrinoids remain unknown, except for B. thetaiotaomicron. In that species, Degnan et al. 1 used the studies men- tioned above to infer four aspects of uptake. Receptor design on a line. Begin with an overly sim- ple case. Imagine that we can locate the potential up- take properties of each corrinoid type along a single dimension. Given the corrinoid uptake property loca- tions along the line, how should the uptake receptor array be designed to maximize the beneﬁt of uptake? To answer that question about receptor array design, we need to add some assumptions. First, any one of the three distinct receptors takes up canonical B12 (cobalamin) suﬃciently to confer full growth rate. In a mutant strain with all three distinct receptors knocked out, reintroduction of any one of those receptors fully restores growth in vitro in the presence of cobalamin. Set constant concentrations for free corrinoids. Suppose there is a ﬁxed cost for encoding and pro- ducing each additional receptor type. Assume there is a ﬁxed total number of surface receptors produced, so that more of one type means less of other types. Let there be a diminishing beneﬁt of total uptake. Locate each potential receptor along the corrinoid line. The uptake rate of a receptor for a particular corrinoid di- minishes with the distance between the receptor and the corrinoid location along the line. Second, although in vitro growth by an isolated strain appeared to be invariant, direct in vitro compe- tition between diﬀerent mutant strains revealed dif- ferences in uptake of cobalamin. Pairwise compe- titions were conducted between wild-type, with all three receptors, and the three mutant types that ex- press only one of the receptors. The mutants are la- beled btuB1, btuB2, and btuB3, for the single receptor expressed. The growth of btuB2 was nearly identical to wild-type growth, btuB3 was mildly outcompeted by wild-type, and btuB1 was strongly outcompeted by wild-type. A further experiment showed that btuB3 outperforms btuB1 in direct competition. Design of corrinoid receptor arrays Uptake of corrinoids can strongly inﬂuence competi- tive success and community dynamics. Three aspects seem important: corrinoids are diverse, receptors dif- fer in their rate of uptake for diﬀerent corrinoids, and corrinoids can be remodeled into native form. Diﬀerent biochemical properties. Particular bacteria require speciﬁc corrinoid forms17–19. Thus, diﬀer- ent corrinoids have diﬀerent biochemical properties and may not be functionally interchangeable, at least in certain species. These observations lead to ques- tions about the sequence of events in corrinoid diver- gence. Did the corrinoid diﬀerences arise initially to catalyze diﬀerent biochemical transformations? Or did the corrinoid diﬀerences arise initially by another process, such as escape from viral attack of receptors, Some species express more than one receptor type. How does natural selection design the array of uptake receptors expressed by such species? Here, I brieﬂy sketch a conceptual frame for future theory and experiment. 4 git • master @ arXiv-2.0-0::8ec16e7-2017-08-21 (2017-08-22 01:10Z) • safrank . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted August 22, 2017. ; https://doi.org/10.1101/144691 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted August 22, 2017. ; https://doi.org/10.1101/144691 doi: bioRxiv preprint widely diﬀering eﬃcacies. followed by corresponding changes in the molecules and metabolic processes that depend on corrinoids? Third, ﬁve diﬀerent corrinoids plus cobalamin were used for six direct in vitro competitions between btuB1 and btuB3. The receptor btuB1 won two of the six competitions, and btuB3 won the other four. Chance events and drift. Corrinoid forms may initially have changed by a sequence of minor alterations, each by itself with little eﬀect on ﬁtness. Over time, some changes may have led to corresponding changes in the molecules that react with the corrinoids. Evo- lutionary drift among lineages possibly led to diverse corrinoid forms. It seems unlikely that the total diver- sity of corrinoids arose by such chance events, given the strong potential ﬁtness eﬀects of diversity listed above. However, chance probably plays some role in aspects of diversity. Design of corrinoid receptor arrays Fourth, in vivo competition in germ-free mice con- ﬁrmed that btuB2 has greatest eﬃcacy for uptake of cobalamin. In particular, wild-type with all three re- ceptors outcompeted a mutant with btuB2 knocked out and the other two receptors intact. The key points are: multiple receptors exist, each receptor takes up a variety of corrinoids, and the competitive performance of each receptor varies by corrinoid type. The ability of a receptor to take up more than one corrinoid may occur because diﬀerent corrinoid structures are partially constrained by their role as cofactors in particular biochemical transfor- mations. These points provide a basis for evaluating how diﬀerent processes shape the characteristics of receptors arrays. The following paragraphs list some candidate processes. A later section discusses ways in which to approach experimental and comparative tests. How are the properties of receptors tuned to maxi- mize the uptake beneﬁts of an array? Thus, all three receptors can take up cobalamin, but do so with With numerical values for these assumptions, we could calculate the optimal receptor array design to maximize the beneﬁts of corrinoid uptake. An opti- mal design would specify the number of diﬀerent re- ceptor types, the number of surface receptors of each 5 git • master @ arXiv-2.0-0::8ec16e7-2017-08-21 (2017-08-22 01:10Z) • safrank . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted August 22, 2017. ; https://doi.org/10.1101/144691 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted August 22, 2017. ; https://doi.org/10.1101/144691 doi: ioRxiv preprint then rapid scavenging during those rare periods may determine success. type, the location of each receptor type along the line, and the best tradeoﬀbetween the maximum uptake rate and the diminishing uptake rate with distance between corrinoid and receptor. However, the pur- pose is not to make a speciﬁc calculation, for which we have left out many obviously crucial aspects. In- stead, these assumptions begin the sketch for this complex problem. We continue to ﬁll in the sketch and to build the conceptual frame by which we may approach this problem. Rare pulses may occur if there are causes of widespread cell death, such as viral epidemics. If rare pulses of diﬀerent corrinoids happen in a relatively uncorrelated way, then cells may gain by a broad ar- ray of receptors. By contrast, highly correlated pulses may favor cellular types to concentrate on single re- ceptors tuned to particular corrinoids. Conditional versus continuous expression. Turning re- ceptor expression on or oﬀin response to the avail- ability of matching corrinoids may be advantageous. However, such conditional expression may miss tak- ing up rare pulses or otherwise ﬂuctuating concentra- tions. The beneﬁt of conditional expression depends on the availability of information about external con- centrations, the cost of sensing and integrating that information, the cost of turning on expression, and the timescales of ramping up expression and turning it oﬀrelative to the frequency of corrinoid ﬂuctua- tions. Uptake tradeoﬀs. How are the properties of receptors tuned to maxi- mize the uptake beneﬁts of an array? Distance along a line provides a simple way to specify tradeoﬀs. The closer an up- take receptor moves toward a particular corrinoid, the stronger that receptor’s uptake of approaching corrinoids, and the weaker that receptor’s uptake of receding corrinoids. We could increase the number of dimensions or alter the topology in which we locate receptors and corrinoids. Or we could directly specify how changes in a receptor aﬀect the uptake tradeoﬀs among the set of available corrinoids. Production, decay and loss. The availability of partic- ular free corrinoids depends on the production rate by primary producers or the remodeling rate by sec- ondary consumers. Availability also depends on the decay rate when free and when sequestered within cells, the sequestration time within cells before re- lease by cell death, the loss to a community locale by outﬂow, and the gain by inﬂow28. Corrinoid form in relation to uptake. Biochemical function shapes corrinoid form. The uptake proper- ties of free corrinoids by cellular receptors may also inﬂuence their form. Consider what happens when a primary producer or remodeler of corrinoids dies and releases its molecules. If genetically related individu- als of the same species take up those free corrinoids, any modiﬁcation of corrinoid form that increases such uptake is favored by kin selection29,30. Competition for free corrinoids. Uptake of particular corrinoids by one cellular type reduces the availabil- ity of those corrinoids for other cellular types. Thus, the rate of uptake by a cellular type and the abun- dance of that type may inﬂuence the growth dynam- ics of the other types in the community. The growth dynamics in turn inﬂuence the uptake and release of free corrinoids. If other species with positive mutualistic feed- backs on the primary producer take up the corrinoids, natural selection favors increase in such uptake. If other species with negative competitive feedbacks on the primary producer take up the corrinoids, natural selection favors reduction in such uptake. Those pos- itive and negative consequences of uptake can inﬂu- ence the biochemical form of corrinoids and the up- take properties of associated receptors. Fluctuation and the timescales of acquisition and use. The availability of free corrinoids likely varies over time and space. The tuning of receptor arrays will de- pend on those ﬂuctuations in relation to the temporal and spatial scales of various other processes. How are the properties of receptors tuned to maxi- mize the uptake beneﬁts of an array? For ex- ample, rapidly ﬂuctuating concentrations matter little if the timescale for acquisition and internal storage within cells is relatively long. Alternatively, if cor- rinoids tend to be released in low frequency pulses, Siderophores Organisms require iron for many metabolic pro- cesses. Available iron for uptake can limit microbial growth33. Many microbes secrete siderophores to chelate free iron. Cells take up external siderophore- iron complexes through speciﬁc surface receptors and transport mechanisms. The full problem of uptake receptor design for corrinoids extends the signal detection problem by al- lowing, in eﬀect, competition for the signal. Uptake, or sensor measurement, by one receptor reduces the available signal for uptake or measurement by other receptors. Additionally, successful uptake leads to an increase in the abundance of the associated receptor by population dynamics and natural selection, caus- ing a complex competitive game-like quality to the problem of uptake. Siderophores and corrinoids share certain aspects of uptake, diversity, and competitive consequences. The receptor and transport systems for siderophores and corrinoids derive from the same family of ABC transporters34. The close homology often makes it diﬃcult to distinguish siderophore from corrinoid transporters by amino acid sequence17. Although this complex uptake game transcends the signal detection problem, it is useful to keep in mind the core similarities. Many aspects of the up- take problem and the sensor design problem depend on the same issues of sensitivity, tradeoﬀs, and ﬂuctu- ations. By recognizing the abstract structure of the re- ceptor array design problem, one can take advantage of the existing theory, develop new theory that broad- ens understanding for a wide range of problems, and consider uptake receptor design in biology as part of a broader subject of sensor and uptake properties of organisms. Diverse siderophores occur. A species may take up a variety of its own secreted siderophores and various siderophores secreted by other species5,35. Individ- ual cells may express multiple uptake receptors with diﬀerent speciﬁcities. Siderophores mediate competi- tion for uptake of free iron, which can determine the ﬁtness of competing types36. For example, Strepto- myces coelicolor increases secretion of at least 12 dis- tinct siderophores in response to competition by sid- erophores secreted by ﬁve related bacterial species37. Other studies also infer large repertoires of sider- ophores deployed by certain species. Baars et al. 38 inferred over 35 distinct metal-binding molecules se- creted by the bacterium Azotobacter vinelandii. Those molecules are primarily siderophores that bind iron, although binding of other metals occurs. Cornelis and Bodilis 39 inferred 16 siderophore receptors in Pseu- domonas syringae and 42 in P. ﬂuorescens Pf5. Theory Many factors inﬂuence receptor array design. Those factors and their potentially complex interactions suggest a rich subject for theoretical development. This theoretical topic generalizes the important un- 6 git • master @ arXiv-2.0-0::8ec16e7-2017-08-21 (2017-08-22 01:10Z) • safrank . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted August 22, 2017. ; https://doi.org/10.1101/144691 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted August 22, 2017. ; https://doi.org/10.1101/144691 doi: ioRxiv preprint solved engineering problem of multisensor array de- sign for signal detection and estimation31,32. sory measurement of glycan availability. The greater complexity of glycan systems compared with corri- noid systems may reﬂect the greater diversity of gly- cans. In addition, the timescale for which cells must acquire glycans diﬀers from the timescale for which cells must acquire corrinoids. The comparison of dif- ferent systems provides insight into the problems of sensing, uptake, and conditional response. Sensor design for signal detection is roughly sim- ilar to receptor design for uptake of free corrinoids. In sensor design, one assumes that uptake or mea- surement does not inﬂuence concentration or signal intensity. Independence with respect to uptake would approximately hold when uptake of free corrinoids is relatively small compared with other sources of out- ﬂux loss. Other sources of outﬂux loss may include outﬂow or decay or uptake by a ﬁxed entity. Signal detection and uptake are roughly described as prob- lems of tuning sensitivity with respect to certain costs and beneﬁts. Speciﬁcity of uptake receptors Siderophore and corrinoid uptake may diﬀer with regard to speciﬁcity. Siderophore receptors are of- ten described as speciﬁc. For example, Rabsch and Winkelmann 40 state: “Every siderophore utilized by Escherichia coli has its corresponding outer mem- brane receptor: ferric enterobactin (FepA), ferric cit- rate (FecA), ferrichrome (FhuA), coprogen (FhuE), aerobactin (Iut) and other catecholates (Cir and Fiu) (Hantke 1990).” Determination of speciﬁcity depends on context. For example, siderophores comprise many distinct molecular families, each family with diverse forms. Observed speciﬁcity often means that a receptor takes up one of the tested siderophore families relatively well and the other test families relatively ineﬃciently or not at all. Such observations do not rule out re- ceptors that can take up closely related siderophores, perhaps with diﬀering eﬃcacies. I do not know of studies that have comprehensively measured the dif- ferent uptake eﬃcacies of receptors for a variety of closely related siderophores. Overall, the diﬀering constraints on form and function suggest that siderophores are likely to be more diverse and have greater speciﬁcity of uptake receptors than corrinoids. Indeed, siderophores com- prise a wide diversity of families with diﬀering forms. Comparative study of the diversity and uptake speci- ﬁcity of siderophores and corrinoids would be valu- able. Siderophores Individ- ual species can often take up siderophores produced by other species. For example, P. ﬂuorescens encodes a signiﬁcant repertoire of receptors for cross-species uptake35. All of that makes for an interesting theoretical subject. But how can such theory help to understand corrinoid uptake and related problems of siderophore and glycan uptake? Before turning to that practical question, which concerns the design of experiments and comparative tests, I ﬁrst summarize aspects of siderophore and glycan biology. Siderophore and glycan uptake share many sim- ilarities with corrinoid uptake, but also key diﬀer- ences. For example, glycan receptor systems have dis- tinct molecular components for uptake and for sen- 7 git • master @ arXiv-2.0-0::8ec16e7-2017-08-21 (2017-08-22 01:10Z) • safrank . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted August 22, 2017. ; https://doi.org/10.1101/144691 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted August 22, 2017. ; https://doi.org/10.1101/144691 doi: oRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted August 22, 2017. ; https://doi.org/10.1101/144691 doi: bioRxiv preprint The following subsections compare four aspects of siderophore and corrinoid receptor arrays. creted. Instead, they function as cofactors for com- plex biochemical transformations. The special bio- chemical requirements of cofactor action shape cor- rinoid form into large, intricate molecules. Free cor- rinoids probably exist only through release after cell death. To the extent that corrinoid diversity may be shaped by competition for uptake, corrinoid form may be constrained by function as a cofactor and by the high expense for producing the corrinoid’s intricate molecular structure. However, certain parts of a cor- rinoid molecule may be more easily modiﬁed than other parts, providing some opportunity for compe- tition to shape corrinoid structure in relation to re- ceptor binding. Conditional versus continuous expression of receptors The game-like competition for iron-siderophore complexes likely shapes the frequency spectrum of ﬂuctuations in availability in ways that diﬀer from corrinoids. Primary production of corrinoids arises mainly for the internal use by the producers, perhaps also augmented by any beneﬁt of released molecules that are taken up by genetic relatives or by mutualist species. By contrast, primary production of sidero- phores arises mainly for subsequent uptake. Cells may turn on or increase expression of receptors in response to external availability of matching types. The beneﬁts of such conditional expression may diﬀer between corrinoids and siderophores. For corrinoids, the beneﬁts of conditional expres- sion may be limited. If cells require relatively few corrinoid molecules, then it may be more important to capture some molecules when available rather than to ramp up expression to capture a large number of molecules. If the decay rate for pulses of external availability is faster than the ramp up time for expres- sion, then conditional expression may often miss the opportunity for capturing molecules during sporadic pulses of availability. If a particular siderophore is being produced only in rare pulses, then additional production may be favored because of the game-like competitive ad- vantage for rare types associated with rare recep- tors43,44. Such increase in production of rare types may tend to smooth out the frequency spectrum of available siderophores. The point here is not the par- ticular pattern that results from the game-like dy- namics, which may depend on many processes, but the fact that the siderophore pattern of ﬂuctuations likely has a stronger game-like quality than the corri- noid pattern of ﬂuctuations. By contrast, two aspects of siderophore biology may favor conditional expression of siderophore re- ceptors. First, siderophores are actively secreted, whereas corrinoids are passively released. Thus, ex- ternal availability of siderophore-iron complexes de- pends in part on the secretion rate. The game-like dynamics of competition shape secretion rates, which may lead to ﬂuctuating availability. The timescale of ﬂuctuations in secreted molecules is likely to be longer than the timescale for ramp up of conditional receptor expression, providing a potential beneﬁt for conditionally increased expression. How should a receptor array be tuned to the ﬂuc- tuations of availability across the diverse range of sid- erophore types? Fluctuation and the timescales of acquisition and use CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted August 22, 2017. ; https://doi.org/10.1101/144691 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted August 22, 2017. ; https://doi.org/10.1101/144691 doi: bioRxiv preprint Fluctuation and the timescales of acquisition and use Consider the processes that shape the spatiotempo- ral frequency spectrum of availability. For corrinoids, free molecules come from death of primary prokary- otic producers and release by secondary consumers. Free molecules vanish by uptake, intrinsic decay, and outﬂow. Fluctuations may follow rare low frequency pulses or common high frequency pulses or a mix- ture of diﬀerent frequencies. Receptor array design will tune to the frequency pattern of availability with respect to the intrinsic cellular timescales of acquisi- tion, use, and internal loss. A corrinoid receptor can take up diﬀerent forms with varying eﬃcacy, as summarized in the prior sec- tion. However, that conclusion followed from a single detailed study of one bacterial species and a relatively small number of corrinoid forms1. Thus, the available studies only provide a hint that siderophore receptors may be more speciﬁc than corrinoid receptors. Diﬀerences in siderophore and corrinoid biology provide clues about potential diﬀerences in speci- ﬁcity. Siderophores are secreted to bind free iron or to take up external iron from other iron-binding molecules. Siderophore receptors may be tuned to take up native siderophores secreted by the same cell or nonnative siderophores secreted by other cells. Di- versity of form may be inﬂuenced primarily by extra- cellular scavenging in diﬀerent physical and compet- itive environments42. Speciﬁcity of cellular uptake in relation to competition may favor diﬀerentiation into private types and increased diversity6 see earlier sec- tion Why are there diﬀerent types of corrinoid?. Siderophore receptor arrays face the same design issues. However, the ﬂuctuation rhythms likely diﬀer from corrinoids. Available iron inﬂows and outﬂows may arise from organic and inorganic sources. The diversity and abundance of various secreted sidero- phores mediate the competition among siderophores for chelating iron, the ways in which the physical en- vironment shapes the ﬂux of iron-siderophore com- plexes, and the competition for cellular uptake of those complexes. By contrast, corrinoids do not appear to be se- 8 git • master @ arXiv-2.0-0::8ec16e7-2017-08-21 (2017-08-22 01:10Z) • safrank . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted August 22, 2017. ; https://doi.org/10.1101/144691 doi: bioRxiv preprint . Conditional versus continuous expression of receptors That remains an open question, one that takes us again to the broader issue of how to de- sign arrays of sensors and uptake receptors for ﬂuc- tuating inputs. There has been some discussion of this topic in relation to cellular receptors45. The case of siderophores is particularly interesting because of the diversity of forms, but also is particularly com- plex because of the competitive, game-like nature of uptake and because production may be inﬂuenced by iron availability and siderophore concentrations. Second, the diversity of siderophore types ap- pears to be much wider than the diversity of corrinoid types. Thus, cells may alter expression proﬁles of re- ceptors in relation to the spectrum of available sidero- phores. Certain bacterial species appear to adjust the spectrum of their secreted siderophores in relation to speciﬁc competitors37. When such conditional ad- justments in secretions occur, associated adjustments in receptor expression seem likely. Several experimental studies of siderophore com- petition have been published recently4,36. However, I do not know of any experimental studies that have ex- plicitly focused on the frequency spectrum of ﬂuctu- ations in iron availability and physical aspects of the environment that inﬂuence uptake. It would be in- teresting to consider the various costs and beneﬁts of alternative receptor array designs under various ex- periment settings. Various physical and external aspects also inﬂu- ence the relevant timescales for available iron and sid- erophores. Diﬀusion rates and extrinsic causes of in- ﬂow and outﬂow aﬀect ﬂuctuations and availability. The rates at which cells can sense and alter receptor expression proﬁles must be considered in relation to the rates of change in availability. There are few observations about conditional ex- pression of siderophore receptors46. I presented 9 9 git • master @ arXiv-2.0-0::8ec16e7-2017-08-21 (2017-08-22 01:10Z) • safrank . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted August 22, 2017. ; https://doi.org/10.1101/144691 doi: bioRxiv preprint inverse public goods traits can be favored. these speculative comments to stimulate the collec- tion of basic data and the development of new theory. Competition and inverse public goods Are cells simply foraging for their growth require- ments? Or do cells increase receptor expression to take up excess molecules and competitively reduce the growth of their neighbors? Reducing availability for competitors is an inverse public goods problem. Siderophores are a public good4. Once secreted, they become publicly available for uptake by any cell with an appropriate receptor. Excess uptake of siderophores reduces availability of a public good. The shaping of inverse public goods uptake by natural selection has several aspects. Conditional versus continuous expression of receptors Overall, competition will depend on interactions between abundance of cellular types, genetic relat- edness between cellular types, availability of free molecules for uptake, rates of uptake by diﬀerent genotypes, costs of excess uptake, and the recycle rate of molecules taken up in excess. p y Dumas et al. 47 presented an interesting study in which Pseudomonas aeruginosa switches its sidero- phore secretion between alternative types. Severe iron limitation triggers expression of the highly ef- ﬁcient iron scavenging pyoverdine siderophore. As iron availability increases, expression switches to the less eﬃcient pyochelin siderophore. Pyoverdine is metabolically more costly to produce than pyoche- lin. Apparently, increasing iron availability causes P. aeruginosa to switch from the relatively eﬃcient and costly pyoverdine to the relatively ineﬃcient and cheap pyochelin. This study did not address condi- tional expression of receptors. However, it did show how a species may alter its siderophore expression in response to changing external conditions. Interference of iron availability occurs as a host strategy to control pathogens49. Mammals some- times increase their sequestration of iron in response to infection, reducing the available iron for invading pathogens. Thus, interference competition over iron is plausible. However, the host-pathogen situation diﬀers from interference competition between diﬀer- ent microbes. Competitive excess uptake may occur diﬀerently for siderophores and corrinoids. In siderophores, the diversity and speciﬁcity of types may be greater than for corrinoids. If so, excess competitive uptake of siderophores may beneﬁt more strongly from a greater range of expressed receptor types rather than a greater level of expression for particular receptors. By contrast, excess competitive uptake of corrinoids may be more strongly inﬂuenced by upregulating the level of expression for a few receptor types. Receptor diversity and speciﬁcity Siderophore’s game-like dynamics for external iron scavenging, receptor binding, secretion rates, and ﬂuctuating abundances complicate the chal- lenges of uptake receptor array design. Cells of- ten take up a variety of siderophores produced by other species in addition to their own native forms. The frequency spectrum for ﬂuctuations of exter- nal availability sets the key challenge, as for corri- noids. The timescales of ﬂuctuating availability must be measured against the intrinsic cellular timescales for conditionally altering the deployment of various receptors and for varying production of native sider- ophores. Each Bacteroidetes Polysaccharide Utilization Locus (PUL) comprises multiple co-regulated genes. A PUL provides functions to acquire and digest complex carbohydrates. Among Bacteroides thetaiotaomicron, B. ovatus and B. cellulosilyticus WH2, each has ap- proximately 100 PULs. The set of PULs diﬀers sig- niﬁcantly between species pairs10. B. thetaiotaomi- cron and B. ovatus devote approximately 18% of their genomes to PULs53. Speciﬁcity may be inferred in various ways. Up- regulation of particular PULs in response to certain glycans indicates speciﬁcity53,54. Binding aﬃnity of surface receptors aﬀects speciﬁcity. A PUL also en- codes several components that capture and digest gly- cans and that alter the PUL expression level. Func- tional speciﬁcity of digestion and response may de- pend on the binding speciﬁcities of the diﬀerent com- ponents. The following section discusses receptor array de- sign for glycans. Those complex carbohydrates form diverse biochemical structures. The challenges for glycan uptake diﬀer from the previous examples. For corrinoids, cells need relatively small amounts, which are not consumed internally. Diﬀerent corrinoids share basic structure, with relatively modest variety around the basic form. For siderophores, cells need a modest amount of iron. The diversity of siderophore types arises primarily through game-like competitive dynamics. One or more surface molecules initially capture external glycans. Cell surface glycoside hydrolases often bind and partially digest the glycan before transport across the outer membrane. Transporter molecules may inﬂuence rate of uptake and func- tional speciﬁcity of the PUL. Once across the outer membrane, further enzymes bind and digest the gly- can components in the periplasmic space between the outer and inner membranes. PULs typically en- code carbohydrate sensors and transcription factor regulators that control expression level. Individual PUL components may bind a variety of glycans or be highly speciﬁc. Challenges for uptake receptor array design This section brieﬂy summarizes the key challenges of uptake receptor array design for corrinoids and sider- ophores. Those challenges provide focus for the next example of glycans. First, a cell that takes up excess molecules reduces availability for genetic relatives and for competitors. If the harm to genetic relatives is less than the harm to competitors, then the trait may be favored. Corrinoid uptake presents a relatively simple challenge. A cell needs a small number of corrinoid molecules. A cell can take up its preferred type of cor- rinoid. Or it can take up a variant corrinoid, which can either be used directly or remodeled into usable form. Second, excess uptake provides beneﬁts to a genotype in relation to the abundance of that geno- type. When rare, excess uptake by a clone has lit- tle eﬀect on availability for competitors. The costs of excess uptake may be relatively insensitive to abun- dance, including the costs of maintaining internal iron homeostasis48. Thus, rare types may lose more than they gain by excess uptake. When abundant, ex- cess uptake by a clone may signiﬁcantly reduce avail- ability for competitors. If cells in a clone can take up excess molecules in a way that reduces competitor growth more than it reduces clone-mate growth, then I discussed a variety of design issues for corrinoid uptake, including the number, speciﬁcity, and condi- tional expression of receptors. I emphasized that ar- ray design likely depends on the frequency spectrum for ﬂuctuations of external availability. The availabil- ity spectrum must be considered relative to various cellular timescales. Potentially important timescales 10 git • master @ arXiv-2.0-0::8ec16e7-2017-08-21 (2017-08-22 01:10Z) • safrank . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted August 22, 2017. ; https://doi.org/10.1101/144691 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted August 22, 2017. ; https://doi.org/10.1101/144691 doi: bioRxiv preprint Glycans include decay of internal molecules and ramp up time for conditional increase of receptor expression. Some bacteria feed only on simple sugars. Others can break down the most recalcitrant glycan ﬁbers, such as rhamnogalacturonan II, a signiﬁcant cell wall com- ponent of some fruits50. I focus on the Bacteroidetes group, which includes well studied systems of glycan uptake and digestion. I limit my discussion to a few speciﬁc challenges of receptor array design, broad- ening my prior discussions of corrinoids and sidero- phores. Many excellent reviews of glycan catabolism have been published recently e.g.,10,51,52. Siderophore uptake presents a challenge of in- termediate complexity. A cell may obtain necessary iron by uptake of iron-siderophore complexes. Sider- ophores primarily function to chelate iron and bind to cellular receptors for uptake. Siderophores are more diverse and perhaps more speciﬁc with respect to up- take receptor binding than corrinoids. That greater diversity and speciﬁcity likely arises because sider- ophores are primarily designed for uptake, whereas corrinoids are primarily designed for their biochem- ical function as cofactors for complex biochemical transformations. Receptor diversity and speciﬁcity Overall, the diﬀerent steps of bind- ing, digestion, transport, and sensing combine into a By contrast with corrinoids or iron, the glycans themselves are structurally very diverse. Cells need a continuous supply of external energy which, for some species, comes primarily in the form of diverse gly- cans. The binding and initial breakdown of glycans are relatively complex challenges. The next section considers those challenges of glycan uptake in rela- tion to the previous examples. 11 git • master @ arXiv-2.0-0::8ec16e7-2017-08-21 (2017-08-22 01:10Z) • safrank . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted August 22, 2017. ; https://doi.org/10.1101/144691 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted August 22, 2017. ; https://doi.org/10.1101/144691 doi: ioRxiv preprint series of ﬁlters that enhances speciﬁcity55,56. Binding speciﬁcity of individual components is an active area of research50,57–59. provide cross-feeding beneﬁts to other species? Or is release of partially digested components simply a neutral consequence of the selﬁsh processing of com- plex food sources? Surface glycan binding proteins from diﬀerent PULs may interact. A binding protein may initially capture a glycan for which its PUL lacks matching digestive enzymes. By holding the glycan near the cell surface, the binding protein of another PUL may increase its capture rate. If that sort of interaction does occur, then the binding properties of surface molecules and the expression of receptor arrays may be aﬀected. Rakoﬀ-Nahoum et al. 62 presented an example of mutually beneﬁcial cross-feeding. Bacteroides ovatus (Bo) releases partially digested components that en- hance the growth of a partner species, B. vulgates (Bv). In turn, an increase in Bv enhances the growth of Bo. The details provide insight into design aspects of binding, uptake, and processing of food sources. Bo produces two cell surface glycoside hydrolases that partially digest inulin, a complex structure of fruc- tose polymers. Knockouts of those two surface en- zymes had either no detectable eﬀect on growth of Bo or, under certain conditions, improved growth. Receptors for partially digested components Large carbohydrates initially bind to cell surface re- ceptors. Cell surface enzymes partially digest those large molecules before transport across the outer cell membrane. Some of the partially digested compo- nents may release before transport. Bacteroides ovatus releases partially digested com- ponents of various xylans60. In one experiment, wild- type cells grew on the complexly structured corn glu- curonoarabinoxylan. Knockout of a single extracellu- lar glycoside hydrolase prevented growth. That par- ticular enzyme digests the full corn carbohydrate into relatively complex components. Culture with wild- type cells allowed the mutant to grow. Apparently, the wild-type released partially digested components that could be processed by the mutant. This study suggests that partially digested components, when re- leased, may be taken up by the same cell or by other cells of the same species. The partially digested inulin components released by Bo enhance the growth of a partner species, Bv. In co-culture, the partner Bv grew signiﬁcantly bet- ter with wild-type Bo than with a knockout that ex- presses only one of the surface enzymes. Presumably, the Bo knockout released fewer partially digested in- ulin components, decreasing the growth of the Bv partner. How does Bo itself gain a beneﬁt by releasing par- tially digested components that aid the partner Bv? In a co-culture of Bo wild-type and a mutant Bo with both surface enzymes knocked out, addition of the partner Bv enhanced growth of wild-type Bo relative to the knockout. Release of partially digested compo- nents by wild-type Bo cells enhanced growth of par- ticular Bv cells that, in turn, beneﬁtted preferentially the wild-type rather than the mutant Bo cells. Rogowski et al. 60 also studied uptake of partially digested components by another species. The rela- tively simple carbohydrates wheat arabinoxylan and birch glucuronoxylan supported growth of B. ovatus but not Biﬁdobacterium adolescentis. Co-culture of these species allowed growth of B. adolescentis, sug- gesting release of partially digested components by B. ovatus. A return evolutionary beneﬁt to Bo occurs only if the partner enhances growth of the Bo genotype that released the components26. How might such prefer- ential return beneﬁt occur? Spatial association is pos- sible. The partially digested components released by wild-type Bo may enhance growth of neighboring Bv cells. Receptor diversity and speciﬁcity When cultured by itself, Bo appears to grow best by taking up the full inulin molecule rather than the partially digested components produced by these two surface enzymes. Receptors for partially digested components Those more vigorous Bv cells may return a ben- eﬁt to their neighboring Bo cells, which include a dis- proportionate share of the cells that initially released Receptor arrays may be partly designed to take up components released by the same cell, by other cells of the same species, or by other species. Release of partially digested components provides beneﬁts to other cells, possibly of a diﬀerent species61. Is ex- tracellular binding and digestion partly designed to 12 git • master @ arXiv-2.0-0::8ec16e7-2017-08-21 (2017-08-22 01:10Z) • safrank . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted August 22, 2017. ; https://doi.org/10.1101/144691 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted August 22, 2017. ; https://doi.org/10.1101/144691 doi: ioRxiv preprint drates in various ways. For levan, a complex fruc- tose polymer, cells upregulate expression of the levan- speciﬁc PUL in response to the simple fructose compo- nents64. The levan-speciﬁc PUL includes the various functions needed to transform external levan poly- mers to simple intracellular fructose. Initially, an outer membrane surface enzyme breaks levan into large pieces. Outer membrane receptor and trans- port components move the polysaccharide pieces into the periplasmic space between the outer and inner membranes. Additional enzymes in the periplasmic space digest the polysaccharides into simple fruc- tose sugars. A surface molecule on the inner mem- brane senses free fructose in the periplasmic space and triggers upregulation of the PUL. Another surface molecule on the inner membrane transports fructose into the cell. the partially digested components. In general, mu- tually beneﬁcial cross feeding relationships provide a fascinating aspect of uptake receptor design. How- ever, it is challenging to obtain evidence of return beneﬁts directly to the genotype that initially releases partially digested components. Release of partially digested components often arises as an inevitable consequence of leakage at the cell surface by cells tuned to maximize their own self- ish growth. Rakoﬀ-Nahoum et al. 62 showed that B. thetaiotaomicron cells release partially digested com- ponents of the glycans amylopectin and levan. Receptors for partially digested components Knock- outs of the surface glycoside hydrolases for each gly- can gained when grown with wild-type, indicating mutant uptake of partially digested components re- leased by wild-type. In direct competition, wild-type outcompeted the knockouts, suggesting that extracel- lular digestion provides a direct beneﬁt to the cell. Any indirect beneﬁt to neighboring mutants is simply a consequence of leaking partially digested compo- nents that the cell fails to take up. For arabinan, a complex arabinose polymer, cells detect and integrate three distinct signals that reg- ulate expression65. First, outer membrane sur- face molecules break arabinan into oligosaccharide fragments and transport those fragments into the periplasmic space. A surface molecule on the inner membrane senses those oligosaccharides and stim- ulates expression of the arabinan utilization locus. Separately, those oligosaccharides are broken down and transported into the cell, where they become free arabinose sugars. Another study showed that B. thetaiotaomicron limits its release of partially digested components of the yeast cell wall glycan α-mannan63. Extracellular digestion makes large oligosaccharides, almost all of which are taken up by the cell. The observed variation in the leakage of partially digested components raises the problem of how cells tune their processes of digestion and uptake. In some cases, tuning emphasizes direct uptake and growth beneﬁts to the cell. In other cases, tuning may include indirect beneﬁts gained by releasing components to aid the growth of other cells. Second, an intracellular sensor molecule detects arabinose. That internal arabinose sensor is part of a distinct arabinose utilization locus. Stimulation of the internal sensor by arabinose represses expression of both the arabinan and the arabinose utilization loci. Third, a distinct internal molecule upregulates ex- pression of both the arabinan and the arabinose uti- lization loci. The signal that stimulates this third regulator is not known. It could be part of the sys- tem that regulates the preference for diﬀerent food sources when multiple carbohydrates are present. Sensors and conditional response A Bacteroidetes PUL typically expresses its compo- nents at a low level. A PUL-speciﬁc sensor detects par- ticular carbohydrates and upregulates PUL expres- sion. Cells exposed to mixtures of carbohydrates pri- oritize upregulated expression of some PULs over oth- ers. Hierarchical regulation diﬀers between species, causing distinct carbohydrate utilization patterns10. I brieﬂy describe a few examples. I then discuss the general problem of how cells perceive and respond to various environments. In summary, free fructose sugars stimulate ex- pression of the levan utilization locus, whereas free arabinose represses expression of the arabinan uti- lization locus. Cells apparently perceive and respond to distinct carbohydrates in diﬀerent ways. B. thetaiotaomicron detects diﬀerent carbohy- 13 git • master @ arXiv-2.0-0::8ec16e7-2017-08-21 (2017-08-22 01:10Z) • safrank . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted August 22, 2017. ; https://doi.org/10.1101/144691 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted August 22, 2017. ; https://doi.org/10.1101/144691 doi: oRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted August 22, 2017. ; https://doi.org/10.1101/144691 doi: bioRxiv preprint Hierarchy of preferred types the same arabinose oligosaccharide sensor of the ara- binan PUL. Bacteroidetes species upregulate speciﬁc PULs in re- sponse to particular carbohydrates. Mixtures of car- bohydrates do not stimulate all matching PULs. In- stead, cells prioritize the usage of some carbohy- drates over others53,66–70. B. thetaiotaomicron prefers a variety of dietary carbohydrates over host mucins. The dietary carbo- hydrates repress expression of the mucin PULs66,68. By contrast, B. fragilis and B. massiliensis often pre- fer mucins over the limited range of dietary carbo- hydrates that they can digest. B. massiliensis mostly expresses the opposite preferences from B. thetaio- taomicron when presented with starch paired with various mucins. These related species share a com- mon starch PUL and perhaps also components of the regulatory system that controls hierarchical expres- sion. Nevertheless, these species have evolved diﬀer- ent preferences for various carbohydrates. Schwalm et al. 71 grew B. thetaiotaomicron in a mixture of the polysaccharides chondroitin sulfate and arabinan. The presence of chondroitin sulfate repressed expression of the arabinan PUL. Cells ﬁrst consumed chondroitin sulfate, then upregulated ex- pression of the arabinan PUL and switched to using that food source. Increased expression of the arabinan PUL re- quires stimulation of a periplasmic sensor by arabi- nose oligosaccharides, as described in the prior sec- tion. The presence of chondroitin sulfate repressed phosphorylation of the periplasmic sensor by ara- binose oligosaccharides. Repressed phosphorylation prevents signal transduction and increased expres- sion of the arabinan PUL. Deep learning Classiﬁcation and response deﬁne the two primary challenges of deep learning research. Computer vi- sion and voice recognition transform inputs into a best guess for a matching object among a set of possibilities—a classiﬁcation that maps various inputs to particular outputs. Classiﬁcation may be the ﬁnal goal. Or, classiﬁcation may provide the basis for re- sponding to the environment. For example, when the system classiﬁes the visual input as an increasing de- viation from a target, then the system may adjust its future trajectory to be closer to the target. We may ask similar comparative questions about the sensing and regulation of corrinoid and sidero- phore uptake. How are receptor uptake arrays de- signed in relation to the diversity of inputs? How do ﬂuctuations in inputs inﬂuence design? How do timescales of uptake, usage, and need inﬂuence de- sign? What sorts of correlated signals provide in- formation about external availability and internal state? When does it pay to express receptors at a constant level rather than adjust in response to per- ceived changes in availability? Previous studies of up- take provide a basis for these more advanced ques- tions1,78. Recent breakthroughs in computation and ma- chine learning pervade modern life. New computa- tional classiﬁcation and response systems often out- perform humans. The new concepts and methods comprise deep learning. The learning simply means using data, or past experience, to improve stimulus- response performance. The deep qualiﬁer refers to the computational method that triggered the revolution- ary advances in performance79,80. Perception and response CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted August 22, 2017. ; https://doi.org/10.1101/144691 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted August 22, 2017. ; https://doi.org/10.1101/144691 doi: ioRxiv preprint lar growth rate and for temporally ﬂuctuating signals correlated with food availability. Those additional sensors may link into the regulatory control of car- bohydrate usage. sify inputs and how to respond in order to achieve a speciﬁed goal. In engineering, control theory an- alyzes how various input signals should be trans- formed into actions that minimize some measure of distance between system output and an optimal goal. The Appendix brieﬂy summarizes the relevant litera- tures. Bacteria vary greatly in the diversity of carbo- hydrates that each species can take up and digest. That variability provides great opportunity for com- parison. How does the sensory array scale with the breadth of carbohydrate food sources? What sorts of diﬀerent environmental signals and sensors ﬂow into the regulatory control of carbohydrate usage? How do the timescales of signal ﬂuctuations shape the design of sensors and regulatory control? Many reviews summarize current knowledge of preferential resource use in bacteria74–77. In all cases, the essential problem concerns two transformations. First, sensory input leads to a clas- siﬁcation of the environmental state. Second, the in- ferred environmental state leads to an appropriate re- sponse. Perception and response Some bacteria can digest diverse carbohydrates. To perceive the availability of various carbohydrates, many separate subsystems must be expressed at a low level. For example, B. thetaiotaomicron may not be able to detect arabinan without expressing a low level of the arabinan-speciﬁc PUL. That PUL includes the extracellular binding, digestion, transport, and sen- sor that may be needed to detect arabinan availabil- ity. Schwalm et al. 71 analyzed B. thetaiotaomicron growth on 55 pairwise combinations of 11 polysac- charides. They observed an overall tendency ﬁrst to consume polymers not composed of arabinose or fructose sugars, followed by polymers of those sug- ars. In some pairs, B. thetaiotaomicron preferred nei- ther polysaccharide over the other, consuming both polysaccharides during growth. Cells must integrate the various signals of chang- ing availability for glycans, partially digested compo- nents, or simple sugars. B. thetaiotaomicron has a large sensor array. Its various PULs encode 32 dis- tinct hybrid two-component sensors73. Those inner membrane sensors detect speciﬁc carbohydrate avail- ability in the periplasmic space and transduce a signal into the cell. B. thetaiotaomicron’s PULs also encode numerous carbohydrate sensors of the σ/anti-σ fac- tor family and some additional susR family sensors. How do cells integrate all of those inputs to classify the state of the environment? When paired with arabinan, the preferred polysaccharides chondroitin sulfate, pectic galactan, polygalacturonic acid and rhamnogalacturonan I re- pressed expression of the arabinan PUL. Presumably, depletion of the preferred polysaccharide repressed the preferred PUL and upregulated the PUL for ara- binan. Lynch and Sonnenburg 72 demonstrated the pref- erence of B. thetaiotaomicron for arabinan relative to a host mucin, a glycoprotein component of intesti- nal mucus. Stimulation of the arabinose oligosac- charide sensor of the arabinan PUL repressed expres- sion of the mucin-related PUL. Thus, B. thetaiotaomi- cron prefers various polysaccharides over arabinan and prefers arabinan over certain host mucins. Each preference associates with transcriptional repression of the nonpreferred source, mediated in this case by For cells, classiﬁcation eﬀectively means the way in which perceived information transforms regula- tory control pathways to alter the expression of var- ious subsystems. For glycans, how do particular in- puts regulate hierarchical expression of the PUL sub- systems? The inputs include the various carbohy- drate sensors. Other inputs likely play an important role. For example, cells may have sensors for cellu- 14 git • master @ arXiv-2.0-0::8ec16e7-2017-08-21 (2017-08-22 01:10Z) • safrank . Deep learning and control theory interpret or assign signiﬁcant causality to particu- lar connections. Properties such as feedback may be present in a network. But such properties may be dif- fuse aspects of network architecture rather than sim- ple aspects that can be traced between a few nodes. In deep learning networks, recurrence general- izes the notion of feedbacks. Roughly speaking, re- currence means that the state of a node depends on both current inputs and previous inputs. Feedback is one way to ﬂow an older input to a node. Recurrence broadly includes the variety of diﬀuse architectural ways in which a network can remember and combine past inputs to drive the state of various internal nodes and outputs. I illustrate the problem with the following exam- ple. Suppose gut bacteria receive inputs associated with wine consumption. Is that wine a ﬁnal evening glass, to be followed by many hours of fasting? Or is that the ﬁrst glass before a multicourse dinner that will continue over several hours? Although recurrence is a simple principle, design- ing large recurrent network architectures that per- form well is an ongoing challenge in the computa- tional study of deep learning. Some work on biologi- cal neural networks also addresses this issue. A bacterium’s capacity to distinguish and respond to those alternatives depends on the design of its reg- ulatory control network. I discuss four aspects of net- work design. Those aspects include the key design features of modern deep learning networks. The design of bacterial networks to regulate receptor arrays faces similar issues of recurrence. Surely, past inputs can be important in distinguish- ing the ﬁnal night’s glass of wine from the ﬁrst glass that starts a large meal. A bacterial network’s degree and form of recurrence likely depends on the breadth of diﬀerent carbohydrates that it can take up. To distinguish between a few alternative carbohydrate food sources, simple designs, such as direct feedback loops, often work well. But for broad receptor arrays and ﬂuctuating environments, more diﬀuse networks of the types in deep learning may perform better. Architecture. Network structure describes the pat- tern of connections between inputs and outputs. A simple feedforward architecture ﬂows unidirection- ally from inputs to internal nodes to outputs. In that case, inputs arise from sensors that detect carbohy- drates and outputs regulate expression of the match- ing receptors. Feedback loops reverse directionality. Deep learning and control theory When the availability of a particular nutrient rises, cells may increase the receptors for taking up that nu- trient. However, that simple stimulus-response notion ignores many aspects of cellular biology and the tem- poral structure of ﬂuctuating availability. I discussed those additional aspects in previous sections. But I did not oﬀer a comprehensive conceptual framework in which to analyze the problem. How can we frame the problem to highlight the essential features and provide structure for future analysis? A deep learning system is a computational net- work loosely modeled after a biological neural net- work. A set of nodes takes inputs from the environ- ment. Each input node connects to another set of nodes. Those intermediate nodes combine their in- puts to produce an output that connects to yet an- other set of nodes, and so on. The ﬁnal nodes produce an action. That action classiﬁes the environmental state of the initial inputs or takes an action based on that classiﬁcation. A deep neural network has many layers of nodes between initial inputs and ﬁnal out- puts. Theory from several diﬀerent ﬁelds relates to these issues. In evolutionary biology, phenotypic plas- ticity considers how natural selection shapes an or- ganism’s response to changes in its environment. In computer science, deep learning studies how to clas- 15 git • master @ arXiv-2.0-0::8ec16e7-2017-08-21 (2017-08-22 01:10Z) • safrank . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted August 22, 2017. ; https://doi.org/10.1101/144691 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted August 22, 2017. ; https://doi.org/10.1101/144691 doi: ioRxiv preprint Does deep learning research provide insight into the design of bacterial receptor arrays for the uptake of nutrients? That remains an open question. Several articles have linked problems of cellular perception and response to deep learning (see Appendix). So far, few speciﬁc insights have followed. However, analo- gies between engineering and biological design often provide mutually beneﬁcial insight. Such insight clar- iﬁes both the similarities and the diﬀerences between human-designed and naturally designed systems. Deep learning and control theory For example, greater receptor expression may repress the associated sensors, reducing sensitivity to the sig- nal and limiting further increase in receptor expres- sion. The goal in biology is to understand how the particular structure of the external challenge corre- sponds to the variety of network architectures. By considering the various challenges of glycan, sidero- phore, and corrinoid uptake, we gain a broader per- spective on the role of network architecture. Feedback loops and other simple network motifs have been studied extensively in biology81. Such mo- tifs can be analyzed when they occur as simple con- nections between a few nodes. However, some bacter- ial regulatory networks may have a hundred or more input sensors. For example, B. thetaiotaomicron has dozens and perhaps a hundred carbohydrate sensors. In addition, many other external sensors of the en- vironment and internal sensors of cellular state may produce inputs into the regulatory control of the gly- can uptake receptor array. This large regulatory net- work likely has many connections. Representation and classiﬁcation. A well designed network must represent the environmental inputs and system outputs in an eﬀective way. Eﬀective of- ten means discriminating between likely alternatives that matter for performance. In our wine example, the alternatives concern whether the wine will be fol- lowed by a large meal or a long nighttime fast. Sen- sors only for simple sugars may not allow an accurate representation of attributes such as wine consump- Deep learning analyzes large, highly connected networks. In such networks, it is often diﬃcult to 16 git • master @ arXiv-2.0-0::8ec16e7-2017-08-21 (2017-08-22 01:10Z) • safrank . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted August 22, 2017. ; https://doi.org/10.1101/144691 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted August 22, 2017. ; https://doi.org/10.1101/144691 doi: oRxiv preprint Often, classiﬁcation and response cannot be sepa- rated. For example, an upcoming nighttime fast may describe one attribute of the environment. Deep learning and control theory But re- sponses to that environmental classiﬁcation may vary depending on other environmental attributes, such as availability of host mucins for digestion. We can think of mucins as another attribute of classiﬁcation. However, every environmental aspect is potentially another attribute of classiﬁcation. A meaningful clas- siﬁcation gains its meaning with respect to potential alternative responses. tion, prior fasting period, and time of day. Instead, the sensors must be tuned to build a representation of the problem from meaningful parts. A representational part might be a component of wine. Another part might correlate with time of day, responding to molecules that follow a circadian rhythm. Yet another part might pick up cues about time since the last meal. A cue about a time inter- val may combine a sequence of previous inputs in a recurrent manner. Those high-level concepts may correspond to an internal network node that takes inputs from many individual sensors or from other internal nodes. For example, the individual sensors may detect various simple sugars and complex polysaccharides. The pro- ﬁle of sugars and polysaccharides may be combined together to represent particular types of consump- tion, such as wine versus nonalcoholic fruits. We may combine classiﬁcation and response in the following way. Consider the space of inputs over which the best response remains invariant. Then we can partition the environment into subsets, each with invariant responses. In practice, we want the best cost-beneﬁt tradeoﬀfor sensors that pick up the en- vironmental correlates and that allow estimation of which response-invariant partition includes the cur- rent environment. As information ﬂows from low-level sensors to deeper internal nodes, the representations may take on higher-level aspects of the alternative environment states, such as wine, time of day, and prior fasting pe- riod. At that high level point, the network has eﬀec- tively classiﬁed broad aspects of environmental state. That notion of partitioning the sample space matches the essence of ﬁnding suﬃcient statistics in statistical theory. The concept is relatively simple. Yet making a network that performs with such suﬃciency and invariance is far from trivial in practice. The dif- ﬁculty of applying the concepts in practice brings us back to architecture, representation, and classiﬁca- tion. Deep learning develops the methods that make the simple overarching principles work in application. That practical success suggests that there are simple underlying concepts that remain to be fully under- stood. Deep learning and control theory For glycans, the actual sensors, representations, and classiﬁcations will of course depend on the par- ticular organism, its environment, and the costs and beneﬁts of building a network to link inputs with outputs. The point here does not concern wine and meals, but the fact that we can think about regula- tory control with respect to sensors, representation, and classiﬁcation. These principles deﬁne many of the current challenges and fascinating progress in the study of deep learning. Discussion I highlight key processes, grouped into ﬁve topics. Ideally, each process associates with speciﬁc predic- tions and empirical tests. However, predictions and tests require a clear conceptual structure and some basic facts. The concepts and facts are not yet suﬃ- cient for many aspects. I develop a ﬁrst draft, which leaves gaps. Filling those gaps will advance the sub- ject. Those examples describe the external and inter- nal signal ﬂuctuations over time. For very simple ﬂuctuation patterns, it is relatively easy to track the changes in the time domain. In reality, actual ﬂuctu- ations usually combine many diﬀerent processes act- ing over diﬀerent periods. Rare low frequency bursts arise by occasional epidemics, changes in weather, and so on. Common high frequency ﬂuctuations arise by stochastic processes of death, local ﬂow, and so on. Fluctuation patterns combine those various frequen- cies. analysis. Control theory provides a complementary per- spective. A control system transforms environmental signals into action signals. For example, signals of environmental temperature may be transformed into signals that raise or lower heat output82,83. Fluctuating signals measured over time contain the same information as ﬂuctuating signals measured by the frequency power spectrum. But, for com- plexly ﬂuctuating signals, it is much easier to think about how systems transform signals in terms of the changes in the frequency power spectrum. Control theory emphasizes signal transformation. We can think of a receptor array as transforming sig- nals of external nutrient ﬂuctuations into signals of internal nutrient ﬂow into the cell. Similarly, we can think of nutrient sensors as transforming signals of nutrient ﬂuctuations into internal signals that control receptor deployment. These descriptions of control theory and fre- quency domain analysis are widely used in both en- gineering and systems biology. However, once the equations and technical aspects begin, it is easy to lose sight of the simple underlying qualitative princi- ples. Those simple principles can help greatly in the formation of testable predictions and in the design of experiments to test those predictions. Consider a daily ﬂuctuation of an available nutri- ent. One can think of the rise and fall of the nutrient with time, repeating the cycle in each daily period. How does a sensor transform the external signal into an internal signal? If the sensor integrates informa- tion over a daily period, then the internal signal will be constant. If the sensor integrates information over a few hours, then the internal signal rises and falls each day, but with a shifted peak and lower ampli- tude. Control theory and the frequency domain Comparatively, it is useful to think about the dif- ferent kinds of challenges that arise for glycans, sid- erophores, and corrinoids. How do those challenges lead to particular architectures, representations, and classiﬁcations? Return to the basic problem. A nutrient type comes in diﬀerent forms. Availability ﬂuctuates. How should cells design receptor arrays to capture the various nu- trient forms? When does it pay to gather information with sensors and adjust receptor deployment? When does it pay to use a small static set of receptors, with- out adjustment to changing availability? Response. Sometimes, simple classiﬁcation corre- sponds closely to the ultimate goal. For example, an environmental state that strongly correlates with an upcoming nighttime fast may favor reduced expres- sion of uptake receptors and digestive enzymes. The cell only needs to classify the environment in terms of likelihood of an upcoming fast. Deep learning focuses attention on network archi- tecture, representation, classiﬁcation, and response. That perspective separates the design problem into key components. Separation provides a clear way to parse observable patterns and to develop theory and 17 git • master @ arXiv-2.0-0::8ec16e7-2017-08-21 (2017-08-22 01:10Z) • safrank . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted August 22, 2017. ; https://doi.org/10.1101/144691 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted August 22, 2017. ; https://doi.org/10.1101/144691 doi: ioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted August 22, 2017. ; https://doi.org/10.1101/144691 doi: bioRxiv preprint system, transforms the frequency power spectrum of external signals into a spectrum of internal signals. Those internal signals are, in turn, transformed into a spectrum of action signals. Diversity of available nutrients and matching receptors Diversity arises by divergence of an ancestral type into two. What are the key process of divergence? One can think of a sensor or an uptake recep- tor as a ﬁlter. The ﬁlter passes certain types of ex- ternal ﬂuctuations through as internal signals and blocks other types of ﬂuctuations. For example, rapid high frequency ﬂuctuations may happen too quickly to change the rate of uptake by a receptor or the in- ternal signal passed through by a sensor. By contrast, low frequency changes may pass through such ﬁlters. Function. Diﬀerent corrinoid forms may catalyze dif- ferent biochemical reactions. Diﬀerent siderophore forms may bind iron diﬀerently. Do the molecular changes that alter function also inﬂuence receptor up- take? If so, then functional divergence may lead to re- ceptor array diversity, whereas functional constraints may limit receptor diversity. These examples show that, instead of thinking about ﬂuctuations as changes through time, we can think about them as happening with certain frequen- cies. Each frequency of ﬂuctuation has a correspond- ing intensity or power. A receptor array, as a control Most glycans function independently of the bac- teria that take them up. Mammalian mucins that line the gut may be an exception. If gut mucins were sim- ple and lacked diversity, then bacteria could easily 18 git • master @ arXiv-2.0-0::8ec16e7-2017-08-21 (2017-08-22 01:10Z) • safrank . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted August 22, 2017. ; https://doi.org/10.1101/144691 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted August 22, 2017. ; https://doi.org/10.1101/144691 doi: ioRxiv preprint digest through the mucosal protective layer by using just a few matching mucin utilization gene clusters. However, gut mucins are often highly complex and di- verse. That complexity and diversity prevent bacteria from attacking by use of a few simple gene clusters. ment of the function-binding correlation. Experi- mental evolution studies could select for novel func- tion and examine the correlated response in receptor binding. Diversity of available nutrients and matching receptors For escape, highly abundant species may be un- der more intense selection to vary their receptors than rare species. If so, one expects greater receptor diversity in abundant species. Experimental evolu- tion could vary attack rates for various receptors and study the patterns of receptor diversiﬁcation and as- sociated functional changes. Neutrality. Corrinoid and siderophore forms may initially change by minor alterations with little eﬀect on function. Over time, corresponding changes may arise in matching receptors and in other molecules that interact functionally with the altered forms. Gly- cans usually function independently of bacterial con- sumers. Thus, glycan diversity can be regarded as ﬁxed by external processes. Privacy in common species may drive sequential evolution of new variants. As each new private form rises in abundance, competitors may evolve to take up that form. By contrast, rare species may be more likely to maintain private variants without generat- ing as much sequential novelty. Siderophores may more easily develop private variants than corrinoids, because of the relatively large, costly and complex as- pects of corrinoid function. Escape. Toxins and viruses target common receptors for entry into cells. Rare receptor variants escape at- tack. Rare-type advantage favors diversiﬁcation of re- ceptors. Diversiﬁcation of receptors may drive diver- siﬁcation of corrinoids and siderophores. For glycans, receptor escape may favor uptake of similar glycans by variant receptors or diversiﬁcation of glycan usage between species. A greater correlation between function and re- ceptor binding should limit relative opportunities for generating private variants. Mutualistic cross feeding by private variants may arise more easily in spatially structured environments that allow partners to return beneﬁts to the producing genotype84. Privacy. Genotypes that produce and take up novel variants can escape competition. The potential bene- ﬁts of private variants diﬀer by nutrient type1,6. Privacy. Genotypes that produce and take up novel variants can escape competition. The potential bene- ﬁts of private variants diﬀer by nutrient type1,6. Corrinoid private forms, released at cell death, beneﬁt nearby genetic relatives. A novel variant may also become a private channel to send beneﬁts to mu- tualistic partners. If the private beneﬁt enhances the growth of a partner genotype or species, then the en- hanced partner growth may return a beneﬁt to the original producing genotype. Environmental aspects of resource abundance and nutrient ﬂux rates may inﬂuence diversity. Diversity of available nutrients and matching receptors For example, resource rich and iron poor environments may favor greater siderophore diversity than resource poor and iron rich environments. Number and speciﬁcity of receptor variants per cell Above, I predicted that divergence caused by neutrality or functional aspects would likely lead to less separation between variants than divergence caused by escape or privacy. Less di- vergence between nutrient variants would lead to a greater tendency for single receptors to take up mul- tiple variants. Tradeoﬀs. For a given receptor, increased uptake of a particular nutrient may reduce uptake of variant forms. Such uptake versus speciﬁcity tradeoﬀs inﬂu- ence the design of individual receptors and the design of receptor arrays. Tradeoﬀs between binding aﬃn- ity and speciﬁcity are well known in other systems, such as in antibodies and other aspects of vertebrate immunity85. Individual receptors may be tuned to low aﬃn- ity and broad speciﬁcity. That broad tuning allows a few receptors to take up a relatively wide set of vari- ants. Alternatively, each receptor in an array may be maximally tuned for high uptake rate of a particular variant. That narrow tuning requires more receptors to take up a wide set of variants. The tuning of each receptor depends on the separation between the nu- trient forms and on the tradeoﬀbetween uptake rate and speciﬁcity. If the expression of an additional re- ceptor is costly, then it may pay to locate a single re- ceptor between two separated nutrient variants. That midpoint tuning provides a low rate of uptake for both variants. Comparative genomics and assays of receptor up- take for variants may provide opportunity to test these predictions. The patterns of amino acid substi- tutions in diﬀerent lineages may contain information about the particular kinds of selection or neutrality that led to divergence. Those causes of divergence can be matched to the degree of receptor speciﬁcity. Experimental evolution studies could apply par- ticular selective pressures and analyze the evolution- ary response. For example, how do siderophores and their matching receptors evolve when the receptors change to escape viral attack? Tradeoﬀs lead to evaluation of the costs and ben- eﬁts for changes in design. Presumably, producing an additional uptake receptor must impose some cost. Cells beneﬁt by adding another receptor only when the marginal gain for that new receptor exceeds the cost. Although trivial as stated, notions of cost and beneﬁt can lead to testable hypotheses. Filtering. I have used the word ‘receptor’ for a vari- ety of distinct uptake processes. Uptake processes can often be thought of as sequential ﬁlters. Number and speciﬁcity of receptor variants per cell Siderophores are actively secreted. A private form could be taken up by the secreting cell or its genetic relatives, providing a direct return beneﬁt. Or a pri- vate variant could enhance the growth of a partner in a mutualistic cross-feeding relationship. This section discusses uptake processes that inﬂuence receptor diversity. Separation. A single receptor may be able to take up variant forms of a nutrient. The potential to take up variants depends on how diﬀerent the variants are. That separation between nutrient variants arises by the processes that cause divergence. Processes such as altered function or neutrality drive divergence for reasons that have nothing to do with receptor bind- ing. Those processes of divergence may not cause a large separation with regard to uptake, allowing a single receptor to take up more than one variant. By Glycan forms arise extrinsically. Cells may par- tially digest glycans into novel or rare components. Such private components may be taken up by genetic relatives or by cross-feeding mutualistic partners. Predictions. A few examples illustrate the potential. For function, a weaker correlation between changes in function and changes in receptor binding may lead to greater diversity. Controlled mutational changes to corrinoids and siderophores could allow measure- 19 git • master @ arXiv-2.0-0::8ec16e7-2017-08-21 (2017-08-22 01:10Z) • safrank . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted August 22, 2017. ; https://doi.org/10.1101/144691 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted August 22, 2017. ; https://doi.org/10.1101/144691 doi: oRxiv preprint then remodel those variant forms into their native form17–19. Initial binding and subsequent transport may also be sequential steps. contrast, escape and privacy drive divergence in order to separate variants with regard to receptor binding. In that case, each receptor likely takes up only one variant. Predictions. For separation, receptor speciﬁcity may be inﬂuenced by the processes that cause divergence between nutrient variants. Conditional response, ﬂuctuations and timescales Cells may alter receptor expression in response to nu- trient abundance and cellular need. Nutrient abun- dance and cellular need may ﬂuctuate. Syntrophy. A species may use nutrients released by another species. If a common species releases a high ﬂux of a particular nutrient form, then specialist re- cipients may evolve to have a narrow range of recep- tors. Fluctuations. Relative timescale often determines the consequence of ﬂuctuations. For example, loss of corrinoids probably happens by dilutive cell divi- sion and by relatively slow intrinsic decay. Thus, the timescale of need may be relatively long compared with the timescale of external ﬂuctuations in avail- ability. By contrast, certain species may require a rel- atively steady inﬂux of glycans to fuel metabolism, setting a short timescale for need. Mutualism. A recipient may return mutualistic bene- ﬁts to the genotypes that release nutrients. Such feed- back may require temporal and spatial association be- tween partners. If the nutrients passed between mu- tualist species become abundant, other species may hijack mutualistic ﬂux and destroy the relation. Rar- ity and privacy may tend to protect a mutualism. Sensors. To adjust receptor expression, cells must sense ﬂuctuating nutrient availability and internal need. A sensor may respond directly to a particu- lar nutrient. Or a sensor may respond to correlates of nutrient availability, such as time of day or rate of cellular growth and division. Predictions. Species interactions often have a game- like quality, in which the success of one species de- pends on what other species do. Game dynamics have many feedbacks that make it diﬃcult to predict the full range of outcomes. Here, an outcome concerns the receptor array diversity of each species and the relative abundance of the diﬀerent species. Although overall dynamics may be complex, one can sometimes make clearly deﬁned comparative predictions. Frequencies. Fluctuations and responses occur at various frequencies. For example, stochastic ﬂuctu- ations of nutrient availability may happen relatively frequently, whereas daily ﬂuxes may happen rela- tively infrequently. We may describe ﬂuctuations as either a rise and fall over time or as a combination of frequencies. When using frequencies, one can think of each ﬂuctuating aspect as a signal. Communities in relatively stable environments may evolve a range of specialist and generalist types86. By contrast, ﬂuctuating environments may push communities either to a diversity of specialists or to a small number of generalists. Species interactions As mutualistic pairs become more abundant, their shared nutrients become more susceptible to hijack- ing by other species that do not return beneﬁts to pro- ducers. Hijacking species may become more common with greater abundance of the mutualistic partners. Receptor array expression mediates mutualistic and hijacking relations. Competitive and cooperative interactions may inﬂu- ence the design of receptor arrays and the ﬂow of nu- trients through communities. Interactions may be be- tween diﬀerent species or diﬀerent genotypes within a species. Here, I use ‘species’ to mean interacting types. Specialization. A genotype can gain by taking up a broader range of variant forms. However, compet- ing genotypes or species that specialize on a narrow range may be better at taking up particular nutrient forms. This classic generalist versus specialist trade- oﬀshapes the receptor arrays of individuals species. Number and speciﬁcity of receptor variants per cell It is made available under The copyright holder for this preprint (which was not this version posted August 22, 2017. ; https://doi.org/10.1101/144691 doi: bioRxiv preprint narrow, highly specialized receptor arrays. manipulate nutrient richness to test ideas about how changing marginal costs and beneﬁts alter receptor array design. Mutualistic beneﬁts between partners are more likely in spatially structured habitats. Comparing habitats, well mixed environments may have less po- tential for mutualisms than structured habitats. Number and speciﬁcity of receptor variants per cell For glycans, outer membrane molecules initially bind various ex- ternal polysaccharides. Surface enzymes catalyze partial digestion of speciﬁc polysaccharides. Of those polysaccharides that are partially digested, the result- ing components are transported into the periplasmic space. Further digestion occurs, followed by trans- port of sugars and simple polysaccharides through the inner membrane. Each step ﬁlters the initial cell- surface binding process, altering the rate and speci- ﬁcity of uptake. In some cases, the initial surface di- gestion process may release partially digested compo- nents that are taken up by other receptors. For example, conditional response could lower costs by reducing expression in the absence of match- ing nutrients. Lower cost per receptor may associate with a greater number of receptors. Costs may be measured by competitive assays between strains with diﬀerent expression attributes. In experimental evo- lution, higher costs may be associated with a more rapid loss of receptors in the absence of matching nu- trients. One may also consider how high versus low nu- trient availability alters the relative costs and bene- ﬁts of particular receptors. Predictions about nutri- ent richness and receptor diversity may be tested by comparing species that live in diﬀerent kinds of habi- tats. Additionally, experimental evolution studies can Binding and uptake may be simpler for corrinoids and siderophores than for glycans. However, some multistep ﬁltering does occur in the simpler cases. For example, certain bacteria take up corrinoids that dif- fer from the particular form required by the cell and 20 git • master @ arXiv-2.0-0::8ec16e7-2017-08-21 (2017-08-22 01:10Z) • safrank . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted August 22, 2017. ; https://doi.org/10.1101/144691 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted August 22, 2017. ; https://doi.org/10.1101/144691 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. Regulatory overwiring Predictions. Conditional response becomes less likely as the timescale of internal need increases relative to the timescale of external ﬂuctuation. For example, the ratio of internal to external timescale may tend to be larger for corrinoids than for glycans, favoring greater conditional response for glycans. However, the biology of corrinoid versus glycan uptake diﬀers in many ways, which can make such comparisons dif- ﬁcult to interpret. Some Bacteroides species can take up many diﬀerent glycans. Those species use a broad array of sensors to obtain information about availability. Cells inte- grate information from those sensors to control recep- tor expression. Sensor integration and control likely ﬂow through a highly connected regulatory network. That regulatory wiring provides an excellent model to study the mechanisms by which cells integrate in- formation and respond to their environment. Comparing uptake of diﬀerent glycans by the same species may provide a stronger approach. For example, the availability of one glycan may change primarily by high frequency ﬂuctuations. By contrast, the availability of another glycan may include lower frequency ﬂuctuations, which change over longer timescales. In general, the stronger the low fre- quency components of glycan ﬂuctuation, the greater the advantage of conditional response. Thus, the re- sponse characteristics of diﬀerent receptors should be tuned to the frequency spectrum of availability for the matching nutrient. Species diﬀer in the variety of carbohydrates taken up. The design of regulatory wiring likely dif- fers with diet breadth. For siderophores, cells some- times adjust expression of uptake receptors. For cor- rinoids, little is known about conditional expression of uptake receptors. The diﬀerent nutrient types and the diﬀerent number of nutrient variants taken up by each species provide a broad basis for comparative study of regulatory wiring. To make useful comparative predictions, we need more empirical information and conceptual under- standing. On the conceptual side, I discussed analo- gies with deep learning. That subject focuses atten- tion on network architecture, representation of ex- ternal environmental state within the network, clas- siﬁcation of environmental state, and control of re- sponse. Classic control theory provides methods to op- timize the response performance of a system83,87. Those methods can be used to predict attributes of up- take receptors, such as sensor design and the transfor- mation of signals through the sequence of processes that control expression. Systems biology often uses control theory methods88,89. Uptake arrays provide an excellent model to test such predictions. Conditional response, ﬂuctuations and timescales Sensors transform external signals into internal signals. Transformation alters the frequency spec- trum of the signal. Sensor design can be described in terms of tuning a ﬁlter. For example, a sensor may Species that feed syntrophically on nutrients re- leased by common species may be more likely to have 21 git • master @ arXiv-2.0-0::8ec16e7-2017-08-21 (2017-08-22 01:10Z) • safrank . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted August 22, 2017. ; https://doi.org/10.1101/144691 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted August 22, 2017. ; https://doi.org/10.1101/144691 doi: bioRxiv preprint stimulate increased expression. block high frequency stochastic ﬂuctuations and pass through low frequency daily ﬂuctuations—a low pass ﬁlter. The entire loop of nutrient availability, sensor information, uptake rates, and conditional response can be described in terms of frequency signal trans- formations. Problems of receptor array design may then be considered as an extended aspect of signal processing. A control theory analysis of frequencies would provide insight into the correlation and mutual infor- mation between various signals. The same approach would lead to predictions about the best signals for conditional response. Acknowledgments National Science Foundation grant DEB–1251035 supports my research. Those ideas lead to a simple prediction. When there are few variant nutrient types or environmental states, network architecture may follow simple clas- sical control loops that can be easily parsed. As the number of variants or states increases, networks may become large and diﬀusely connected multilayer sys- tems. The large networks may become overwired, in the sense that they become more densely connected than would be designed by an engineer following classic control theory principles90. 1. P. H. Degnan, N. A. Barry, K. C. Mok, M. E. Taga, and A. L. Goodman, “Human gut microbes use multiple transporters to distinguish vitamin B12 analogs and compete in the gut,” Cell Host & Mi- crobe, vol. 15, pp. 47–57, 2014. 2. D. A. Rodionov, A. G. Vitreschak, A. A. Mironov, and M. S. Gelfand, “Comparative genomics of the vitamin B12 metabolism and regulation in prokaryotes,” Journal of Biological Chemistry, vol. 278, no. 42, pp. 41 148–41 159, 2003. 2. D. A. Rodionov, A. G. Vitreschak, A. A. Mironov, and M. S. Gelfand, “Comparative genomics of the vitamin B12 metabolism and regulation in prokaryotes,” Journal of Biological Chemistry, vol. 278, no. 42, pp. 41 148–41 159, 2003. Many examples of basic control loops arise in sim- ple cellular controls88,89,91. Larger eukaryotic regula- tory networks often seem densely connected and per- haps overwired. In bacteria, it would be interesting to develop a strong comparative analysis of network architecture in relation to changes in the dimension- ality and complexity of environmental challenge. For example, E. coli has a relatively simple regulatory sys- tem to control response to the availability of a small number of carbohydrate food sources92. How does the architecture of control change in species that can use an increasing number of diﬀerent carbohydrate food sources? 3. Y. Zhang, D. A. Rodionov, M. S. Gelfand, and V. N. Gladyshev, “Comparative ge- nomic analyses of nickel, cobalt and vita- min B12 utilization,” BMC Genomics, vol. 10, no. 1, p. 78, 2009. [Online]. Available: http://dx.doi.org/10.1186/1471-2164-10-78 4. S. A. West, S. P. Diggle, A. Buckling, A. Gard- ner, and A. S. Griﬃn, “The social lives of mi- crobes,” Annual Review of Ecology and Systemat- ics, vol. 38, pp. 53–77, 2007. 5. R. Chakraborty, V. Braun, K. Hantke, and P. Cor- nelis, Eds., Iron Uptake in Bacteria with Empha- sis on E. coli and Pseudomonas. Acknowledgments New York: Springer Science & Business Media, 2013. Regulatory overwiring Deep learning emphasizes the kinds of architec- ture and representation that allow networks to learn or to evolve by trial and error. How can we develop the vague analogies between deep learning and the regulatory wiring of cellular response? That remains an open problem. Ideally, we will develop strong com- parative predictions. Here is a rough example. For example, why is expression of some glycan uptake systems stimulated by availability of the sugar components, whereas other systems only respond to the full glycan or large components of it? The simple sugar fructose simulates expression of the uptake sys- tem for levan, a complex fructose polysaccharide. By contrast, free arabinose does not stimulate expression of the arabinan uptake systems. Only oligosaccharide components of arabinan arising from partial digestion When the number of inputs and alternative envi- ronmental states is small, regulatory wiring may fol- low simple control loops such as basic feedback. As the number of inputs and alternative environmental states rises, regulatory wiring may become more dif- 22 git • master @ arXiv-2.0-0::8ec16e7-2017-08-21 (2017-08-22 01:10Z) • safrank . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted August 22, 2017. ; https://doi.org/10.1101/144691 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted August 22, 2017. ; https://doi.org/10.1101/144691 doi: bioRxiv preprint design problems arise in control theory and artiﬁ- cial intelligence. The joint study of cellular design, control, and artiﬁcial intelligence delivers synergistic beneﬁts. fuse and densely connected among a large number of nodes. In densely connected networks, it is often diﬃcult to trace simple pathways of signal transmis- sion and causality. Changes in environmental state cause diﬀuse changes in the initial layers of the net- work. Processing of signals through the network may cause later network layers to encode representations for components of environmental state. Conclusions Forano, “Microbial degradation of com- plex carbohydrates in the gut,” Gut Microbes, vol. 3, no. 4, pp. 289–306, 2012. 10. J. M. Grondin, K. Tamura, G. Déjean, D. W. Abbott, and H. Brumer, “Polysaccharide Uti- lization Loci: Fuelling microbial communities,” Journal of Bacteriology, vol. 00, p. 00, 2017. 20. M. J. Gray and J. C. Escalante-Semerena, “The cobinamide amidohydrolase (cobyric acid- forming) CbiZ enzyme: a critical activity of the cobamide remodelling system of Rhodobacter sphaeroides,” Molecular Microbiology, vol. 74, no. 5, pp. 1198–1210, 2009. 11. J. R. Roth, J. G. Lawrence, and T. A. Bobik, “Cobalamin (coenzyme B12): synthesis and bi- ological signiﬁcance,” Annual Reviews in Micro- biology, vol. 50, pp. 137–181, 1996. 21. R. H. Allen and S. P. Stabler, “Identiﬁcation and quantitation of cobalamin and cobalamin ana- logues in human feces,” The American Journal of Clinical Nutrition, vol. 87, no. 5, pp. 1324– 1335, 2008. 12. R. G. Matthews, “Cobalamin-and corrinoid- dependent enzymes,” Metal Ions in Life Sciences, vol. 6, pp. 53–114, 2009. 13. S. A. Sanudo-Wilhelmy, L. Gomez-Consarnau, C. Suﬀridge, and E. A. Webb, “The role of B vitamins in marine biogeochemistry,” Annual Review of Marine Science, vol. 6, pp. 339–367, 2014. 22. C. Bradbeer, M. L. Woodrow, and L. I. Khalifah, “Transport of vitamin B12 in Escherichia coli: common receptor system for vitamin B12 and bacteriophage BF23 on the outer membrane of the cell envelope.” Journal of Bacteriology, vol. 125, no. 3, pp. 1032–1039, 1976. 14. F. Koch, M. A. Marcoval, C. Panzeca, K. W. Bru- land, S. A. Sanudo-Wilhelmy, and C. J. Gob- ler, “The eﬀect of vitamin B12 on phytoplankton growth and community structure in the Gulf of Alaska,” Limnology and Oceanography, vol. 56, no. 3, pp. 1023–1034, 2011. 23. D. R. Di Masi, J. C. White, C. A. Schnaitman, and C. Bradbeer, “Transport of vitamin B12 in Escherichia coli: common receptor sites for vita- min B12 and the E colicins on the outer mem- brane of the cell envelope,” Journal of Bacteriol- ogy, vol. 115, no. 2, pp. 506–513, 1973. 15. M. R. Droop, “Vitamins, phytoplankton and bacteria: symbiosis or scavenging?” Journal of Plankton Research, vol. 29, no. 2, pp. 107–113, 2007. 24. R. J. Kadner and P. Bassford, “Relation of cell growth and colicin tolerance to vitamin B12 up- take in Escherichia coli,” Journal of Bacteriology, vol. 129, no. 1, pp. 254–264, 1977. 16. P. H. Degnan, M. E. Taga, and A. Conclusions I emphasized ﬁve aspects of receptor arrays. Diver- siﬁcation of nutrients sets the challenge for receptor uptake. Receptor speciﬁcity delimits the component properties for the array. Species interactions focus each strain on a subset of the available nutrient di- versity. Fluctuations enhance the value of sensors and conditional adjustment of receptor deployment. Reg- ulatory wiring integrates sensor information and con- trols the response to ﬂuctuations. 6. R. Niehus, A. Picot, N. M. Oliveira, S. Mitri, and K. R. Foster, “The evolution of siderophore production as a competitive trait,” Evolution, vol. 00, pp. 00–00, 2017. 7. J. E. Loper and M. D. Henkels, “Utilization of heterologous siderophores enhances levels of iron available to Pseudomonas putida in the rhi- zosphere,” Applied and Environmental Microbi- ology, vol. 65, pp. 5357–5363, 1999. 8. E. C. Martens, N. M. Koropatkin, T. J. Smith, and J. I. Gordon, “Complex glycan catabolism by the human gut microbiota: the Bacteroidetes Corrinoids, siderophores, and glycans provide op- portunity for broad comparative study. 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It is made available under The copyright holder for this preprint (which was not this version posted August 22, 2017. ; https://doi.org/10.1101/144691 doi: bioRxiv preprint Conclusions Hantke, “Dihydroxybenzolyserine — a sider- ophore for E. coli,” FEMS Microbiology Letters, vol. 67, no. 1-2, pp. 5–8, 1990. 31. A. Bicchi and G. Canepa, “Optimal design of multivariate sensors,” Measurement Sci- ence and Technology, vol. 5, no. 4, pp. 319–322, 1994. [Online]. Available: http: //stacks.iop.org/0957-0233/5/i=4/a=001 42. R. Kümmerli, K. T. Schiessl, T. Waldvogel, K. McNeill, and M. Ackermann, “Habitat struc- ture and the evolution of diﬀusible siderophores in bacteria,” Ecology Letters, vol. 17, no. 12, pp. 1536–1544, 2014. 32. K. J. Johnson and S. L. Rose-Pehrsson, “Sensor array design for complex sensing tasks,” Annual Review of Analytical Chemistry, vol. 8, pp. 287– 310, 2015. 43. W. Lee, M. Van Baalen, and V. A. 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It is made available under The copyright holder for this preprint (which was not this version posted August 22, 2017. ; https://doi.org/10.1101/144691 doi: bioRxiv preprint 2007. . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted August 22, 2017. ; https://doi.org/10.1101/144691 doi: bioRxiv preprint Appendix This section lists key references for various topics. Phenotypic plasticity analyzes an individual’s adjust- ment of its phenotype in response to the environ- ment. In this article, I considered how cells may ad- just the expression level of diﬀerent uptake receptors in response to nutrient availability. That sort of re- sponsive phenotypic plasticity is a large subject in ecology and evolutionary biology. For example, when predators are abundant, an individual may grow pro- tective spines93. Organisms often induce various de- fenses in response to signals correlated with attack. Several books cover broad aspects of phenotypic plas- ticity and its consequences94–96. 92. K. Kochanowski, L. Gerosa, S. F. Brunner, D. Christodoulou, Y. V. Nikolaev, and U. Sauer, “Few regulatory metabolites coordinate expres- sion of central metabolic genes in Escherichia coli,” Molecular Systems Biology, vol. 13, no. 1, p. 903, 2017. 93. R. Tollrian and C. D. Harvell, Eds., The Ecology and Evolution of Inducible Defenses. Princeton University Press, 1999. 94. M. Pigliucci, Phenotypic Plasticity: Beyond Na- ture and Nurture. Baltimore, Maryland: Johns Hopkins University Press, 2001. 95. M. J. West-Eberhard, Developmental Plasticity and Evolution. New York: Oxford University Press, 2003. Control theory has played an important role in systems biology. The theory arose in engineer- ing83,87,97,98. Many studies of cellular regulatory con- trol have adopted the conceptual framework of engi- neering control88,89,91,99,100. Roughly speaking, one can think of control theory in systems biology as the theoretical and mechanistic study of cellular pheno- typic plasticity. 96. T. J. DeWitt and S. M. Scheiner, Phenotypic Plas- ticity: Functional and Conceptual Approaches. New York: Oxford University Press, 2004. 97. D. E. Davison, J. Chen, S. R. Ploen, and D. S. Bernstein, “What is your favorite book on clas- sical control? responses to an informal survey,” IEEE Control Systems, vol. 27, no. 3, pp. 89–99, Deep learning comprises recent advances in com- 28 git • master @ arXiv-2.0-0::8ec16e7-2017-08-21 (2017-08-22 01:10Z) • safrank . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted August 22, 2017. ; https://doi.org/10.1101/144691 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. Appendix It is made available under The copyright holder for this preprint (which was not this version posted August 22, 2017. ; https://doi.org/10.1101/144691 doi: ioRxiv preprint ulatory control in the deployment of cellular recep- tor arrays, deep learning provides potentially useful analogies, insights, and computational methods. puter learning systems and artiﬁcial intelligence. Those advances primarily concern techniques that greatly improve performance of computer algorithms for recognition, classiﬁcation, and response to broad classes of inputs and challenges. The speciﬁc as- pects of deep concern artiﬁcial systems loosely analo- gous to biological neural networks. In such networks, depth expresses the number of levels of the network that connect between input nodes and output nodes. Deep networks have many levels of connectedness. The advances have to do with network architecture, representation of information, training of networks, and computer algorithms79,80. For the study of reg- Cellular perception concerns cellular sensors and the use of information to regulate cellular expression and phenotype. With regard to this article, recent literature includes various analogies of cellular per- ception with aspects of artiﬁcial intelligence and deep learning. I believe these kinds of analogies may even- tually be developed in a useful way. However, the work so far has accomplished little beyond establish- ing the possible relations between disciplines101–103. 29 git • master @ arXiv-2.0-0::8ec16e7-2017-08-21 (2017-08-22 01:10Z) • safrank
https://openalex.org/W2898037283	https://europepmc.org/articles/pmc6585806?pdf=render	English	null	On the reconceptualization of Alzheimer’s disease	Bioethics	2,018	cc-by	11,759	Received: 28 December 2017 \| Revised: 25 June 2018 \| Accepted: 31 July 2018 Received: 28 December 2017 \| Revised: 25 June 2018 \| Accepted: 31 July 2018 Received: 28 December 2017 \| Revised: 25 June 2018 \| Accepted: 31 July 2018 DOI: 10.1111/bioe.12516 DOI: 10.1111/bioe.12516 K E Y W O R D S K E Y W O R D S Alzheimer’s disease, biomarkers, dementia, disease concepts, early diagnosis, pragmatism Maartje H. N. Schermer1 \| Edo Richard2 Maartje H. N. Schermer1 \| Edo Richard2 1Department of Medical Ethics and Philosophy of Medicine, University Medical Center, Rotterdam, The Netherlands 2Department of Neurology, Radboud University Medical Center, Nijmegen, The Netherlands Abstract In the hope of future treatments to prevent or slow down the disease, there is a strong movement towards an ever‐earlier detection of Alzheimer’s disease (AD). In conjunction with scientific developments, this has prompted a reconceptualization of AD, as a slowly progressive pathological process with a long asymptomatic phase. New concepts such as ‘preclinical’ and ‘prodromal’ AD have been introduced, raising a number of conceptual and ethical questions. We evaluate whether these new con‐ cepts are theoretically defensible, in light of theories of health and disease, and whether they should be understood as disease or as an at‐risk state. We introduce a pragmatic view on disease concepts and argue that an evaluation of the reconceptu‐ alization of AD should also take its aims and effects into account, and assess their ethical acceptability. The reconceptualization of AD is useful to coordinate research into preventive strategies, and may potentially benefit future patients. However, in the short term, early detection and labelling of ‘preclinical AD’ can potentially harm people. Since there is no treatment available and the predictive value is unclear, it may only create a group of ‘patients‐in‐waiting’ who may suffer from anxiety, uncer‐ tainty and stigmatization, but will never actually develop dementia. We conclude that only if the promise of preventive medication materializes, will the reconceptualiza‐ tion of AD turn out unequivocally to be for the better. Otherwise, the reconceptual‐ ization may do more harm than good. \| wileyonlinelibrary.com/journal/bioe © 2018 The Authors Bioethics Published by 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. Correspondence Correspondence Maartje H. N. Schermer, Department of Medical Ethics and Philosophy of Medicine, Erasmus MC University Medical Center, PO Box 2040, 3000 CA Rotterdam, The Netherlands. 2 \| FROM ALOIS ALZHEIMER TO CURRENT DISEASE CRITERIA Historically, the notion of AD has changed considerably since Alois Alzheimer first described the case of Auguste D who developed ‘pre‐ senile dementia’ in her early fifties, in 1907. For decades, AD was considered a rare form of dementia with an onset at relatively young age. This as opposed to the common ‘senile dementia’, which was considered to be attributable to cerebral atherosclerosis. It was not until the 1970s that neurologist Robert Katzman first suggested that most dementia cases, also in later life, should be considered as AD.4 This led to a radical change in disease concepts and a dramatic in‐ crease in interest of the scientific world in dementia. In the 1980s the proteins of which the plaques and tangles Alois Alzheimer de‐ scribed consisted were identified as amyloid‐β and tau, respectively. This further enhanced research and fueled the hope for earlier diag‐ nosis and potential treatment. This reconceptualization of AD, and the concomitant emerging use of biomarkers aiming to detect biological evidence of a presumed pathological process, raise a number of conceptual and ethical ques‐ tions. First, from a theoretical perspective it is questionable whether a state with abnormal biomarkers but without overt clinical symp‐ toms should be considered as a disease, or rather as an at‐risk state. For the persons it concerns, the distinction between risk prediction and a very early diagnosis of a much‐feared disease may not be that clear. This is further complicated by the fact that it is still contested how accurately biomarkers can predict future symptomatic disease. Hence, the exact meanings of biomarker‐based categorizations of preclinical and prodromal AD are unclear. Second, from a moral perspective, the unclear predictive abili‐ ties of current biomarker tests and the current lack of meaningful treatment and prevention options – a lack of actionability – make it questionable whether the categorization of people as having an asymptomatic early phase of AD is defensible. It could create a group In parallel, clinical concepts started shifting, and the early dis‐ ease stages, before the onset of dementia – which is defined by im‐ pairment in daily functioning – got more attention. 1 \| INTRODUCTION 1 of ‘patients in waiting’ who may suffer from anxiety and fear for the future, but may never actually develop dementia. Third, the motives for aiming at early detection are not always clear. Of course there is the intrinsic motivation of clinicians and re‐ searchers alike to stop the progression or even prevent the onset of dementia. Another driving factor is constant technological ad‐ vancement, leading to the ability to detect molecular disease mark‐ ers in body fluids as well as on neuroimaging. This technological advancement brings inevitable financial incentives, both for those who develop the biomarker tests (‘diagnostic tests’) and for those who foresee a large target population of people with an early stage of a disease for their new drugs. The persons it concerns are often driven by fear or the hope that something may be done to prevent a feared future with dementia; this may render them vulnerable to misconceptions and unrealistic expectations. In the hope of future treatments to prevent or slow down the dis‐ ease, there is a strong movement towards an ever‐earlier detection of Alzheimer’s disease (AD). It is believed that brain changes pre‐ sumed eventually to lead to dementia start to develop years to de‐ cades before clinical symptoms of dementia occur.1 It seems attractive to develop treatments that can stop or slow down these changes as early as possible, even before symptoms of cognitive im‐ pairment arise. It is now possible to detect certain biomarkers – pro‐ teins in cerebrospinal fluid (CSF) and protein depositions on neuroimaging – that are presumed to reflect the early brain changes that may eventually lead to clinical dementia, in persons with no or only mild cognitive impairment. Biomarkers are thus increasingly seen as means for early detection of the disease.2 In this article we aim to address these important conceptual and ethical questions that result from the recent reconceptualization of AD. We will discuss whether the new concepts of preclinical and prodromal AD are theoretically defensible and ethically desirable and consider their implications for medical practice. In line with these scientific developments, a reconceptualization of the notion of AD is taking place. Instead of being defined by the clinical syndrome of dementia, AD is more and more depicted as a well‐defined slowly progressive pathological process with a long asymptomatic phase. 3Sperling, R. A., Aisen, P. S., Beckett, L. A., Bennett, D. A., Craft, S., Fagan, A. M., … Phelps, C. H. (2011). Toward defining the preclinical stages of Alzheimer’s disease: recommenda‐ tions from the National Institute on Aging–Alzheimer’s Association workgroups on diag‐ nostic guidelines for Alzheimer’s disease. Alzheimer’s Dementia, 7(3), 280–292; Dubois, B., Feldman, H. H., Jacova, C., Cummings, J. L., Dekosky, S. T., Barberger‐Gateau, P., … Scheltens, P. (2010). Revising the definition of Alzheimer’s disease: A new lexicon. Lancet Neurology, 9, 1118–1127; Dubois, B., Feldman, H. H., Jacova, C., Hampel, H., Molinuevo, J. L., Blennow, K., … Cummings, J. L. (2014). Advancing research diagnostic criteria for Alzheimer’s disease: The IWG‐2 criteria. Lancet Neurology, 13, 614–629. 1 \| INTRODUCTION A new lexicon has even been proposed and new concepts and definitions have been introduced, such as ‘preclin‐ ical’ and ‘prodromal’ AD in those with no or only mild symptoms.3 While currently mainly used in research, these new concepts of pre‐ clinical and prodromal AD, as well as the use of biomarkers that de‐ fine these ‘conditions’, which were originally intended for research purposes, are now gradually entering clinical practice. 1Scheltens, P., Blennow, K., Breteler, M. M., de Strooper, B., Fisoni, G. B., Salloway, S., & Van der Flier, W. M. (2016). Alzheimer’s disease. Lancet, 388, 505–517. 4Fox, P. (1989). From senility to Alzheimer’s disease: The rise of the Alzheimer’s disease movement. The Milbank Quarterly, 67(1), 58–102 5Whitehouse, P. J. (2007). Mild cognitive impairment – A confused concept? Nature Clinical Practice. Neurology, 3(2), 62–63; Petersen, R. C. (2007). Mild cognitive impairment: current research and clinical implications. Seminars in Neurology, 27(1), 22–31. 4Fox, P. (1989). From senility to Alzheimer’s disease: The rise of the Alzheimer’s disease movement. The Milbank Quarterly, 67(1), 58–102 5Whitehouse, P. J. (2007). Mild cognitive impairment – A confused concept? Nature Clinical Practice. Neurology, 3(2), 62–63; Petersen, R. C. (2007). Mild cognitive impairment: current research and clinical implications. Seminars in Neurology, 27(1), 22–31. THE CASE insecure and lets his wife take more decisions than he used to. On formal cognitive testing he has slight memory impairment, but no impairment in other cognitive domains such as executive function‐ ing, language or visuospatial functioning. A 75‐year‐old man visits a memory clinic because of memory com‐ plaints over the past 6 months. His wife was the first to notice. He used to have a fantastic memory, but now he sometimes forgets con‐ versations they had. He also has a tendency to repeat a story he has told not so long ago. The couple live independently and have an ac‐ tive social life. The ‘patient’ plays tennis in a near‐by village, where he travels to by car or bike, depending on the weather. They have al‐ ways done the financial administration of their household together. This has not really changed, although he has become slightly This person clearly does not have dementia. In spite of some mem‐ ory impairment he has almost normal and independent daily func‐ tioning. But the question is whether he is showing signs of a very early stage of a devastating brain disease that will eventually lead to dementia. Should we aim to label this person as having a disease at this point? And can we actually predict with sufficient accuracy if and when this person will eventually develop dementia? And if so, would this person benefit from this prediction? © 2018 The Authors Bioethics Published by 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, id d h i i l k i l i d Bioethics. 2019;33:138–145. 138 SCHERMER and RICHARD 139 State Biomarker presence NIA‐AA disease definition IWG disease definition No cognitive impairment + Preclinical AD stage 1–2 Asymptomatic at risk for AD Subtle cognitive impairment + Preclinical AD stage 3 MCI + MCI due to AD Prodromal AD − MCI unlikely due to AD MCI Dementia + Probable AD dementia AD dementia − Dementia unlikely due to AD Dementia, not due to AD Note. AD = Alzheimer’s disease; MCI = mild cognitive impairment. Note. AD = Alzheimer’s disease; MCI = mild cognitive impairment. not certain that the brain changes that define the condition do inev‐ itably lead to symptoms. It is known that a considerable proportion of people dying in old age without cognitive impairment have a sub‐ stantial load of amyloid‐β and abnormal tau protein, considered to be the hallmarks of AD, in their brains.8 In contrast, these changes are not always found in those dying at a greater age with clear symptoms of dementia, which was clinically diagnosed as Alzheimer’s demen‐ tia.9 Moreover, of those with MCI, only 5–15% develop dementia per year, depending on the population under study. Those with MCI who have these ‘AD biomarkers’ in their brain, do not always develop fur‐ ther cognitive decline, and after several years a considerable propor‐ tion has not developed dementia.10 For people without cognitive impairment, data are scarce, but do suggest that abnormal biomark‐ ers are common and do not indicate imminent dementia in the ma‐ jority of people.11 the disease, shifting from a clinical diagnosis based on functioning to a biological diagnosis with or without cognitive dysfunction. From 2009 onward, this has led to the development of two new conceptual frameworks, by the U.S. National Institute on Aging and the Alzheimer’s Association (NIA‐AA) and an international working group for new research criteria for the diagnosis of AD (IWG).6 According to these frameworks, AD is defined by a pathological pro‐ cess characterized by a specific sequence of brain changes, which develop over a long period of time and may or may not become symptomatic during life. Resulting from these frameworks, a new lexicon and new criteria have been proposed to capture these chang‐ ing conceptualizations. Biomarkers, which can be detected using CSF analysis, magnetic resonance imaging (MRI) scanning and posi‐ tron emission tomography (PET) scanning, play an important role in these definitions (Table 1). The main difference between the two conceptual frameworks is that according to the NIA‐AA criteria one can have AD in the absence of any symptoms, whereas, according to the IWG, people without symptoms can only be labelled as ‘at risk for AD’. Both frameworks consider people with MCI and abnormal biomarkers as having AD, irrespective of whether their cognitive im‐ pairment is progressive and will lead to dementia. This brings us back to our main question: how should we eval‐ uate the reconceptualization of AD? Are the new disease concepts theoretically defensible? And are they ethically desirable? We will start with the first question – which will eventually lead us to the second. So over the last century AD seems to have changed from a rare presenile form of dementia, clinically defined and characterized by the presence of cognitive impairment leading to functional decline, of which a certain diagnosis could only be made post‐mortem, to a common biologically defined condition in older people with or with‐ out cognitive impairment. The current views as represented in Table 1 illustrate the conception of AD as a slowly progressive pathophysiological process that will eventually lead to symptoms that will worsen and ultimately result in full‐blown dementia. This process can presumably be detected early on (before any clinical symptoms are present) and is – also presumably – unidirectional. The often‐invoked metaphor of a cascade (as in the amyloid cascade hy‐ pothesis7) indicates necessary progression in a single direction: inev‐ itable decline. 11Wolfsgruber, S., Polcher, A., Koppara, A., Kleineidam, L., Frölich, L., Peters, O., … Wagner, M. (2017). Cerebrospinal fluid biomarkers and clinical progression in patients with subjec‐ tive cognitive decline and mild cognitive impairment. Journal of Alzheimer’s Disease, 58, 939–950. 10Heister, D., Brewer, J. B., Magda, S., Blennow, K., McEvoy, L. K.; Alzheimer’s Disease Neuroimaging Initiative. (2011). Predicting MCI outcome with clinically available MRI and CSF biomarkers. Neurology, 77, 1619–1628. 8Schneider, J. A., Arvanitakis, Z., Bang, W., & Bennett, D. A. (2007). Mixed brain patholo‐ gies account for most dementia cases in community‐dwelling older persons. Neurology, 69, 24; Savva, G. M., Wharton, S. B., Ince, P. G., Forster, G., Matthews, F. E., Brayne, C.; Medical Research Council Cognitive Function and Ageing Study. (2009). Age, neuropa‐ thology, and dementia. New England Journal of Medicine, 360, 2302–2309. 6Dubois et al. (2014), op. cit. note 3; Sperling et al., op cit. note 3. 7Jack, C. R., Knopman, D. S., Jagust, W. J., Shaw, L. M., Aisen, P. S., Weiner, M. W., … Trojanowski, J. Q. (2010). Hypothetical model of dynamic biomarkers of the Alzheimer’s pathological cascade. Lancet Neurology, 9(1), 119–128. 8Schneider, J. A., Arvanitakis, Z., Bang, W., & Bennett, D. A. (2007). Mixed brain patholo‐ gies account for most dementia cases in community‐dwelling older persons. Neurology, 69, 24; Savva, G. M., Wharton, S. B., Ince, P. G., Forster, G., Matthews, F. E., Brayne, C.; Medical Research Council Cognitive Function and Ageing Study. (2009). Age, neuropa‐ thology, and dementia. New England Journal of Medicine, 360, 2302–2309. 6Dubois et al. (2014), op. cit. note 3; Sperling et al., op cit. note 3. 2 \| FROM ALOIS ALZHEIMER TO CURRENT DISEASE CRITERIA In the late 1990s, ‘mild cognitive impairment’ (MCI) was introduced as an entity de‐ fined by cognitive impairment, but not severe enough to impair daily functioning.5 It became technically possible to assess the presence of amyloid‐β and tau protein in CSF and even using nuclear imaging of the brain. Combining early clinical detection with detecting these biomarkers has triggered a completely new way of thinking about 140 \| SCHERMER and RICHARD TA B LE 1 Simplified representation of the new nomenclature for AD as suggested by the two working groups State Biomarker presence NIA‐AA disease definition IWG disease definition No cognitive impairment + Preclinical AD stage 1–2 Asymptomatic at risk for AD Subtle cognitive impairment + Preclinical AD stage 3 MCI + MCI due to AD Prodromal AD − MCI unlikely due to AD MCI Dementia + Probable AD dementia AD dementia − Dementia unlikely due to AD Dementia, not due to AD Note. AD = Alzheimer’s disease; MCI = mild cognitive impairment. SCHERMER and RICHARD 140 general have been conceptualized in the philosophy of medicine lit‐ erature. Two well‐known and influential theories in this field are those of Christopher Boorse and Lennart Nordenfelt. There is a long‐standing debate between proponents of these two theories, mainly focusing on the question of whether health and disease are value‐free concepts. Boorse and other naturalists claim that they are, while adherents of normativist theories such as Nordenfelt’s claim they are not.12 This difference in approach becomes apparent in the fact that Boorse’s biostatistical theory (BST) considers patho‐ physiological processes to be the objective, natural essence of dis‐ ease, whereas Nordenfelt’s holistic theory of health (HTH) considers the ability to attain vital goals, and hence the impact of clinical symp‐ toms on the lives of people, to be the fundamental aspect. This is also important for the discussion about AD. Boorse’s BST defines health as statistically normal biological functioning of all parts of the organism, contributing to survival and procreation.13 He defines disease as any disturbance of health, and thus as statistically abnormal functioning of one or more parts of the organism. According to this theory, whether ‘preclinical AD’ is a disease depends on the question whether there actually is a pathological process going on in the brain.14 Boorse would say that if the nerve cells, neural networks, or brain tissues involved func‐ tion with less than typical efficiency, this implies that a disease process is present, even if there are no clinical symptoms. This is in line with the proposal of the NIA‐AA to consider the asymptom‐ atic stages of AD as disease states because, according to their view, pathophysiological changes are present in the brain, and these are seen as the essence of the disease. An interesting prob‐ lem here is that with increasing age, amyloid‐β and abnormal tau are present in an increasing number of people with increasing se‐ verity, eventually in up to over half of those over 80, irrespective of cognitive functioning.15 So, if we follow Boorse’s understanding of normal functioning as statistically normal in a specific reference group the presence of amyloid‐β and high abnormal tau levels do not constitute disease but are normal in this age group.16 However, it is not uncommon in medicine to recognize asymptom‐ atic disease – for example asymptomatic cancer, or asymptomatic renal failure – and Nordenfelt would probably agree that these are diseases because over time they do indeed tend to reduce the ability to attain vital goals. The bigger the chance that a pathological process will lead to symptoms within a short timeframe, the more reason there is to call this condition a disease, according to the HTH. With regard to asymptom‐ atic presence of AD biomarkers, however, it is as yet unclear how high the chances of developing symptoms are, while it is clear that not all biomarker‐positive individuals will develop cognitive decline. Therefore we believe that, in line with the HTH and given the present state of scientific evidence, prodromal AD should not be called a disease. In summary, at present the theoretical defensibility of preclin‐ ical AD as being an asymptomatic disease state, as proposed by the NIA‐AA and in line with Boorse’s theory of disease, is doubtful because scientific evidence is insufficient and because it is unclear whether at a certain age it may be a sign of normal aging. The con‐ ceptualization of the IWG, that understands the asymptomatic presence of biomarkers (‘preclinical AD’ according to NIA‐AA) to be a risk factor for developing symptomatic disease is more in line with Nordenfelt’s theory that sets the impact of disease symptoms on a person’s life – sometimes also referred to as ‘illness’19 – cen‐ tre stage. This usage of the term ‘disease’ as referring to symptom- atic disease is more in line with that of ordinary people and clinical practitioners, whereas the NIA terminology, like Boorse’s theory, reflects the pathologist’s and researcher’s perspective. Nordenfelt’s HTH, on the other hand defines health as being in a bodily and mental state that is such that one has the ability to realize all one’s vital goals.17 A disease is a bodily state or process that tends to reduce health, and so tends to prevent people from 18Giaccone, G., Arzberger, T., Alafuzoff, I., Al‐Sarraj, S., Budka, H., Duyckaerts, C., … Winblad, B. (2011). New lexicon and criteria for the diagnosis of Alzheimer’s disease. Lancet Neurology, 10, 298–299. 16In a recent paper, however, Boorse seems open to the idea that young adults should be taken as the reference group for all adults, which would imply that various aspects of ‘normal aging’ should be considered pathological in many cases. See: Boorse, C. (2014). A second rebuttal on health. Journal of Medicine and Philosophy, 39(6), 683–724. 19Hofmann, B. (2002). On the triad disease, illness and sickness. Journal of Medicine and Philosophy, 27(6), 651–673. 3 \| ALZHEIMER’S DISEASE AND THEORIES OF HEALTH AND DISEASE In order to consider the defensibility of the new disease concepts proposed in the AD field, we take a look at how health and disease in 9Of course one could argue that those with Alzheimer changes without dementia simply died before they had the chance to develop symptoms and those with a clinical diagnosis of Alzheimer’s dementia who did not have AD pathology were misdiagnosed during life. Still, these data, as well as the data on MCI, cast doubt on the validity of the amyloid cas‐ cade hypothesis. The current amyloid cascade model is, however, hypothetical. The biological mechanisms are still insufficiently understood and it is \| 141 realizing their vital goals. According to this view, at first glance, ‘preclinical AD’ is not a disease. Since there are no symptoms, the ability to attain vital goals is not compromised, and hence the per‐ son in question can be considered healthy. Several medical com‐ mentators have also stressed that, in the absence of symptoms, preclinical AD should not be considered a disease.18 This view is in line with the proposal of the IWG, which states we should speak of disease only when symptoms are present, i.e., in the prodromal and dementia stages. Asymptomatic presence of AD pathology, in their view, does not constitute disease but rather a risk factor for disease (‘asymptomatic at risk for AD’ as they call this ‘condition’). Even in prodromal AD, when there are only limited symptoms and it is uncertain if and when this will lead to dysfunction in daily life, it is questionable if, according to the HTH, these people should be considered as having a disease. SCHERMER and RICHARD 141 17Nordenfelt 1995, op. cit. note 12; Nordenfelt 2007, op. cit. note 12. 12Boorse, C. (1977). Health as a theoretical concept. Philosophy of Science, 44(4), 542–573; Boorse, C. (1997). A rebuttal on health. In J. M. Humber & R. F. Almeder (Eds.), What is disease? (pp. 3–133). Totowa, NJ: Humana Press; Nordenfelt, L. (1995). On the nature of health. Dordrecht, Netherlands: Kluwer Academic Publishers; Nordenfelt, L. (2007). The concepts of health and illness revisited. Medicine, Healthcare and Philosophy, 10, 5–10. 15Savva et al., op. cit. note 8; Richard, E., Schmand, B., Eikelenboom, P., Westendorp, R. G., & Van Gool, W. A. (2012). The Alzheimer myth and biomarkers research in dementia. Journal of Alzheimer’s Disease, 31(s3), S203–S209. 14As mentioned above, there is some doubt about the accuracy of the amyloid cascade hypothesis, and the question of whether biomarkers do indeed represent pathology, i.e., abnormal physiological functioning. 12Boorse, C. (1977). Health as a theoretical concept. Philosophy of Science, 44(4), 542–573; Boorse, C. (1997). A rebuttal on health. In J. M. Humber & R. F. Almeder (Eds.), What is disease? (pp. 3–133). Totowa, NJ: Humana Press; Nordenfelt, L. (1995). On the nature of health. Dordrecht, Netherlands: Kluwer Academic Publishers; Nordenfelt, L. (2007). The concepts of health and illness revisited. Medicine, Healthcare and Philosophy, 10, 5–10. 13Boorse 1977, op cit. note 12; Boorse 1997, op cit. note 12. 14As mentioned above, there is some doubt about the accuracy of the amyloid cascade hypothesis, and the question of whether biomarkers do indeed represent pathology, i.e., abnormal physiological functioning. 15Savva et al., op. cit. note 8; Richard, E., Schmand, B., Eikelenboom, P., Westendorp, R. G., & Van Gool, W. A. (2012). The Alzheimer myth and biomarkers research in dementia. Journal of Alzheimer’s Disease, 31(s3), S203–S209. 16In a recent paper, however, Boorse seems open to the idea that young adults should be taken as the reference group for all adults, which would imply that various aspects of ‘normal aging’ should be considered pathological in many cases. See: Boorse, C. (2014). A second rebuttal on health. Journal of Medicine and Philosophy, 39(6), 683–724. 17Nordenfelt 1995, op. cit. note 12; Nordenfelt 2007, op. cit. note 12. 13Boorse 1977, op cit. note 12; Boorse 1997, op cit. note 12. 30Richards and Brayne state: ‘There exists a very deep disagreement about what Alzheimer’s disease is. According to the amyloid hypothesis, it is a clinico‐pathological entity consisting of specific brain pathology (plaques and tangles) and a specific set of clinical symptoms. According to other experts, Alzheimer’s disease is a “diffuse clinical syndrome representing the gradual accumulation of multiple pathologies, arising from multiple interlocking risk factors over the life course”.’ Richards, M., & Brayne, C. (2010). What do we mean by Alzheimer’s disease? British Medical Journal, 341, 865–867. Moreover, and very im‐ portant for our present evaluation of the reconceptualization of AD, social–constructivism does not merely focus on health and disease as general concepts, but recognizes that defining and de‐ marcating disease is a human activity; it is always a matter of dis‐ cussion, negotiation, consensus‐seeking and agreement among the experts involved. The processes of redefining AD by the work‐ ing groups mentioned above poses an excellent illustration of such a process.21 Social–constructivists do not attempt to give abstract defini‐ tions of what health and disease really are, but claim that this is contingent on human decisions and agreements.22 The entities we distinguish and the demarcation lines we draw are the result of human activity, not simply given by nature. Another way to put this is to say that diseases are not ‘natural kinds’ but ‘practical kinds’, and can change over time.23 The mere fact that two differ‐ ent groups of experts come up with similar, yet not identical pro‐ posals of how to ‘carve up’ the landscape of AD, illustrates this point. Scientific work as performed in these groups consists of drawing up definitions, agreeing on classifications, and providing argumentative and empirical justification for the choices made in this process. Social–constructivism claims that this is not a matter of ‘discovering’ some underlying true structure of the world, but rather of constructing useful and justifiable concepts and entities. Interestingly, this constructive nature of the reconceptualization of AD appears to be recognized by the medical research commu‐ nity itself: an editorial commentary in the Lancet speaks of ‘organ‐ ising the language of AD’;24 another talks of preclinical and prodromal AD as ‘conceptual constructs’.25 Moreover, it has been argued that various interests, beyond the interest of the ‘patient’, e.g., of pharmaceutical companies and of researchers, are at stake in this (re‐)construction of AD.26 23Hacking, I. (1999). The social construction of what? Cambridge, MA: Harvard University Press; Zachar, P. (2002). The practical kinds model as a pragmatist theory of classification. Philosophy, Psychiatry & Psychology, 9(3), 219–227. 22This does not imply that diseases are merely constructions, or that they are arbitrary. It just claims that the natural world does not by itself determine the way in which we ought to ‘carve her up’; we make decisions about that. 21George, D. R., Qualls, S. H., Camp, C. J., & Whitehouse, P. J. (2013). Renovating Alzheimer’s: ‘Constructive’ reflections on the new clinical and research diagnostic guide‐ lines. The Gerontologist, 53(3), 378–387. 20Kingma, E. (2013). Health and disease: social constructivism as a combination of natural‐ ism and normativism. In H. Carel & R. Cooper (Eds.), Health, illness & disease. Philosophical essays (pp. 37–56). Durham, UK: Acumen. 4 \| A PRAGMATIC APPROACH TO DISEASE CONCEPTS Whether the re ceptualization of AD is defensible is therefore not merely an episte scientific question, but also a normative, moral issue. So, the quest become: what does the reconceptualization of AD aim to achieve 142 SCHERMER and RICHARD 27See for example, Zachar, op. cit. note 23; Agich, G. (1997). Toward a pragmatic theory of disease. In J. M. Humber & R. F. Almeder (Eds.), What is disease? (pp. 219–246). Totowa, NJ: Humana Press; Ross, P. (2005). Sorting out the concept of disorder. Theoretical Medicine and Bioethics, 26, 115–140. Severinsen, M. (2001). Principles behind definitions of dis‐ eases – A criticism of the principle of disease mechanism and the development of a prag‐ matic alternative. Theoretical Medicine, 22, 319–336. 28Petersen, R. C. (2013). Do preclinical Alzheimer’s disease criteria work? Lancet Neurology, 12, 935. 28Petersen, R. C. (2013). Do preclinical Alzheimer’s disease criteria work? Lancet Neurology, 12, 935. 20Kingma, E. (2013). Health and disease: social constructivism as a combination of natural‐ ism and normativism. In H. Carel & R. Cooper (Eds.), Health, illness & disease. Philosophical essays (pp. 37–56). Durham, UK: Acumen. 29Kingma, op. cit. note 20, p. 45. A shortcoming of a purely social–constructivist approach, however, is that it is merely descriptive and not normative. It gives an account of how concepts of disease and disease classification are developed. As such, it offers no criteria to decide whether a certain conceptualization is a good one, or to determine whether one conceptualization, definition, or classi‐ fication is better or worse than another. We therefore propose a prag‐ matic approach to concepts and classifications of disease.27 Philosophical pragmatism understands definitions, concepts and classifications as tools. From this perspective, it makes sense to ask what new definitions or con‐ cepts are supposed to do – what their goal or aim is – and whether they do this well.28 Moreover, it makes sense to look at the, perhaps unin‐ tended or unforeseen, effects that the introduction and usage of these tools has, and take these into account in the evaluation. As Kingma says: ‘the reason we should care about ideas, concepts and classifications is that they have effects’,29 and these effects can be good or bad, desirable or undesirable. Conceptions and classifications of diseases do not exist in a vacuum, but they influence practices, create new realities and have con‐ sequences for people’s lives. As such, they are ethically relevant and hence they can, and should, be evaluated from a moral point of view. Taking a pragmatic approach thus implies that a moral evaluation of the effects of a newly proposed concept, definition or classification, becomes an integral part of the evaluation of its defensibility. Whether the recon‐ ceptualization of AD is defensible is therefore not merely an epistemic, scientific question, but also a normative, moral issue. So, the questions become: what does the reconceptualization of AD aim to achieve and does it do so well? And what are the practical effects and consequences of this reconceptualization and are they desirable? and normativist positions, and about what constitutes the essence of health and disease. We agree, however, with an argument by Kingma20 that a social–constructivist approach to health and dis‐ ease may be more fruitful, because it can unite those traditional positions and move the debate forward. 24Schneider, L. S. (2010). Organising the language of Alzheimer’s disease in light of bio‐ markers. Lancet Neurology, 9, 1045. 4 \| A PRAGMATIC APPROACH TO DISEASE CONCEPTS Moreover, and very im‐ for our present evaluation of the reconceptualization of ial–constructivism does not merely focus on health and as general concepts, but recognizes that defining and de‐ ng disease is a human activity; it is always a matter of dis‐ negotiation, consensus‐seeking and agreement among erts involved. The processes of redefining AD by the work‐ ps mentioned above poses an excellent illustration of such ss.21 al–constructivists do not attempt to give abstract defini‐ what health and disease really are, but claim that this is ent on human decisions and agreements.22 The entities we ish and the demarcation lines we draw are the result of activity, not simply given by nature. Another way to put o say that diseases are not ‘natural kinds’ but ‘practical nd can change over time.23 The mere fact that two differ‐ ups of experts come up with similar, yet not identical pro‐ of how to ‘carve up’ the landscape of AD, illustrates this A shortcoming of a purely social–constructiv that it is merely descriptive and not normative. I concepts of disease and disease classification a offers no criteria to decide whether a certain co one, or to determine whether one conceptualiza fication is better or worse than another. We th matic approach to concepts and classifications o pragmatism understands definitions, concepts a From this perspective, it makes sense to ask wha cepts are supposed to do – what their goal or a do this well.28 Moreover, it makes sense to lo tended or unforeseen, effects that the introdu tools has, and take these into account in the eva ‘the reason we should care about ideas, conce that they have effects’,29 and these effects can or undesirable. Conceptions and classifications o a vacuum, but they influence practices, create ne sequences for people’s lives. As such, they a hence they can, and should, be evaluated from Taking a pragmatic approach thus implies that a effects of a newly proposed concept, definition o an integral part of the evaluation of its defensib SCHERMER and RICHARD rmativist positions, and about what constitutes the essence th and disease. We agree, however, with an argument by 20 that a social–constructivist approach to health and dis‐ ay be more fruitful, because it can unite those traditional ns and move the debate forward. 4 \| A PRAGMATIC APPROACH TO DISEASE CONCEPTS Moreover, and very im‐ t for our present evaluation of the reconceptualization of cial–constructivism does not merely focus on health and as general concepts, but recognizes that defining and de‐ ng disease is a human activity; it is always a matter of dis‐ , negotiation, consensus‐seeking and agreement among erts involved. The processes of redefining AD by the work‐ ups mentioned above poses an excellent illustration of such ss.21 ial–constructivists do not attempt to give abstract defini‐ f what health and disease really are, but claim that this is ent on human decisions and agreements.22 The entities we uish and the demarcation lines we draw are the result of activity, not simply given by nature. Another way to put to say that diseases are not ‘natural kinds’ but ‘practical and can change over time.23 The mere fact that two differ‐ ups of experts come up with similar, yet not identical pro‐ of how to ‘carve up’ the landscape of AD, illustrates this Scientific work as performed in these groups consists of g up definitions, agreeing on classifications, and providing ntative and empirical justification for the choices made in ocess. Social–constructivism claims that this is not a matter overing’ some underlying true structure of the world, but of constructing useful and justifiable concepts and entities. tingly this constructive nature of the reconceptualization A shortcoming of a purely social–constructivist approach, however, is that it is merely descriptive and not normative. It gives an account of how concepts of disease and disease classification are developed. As such, it offers no criteria to decide whether a certain conceptualization is a good one, or to determine whether one conceptualization, definition, or classi‐ fication is better or worse than another. We therefore propose a prag‐ matic approach to concepts and classifications of disease.27 Philosophical pragmatism understands definitions, concepts and classifications as tools. From this perspective, it makes sense to ask what new definitions or con‐ cepts are supposed to do – what their goal or aim is – and whether they do this well.28 Moreover, it makes sense to look at the, perhaps unin‐ tended or unforeseen, effects that the introduction and usage of these tools has, and take these into account in the evaluation. As Kingma says: ‘the reason we should care about ideas, concepts and classifications is that they have effects’,29 and these effects can be good or bad, desirable or undesirable. 20Kingma, E. (2013). Health and disease: social constructivism as a combination of natural‐ ism and normativism. In H. Carel & R. Cooper (Eds.), Health, illness & disease. Philosophical essays (pp. 37–56). Durham, UK: Acumen. 21George, D. R., Qualls, S. H., Camp, C. J., & Whitehouse, P. J. (2013). Renovating Alzheimer’s: ‘Constructive’ reflections on the new clinical and research diagnostic guide‐ lines. The Gerontologist, 53(3), 378–387. 4 \| A PRAGMATIC APPROACH TO DISEASE CONCEPTS It gives an acco concepts of disease and disease classification are developed. offers no criteria to decide whether a certain conceptualizatio one, or to determine whether one conceptualization, definitio fication is better or worse than another. We therefore propo matic approach to concepts and classifications of disease.27 Ph pragmatism understands definitions, concepts and classificatio From this perspective, it makes sense to ask what new definiti cepts are supposed to do – what their goal or aim is – and wh do this well.28 Moreover, it makes sense to look at the, per tended or unforeseen, effects that the introduction and usag tools has, and take these into account in the evaluation. As Ki ‘the reason we should care about ideas, concepts and class that they have effects’,29 and these effects can be good or ba or undesirable. Conceptions and classifications of diseases do a vacuum, but they influence practices, create new realities and sequences for people’s lives. As such, they are ethically re hence they can, and should, be evaluated from a moral poi Taking a pragmatic approach thus implies that a moral evalua effects of a newly proposed concept, definition or classificatio an integral part of the evaluation of its defensibility. Whether ceptualization of AD is defensible is therefore not merely an scientific question, but also a normative, moral issue. So, the become: what does the reconceptualization of AD aim to a does it do so well? And what are the practical effects and con of this reconceptualization and are they desirable? 5 \| EVALUATING THE CONCEPTS OF PRECLINICAL AND PRODROMAL ALZHEIMER’S DISEASE Evaluating the reconceptualization of AD in relation to sc idence is primarily a task for scientists, and one that is being taken on in the Alzheimer’s research literature. touched upon that discussion in Section 2, where we p that the amyloid cascade hypothesis is not unanimously among medical scientists, and different conceptualizatio 30 Kingma, E. (2013). Health and disease: social constructivism as a combination of natural‐ m and normativism. In H. Carel & R. Cooper (Eds.), Health, illness & disease. Philosophical mativist positions, and about what constitutes the essence h and disease. We agree, however, with an argument by 20 that a social–constructivist approach to health and dis‐ ay be more fruitful, because it can unite those traditional s and move the debate forward. 4 \| A PRAGMATIC APPROACH TO DISEASE CONCEPTS The processes of redefining AD by the work‐ groups mentioned above poses an excellent illustration of such ocess.21 Social–constructivists do not attempt to give abstract defini‐ s of what health and disease really are, but claim that this is tingent on human decisions and agreements 22 The entities we A shortcoming of a purely social–co that it is merely descriptive and not norm concepts of disease and disease classif offers no criteria to decide whether a ce one, or to determine whether one conc fication is better or worse than anothe matic approach to concepts and classific pragmatism understands definitions, co From this perspective, it makes sense to cepts are supposed to do – what their g do this well.28 Moreover, it makes sen tended or unforeseen, effects that the tools has, and take these into account in ‘the reason we should care about idea that they have effects’,29 and these effe or undesirable Conceptions and classifi utes the essence an argument by o health and dis‐ those traditional ver, and very im‐ ceptualization of us on health and defining and de‐ s a matter of dis‐ greement among AD by the work‐ ustration of such e abstract defini‐ claim that this is 2 The entities we are the result of other way to put ds’ but ‘practical t that two differ‐ not identical pro‐ D, illustrates this oups consists of A shortcoming of a purely social–cons that it is merely descriptive and not norma concepts of disease and disease classifica offers no criteria to decide whether a cert one, or to determine whether one concep fication is better or worse than another. matic approach to concepts and classificat pragmatism understands definitions, conc From this perspective, it makes sense to as cepts are supposed to do – what their go do this well.28 Moreover, it makes sense tended or unforeseen, effects that the in tools has, and take these into account in t ‘the reason we should care about ideas, that they have effects’,29 and these effect or undesirable. Conceptions and classifica a vacuum, but they influence practices, cre sequences for people’s lives. 4 \| A PRAGMATIC APPROACH TO DISEASE CONCEPTS As such, t hence they can, and should, be evaluate Taking a pragmatic approach thus implies effects of a newly proposed concept, defin an integral part of the evaluation of its de ceptualization of AD is defensible is ther SCHERMER and RICH constitutes the essence r, with an argument by oach to health and dis‐ unite those traditional Moreover, and very im‐ reconceptualization of y focus on health and s that defining and de‐ always a matter of dis‐ and agreement among efining AD by the work‐ lent illustration of such to give abstract defini‐ e, but claim that this is ments.22 The entities we draw are the result of e. Another way to put al kinds’ but ‘practical ere fact that two differ‐ r, yet not identical pro‐ e of AD, illustrates this ese groups consists of ications, and providing or the choices made in A shortcoming of a purely social–constructivist approach, howev that it is merely descriptive and not normative. It gives an account of concepts of disease and disease classification are developed. As su offers no criteria to decide whether a certain conceptualization is a g one, or to determine whether one conceptualization, definition, or cl fication is better or worse than another. We therefore propose a p matic approach to concepts and classifications of disease.27 Philosop pragmatism understands definitions, concepts and classifications as t From this perspective, it makes sense to ask what new definitions or cepts are supposed to do – what their goal or aim is – and whether do this well.28 Moreover, it makes sense to look at the, perhaps u tended or unforeseen, effects that the introduction and usage of t tools has, and take these into account in the evaluation. As Kingma ‘the reason we should care about ideas, concepts and classificatio that they have effects’,29 and these effects can be good or bad, desir or undesirable. Conceptions and classifications of diseases do not ex a vacuum, but they influence practices, create new realities and have sequences for people’s lives. As such, they are ethically relevant hence they can, and should, be evaluated from a moral point of v Taking a pragmatic approach thus implies that a moral evaluation o effects of a newly proposed concept, definition or classification, beco an integral part of the evaluation of its defensibility. 27See for example, Zachar, op. cit. note 23; Agich, G. (1997). Toward a pragmatic theory of disease. In J. M. Humber & R. F. Almeder (Eds.), What is disease? (pp. 219–246). Totowa, NJ: Humana Press; Ross, P. (2005). Sorting out the concept of disorder. Theoretical Medicine and Bioethics, 26, 115–140. Severinsen, M. (2001). Principles behind definitions of dis‐ eases – A criticism of the principle of disease mechanism and the development of a prag‐ matic alternative. Theoretical Medicine, 22, 319–336. 4 \| A PRAGMATIC APPROACH TO DISEASE CONCEPTS Conceptions and classifications of diseases do not exist in a vacuum, but they influence practices, create new realities and have con‐ sequences for people’s lives. As such, they are ethically relevant and hence they can, and should, be evaluated from a moral point of view. Taking a pragmatic approach thus implies that a moral evaluation of the effects of a newly proposed concept, definition or classification, becomes an integral part of the evaluation of its defensibility. Whether the recon‐ ceptualization of AD is defensible is therefore not merely an epistemic, scientific question, but also a normative, moral issue. So, the questions become: what does the reconceptualization of AD aim to achieve and does it do so well? And what are the practical effects and consequences of this reconceptualization and are they desirable? \| normativist positions, and about what constitutes the essence health and disease. We agree, however, with an argument by gma20 that a social–constructivist approach to health and dis‐ e may be more fruitful, because it can unite those traditional itions and move the debate forward. Moreover, and very im‐ tant for our present evaluation of the reconceptualization of social–constructivism does not merely focus on health and ease as general concepts, but recognizes that defining and de‐ cating disease is a human activity; it is always a matter of dis‐ sion, negotiation, consensus‐seeking and agreement among experts involved. 4 \| A PRAGMATIC APPROACH TO DISEASE CONCEPTS The debate about definitions of disease in the philosophy of med‐ icine has been focused primarily on the dispute between naturalist 17Nordenfelt 1995, op. cit. note 12; Nordenfelt 2007, op. cit. note 12. 42 \| SCHERMER an nd normativist positions, and about what constitutes the essence f health and disease. We agree, however, with an argument by ingma20 that a social–constructivist approach to health and dis‐ ase may be more fruitful, because it can unite those traditional ositions and move the debate forward. Moreover, and very im‐ ortant for our present evaluation of the reconceptualization of D, social–constructivism does not merely focus on health and sease as general concepts, but recognizes that defining and de‐ arcating disease is a human activity; it is always a matter of dis‐ ussion, negotiation, consensus‐seeking and agreement among he experts involved. The processes of redefining AD by the work‐ g groups mentioned above poses an excellent illustration of such process.21 Social–constructivists do not attempt to give abstract defini‐ ons of what health and disease really are, but claim that this is ontingent on human decisions and agreements.22 The entities we stinguish and the demarcation lines we draw are the result of uman activity, not simply given by nature. Another way to put his is to say that diseases are not ‘natural kinds’ but ‘practical nds’, and can change over time.23 The mere fact that two differ‐ nt groups of experts come up with similar, yet not identical pro‐ osals of how to ‘carve up’ the landscape of AD, illustrates this oint. Scientific work as performed in these groups consists of rawing up definitions, agreeing on classifications, and providing rgumentative and empirical justification for the choices made in his process. Social–constructivism claims that this is not a matter f ‘discovering’ some underlying true structure of the world, but ther of constructing useful and justifiable concepts and entities. terestingly, this constructive nature of the reconceptualization f AD appears to be recognized by the medical research commu‐ ty itself: an editorial commentary in the Lancet speaks of ‘organ‐ ing the language of AD’;24 another talks of preclinical and rodromal AD as ‘conceptual constructs’.25 Moreover, it has been rgued that various interests, beyond the interest of the ‘patient’, g., of pharmaceutical companies and of researchers, are at stake this (re‐)construction of AD.26 A shortcoming of a purely social–constructivist approach, that it is merely descriptive and not normative. 5.1 \| Aims and goals of the reconceptualization The first evaluative question to be asked from a pragmatic perspec‐ tive is: what are the aims of the new concepts and are these attained? From the articles published by the NIA‐AA and IWG it is apparent that the first aim of introducing the new concepts and classification is to enable and support research in this area. In order to perform research into the pathophysiological processes assumed to underlie AD, to test the amyloid hypothesis and to assess the predictive value of various biomarkers, it is necessary to use a common vocabulary and classification system. Likewise, in order to identify suitable re‐ search participants for prevention and early intervention trials, and to establish end‐points within such trials and compare trial results, a common lexicon is needed. In this respect, the newly proposed defi‐ nitions of preclinical and prodromal AD (and their further subclassifi‐ cations) are partly successful. Although there is no complete consensus, the aim of introducing a new vocabulary to support and align research in the Alzheimer field has been met to a certain extent, considering the establishment of large research programmes with accompanying funding.31 The ultimate goal is of course to enable early detection and intervention in order to prevent or modify the disease, and hence to decrease the number of people suffering from Alzheimer’s dementia. Whether this goal will be successfully at‐ tained, the future will tell. For the moment, no interventions aiming to prevent or modify clinical symptoms of dementia have been successful. A second concern is that defining these new conditions of ‘being at risk’ or having preclinical and prodromal AD – whatever the exact terms that one uses to describe them – is not necessarily in the best interest of the people involved. As Kutschenko states: ‘diagnostic strategies of clinical research, which aim to obtain well‐defined study populations (e.g., invasive screening techniques) may be scientifically useful but not necessarily serve the needs of clinical practice.’34 useful but not necessarily serve the needs of clinical practice. Apart from the burdens of invasive techniques such as lumbar puncture, the condition these new terms describe may lead to a state of uncertainty and fear for the future. 5.1 \| Aims and goals of the reconceptualization Knowing one is ‘at risk for’ or ‘in the early stages of’ AD can do psychological harm – by creating stress, anxiety, or depression – especially because there is nothing one can do to prevent further development of the disease, i.e., the acquired knowledge is not actionable. However, given the current uncertainty regarding the predictive value of biomarkers and given that a number of people with preclinical or prodromal AD, or MCI due to AD, may never develop dementia, much of this psychological stress may actu‐ ally be unnecessary. At this moment, there is insufficient evidence regarding the likelihood of such adverse psychological reactions, and it appears that persons who are voluntarily enrolled in clinical trials do understand that biomarkers confer an uncertain risk and may actively seek out that information.35 However, the interest in knowing one’s biomarkers decreases when people understand the uncertainty and limited actionability of the information.36 37Lineweaver, T. T., Bondi, M. W., Galasko, D., & Salmon, D. P. (2014). Effect of knowledge of APOE genotype on subjective and objective memory performance in healthy older adults. American Journal of Psychiatry, 171, 201–208. \| 143 being told that biomarkers have been detected that may indicate that one is at increased risk of developing dementia within 10–15 years. In dealing with research participants and patients, terminology has dif‐ ferent connotations and effects than in communication among re‐ searchers. Terms chosen should convey a truthful image of the condition of the person, and not cause confusion, unnecessary anxi‐ ety or misconceptions.32 Giving someone a diagnosis can be under‐ stood as a ‘speech act’: it turns a healthy person or research participant into a patient, which has considerable psychological and social conse‐ quences and may be harmful, particularly in the case discussed here, where the person it concerns may never develop any symptoms.33 we will focus on evaluating the aims, goals and effects – both in‐ tended and unintended – of the concepts of preclinical and prodro‐ mal AD, referring to a state in which there is no dementia, but abnormal biomarkers presumably related to AD are present. 35Bemelmans, S. A. S. A., Tromp, K., Bunnik, E. M., Milne, R. J., Badger, S., Brayne, C., … Richard, E. (2016). Psychological, behavioral and social effects of disclosing Alzheimer’s disease biomarkers to research participants: a systematic review. Alzheimer’s Research & Therapy, 8, 46; Mozersky, J., Sankar, P., Harkins, K., Hachey, S., & Karlawish, J. (2018). Comprehension of an elevated amyloid positron emission tomography biomarker result by cognitively normal older adults. JAMA Neurology, 75, 44–50. 36 Milne, R., Diaz, A., Richard, E., Badger, S., Gove, D., Georges, J., … Brayne, C. (2018). Perspectives on communicating biomarker‐based assessments of Alzheimer’s disease to cognitively healthy individuals. Journal of Alzheimer’s Disease, 62, 487–498. 143 31Ritchie, C. W., Molinuevo, J. L., Truyen, L., Satlin, A., Van der Geyten, S., Lovestone, S.; European Prevention of Alzheimer’s Dementia (EPAD) Consortium. (2016). Development of interventions for the secondary prevention of Alzheimer’s dementia: the European Prevention of Alzheimer’s Dementia (EPAD) project. Lancet Psychiatry, 3, 179–186. 32Kutschenko, L. K. (2012). Diagnostic misconceptions? A closer look at clinical research on Alzheimer’s disease. Journal of Medical Ethics, 38(1), 57–59. 32Kutschenko, L. K. (2012). Diagnostic misconceptions? A closer look at clinical research on Alzheimer’s disease. Journal of Medical Ethics, 38(1), 57–59. 31Ritchie, C. W., Molinuevo, J. L., Truyen, L., Satlin, A., Van der Geyten, S., Lovestone, S.; European Prevention of Alzheimer’s Dementia (EPAD) Consortium. (2016). Development of interventions for the secondary prevention of Alzheimer’s dementia: the European Prevention of Alzheimer’s Dementia (EPAD) project. Lancet Psychiatry, 3, 179–186. 5 \| EVALUATING THE CONCEPTS OF PRECLINICAL AND PRODROMAL ALZHEIMER’S DISEASE Evaluating the reconceptualization of AD in relation to scientific ev‐ idence is primarily a task for scientists, and one that is presently being taken on in the Alzheimer’s research literature. We have touched upon that discussion in Section 2, where we pointed out that the amyloid cascade hypothesis is not unanimously accepted among medical scientists, and different conceptualizations of AD, with a less prominent role for amyloid, exist as well.30 Here, however, 22This does not imply that diseases are merely constructions, or that they are arbitrary. It just claims that the natural world does not by itself determine the way in which we ought to ‘carve her up’; we make decisions about that. 23Hacking, I. (1999). The social construction of what? Cambridge, MA: Harvard University Press; Zachar, P. (2002). The practical kinds model as a pragmatist theory of classification. Philosophy, Psychiatry & Psychology, 9(3), 219–227. 24Schneider, L. S. (2010). Organising the language of Alzheimer’s disease in light of bio‐ markers. Lancet Neurology, 9, 1045. 25Jack et al., op. cit. note 7. 26George et al., op. cit. note 21: ‘Many of the authors of the guidelines are consultants to drug companies, and the field itself is strongly influenced by the pharmaceutical industry, whose economic interests powerfully shape and influence human comprehension of bio‐ logical processes.’ SCHERMER and RICHARD 33Ibid., 58. 5.2 \| Effects of the reconceptualization on individuals The next important evaluative question regards the effects of the reconceptualization. These can be both intended or unintended – but sometimes foreseeable – and can manifest at either the indi‐ vidual or societal level. Another worry is that people may come to have a different percep‐ tion of themselves once they know about their condition, start to worry more over normal memory lapses or even develop a nocebo reaction, i.e., show cognitive decline as a result of getting labelled.37 Friends and A first concern here is what happens when the terminology that was introduced in the context of research, and intended primarily for communication between researchers, also gets employed in the clinic and in communication with research participants, patients and their family members, as is often the case. The emotional and social effects of terms chosen to communicate with lay‐people can be consider‐ able; being told one is ‘at risk’ for developing AD is different from being told one has preclinical or asymptomatic AD – although the sit‐ uations these terms aim to describe may be exactly the same. Likewise, being told one is in the early stages of AD is different from 33Ibid., 58. 33Ibid., 58. 5.3 \| Societal effects Finally, we should consider the effects of the reconceptualization on a societal level.40 Depending on the context and the level of public awareness, a large proportion of the elderly population may eventually prove to be ‘at risk for AD’ or even to have (an early stage of) AD. Although population‐based screening programmes for those over 65 do not seem very likely at this moment, other levels of screening for cognitive impairment may become reality, such as in the context of comprehensive geriatric assessments, which are increasingly popular, or opportunistic screening in those who are presumed to be at risk for cognitive impairment due to clinical or demographic characteristics. For the short term, with the current state of affairs, the answer is likely to be ‘no’. Detecting and labelling an uncertain condition of being at risk – although we do not exactly know how big a risk – for developing AD dementia somewhere in the future does little bene‐ fit, as long as the predictive value is unclear and there are no effec‐ tive preventive measures available. The reconceptualization is taking out a mortgage on the future success of a specific research agenda, but it may do so at the expense of current research participants, patients and older people in general. This is not necessarily unethical but we should at least be aware of it, weigh the pros and cons and minimize the negative effects. One way to do this is to be very careful with the vocabulary used to address research participants. As Alzheimer Europe states in a recent report: ‘Careful attention should be paid by researchers to the terminology surrounding what is currently defined as pre‐clinical AD and to its possible impact on research participants and the general public.’43 They recommend, for example, that research participants falling in the preclinical AD category, should be described as ‘being at risk of AD’ rather than as ‘having preclinical AD’, and that researchers should speak of ‘disclosure of risk status’, rather than of ‘diagnosis’.44 Labelling large numbers of people as such will inevitably raise questions about medicalization of aging and age‐related memory problems, since a growing number of people will be diagnosed with AD without clinical dementia or MCI being present; and without being sure whether they would ever develop it. The number of ‘pa‐ tients‐in‐waiting’, as they have been called,41 would increase enor‐ mously. 39Bunnik, E. M., Richard, E., Milne, R., & Schermer, M. H. N. (2018). On the personal utility of Alzheimer’s disease‐related biomarker testing in the research context. Journal of Medical Ethics. https://doi.org/10.1136/medethics-2018-104772 40Karlawish, J. (2011). Addressing the ethical, policy, and social challenges of preclinical Alzheimer disease. Neurology, 77, 1487–1493; Boenink, M., Cuijpers, Y., van der Laan, A. L., van Lente, H., & Moors, E. (2011). Assessing the sociocultural impacts of emerging molec‐ ular technologies for the early diagnosis of Alzheimer’s disease. International Journal of Alzheimer’s Disease, Article ID 184298; Schicktanz, S., Schweda, M., Ballenger, J. F., Fox, P. J., Halpern, J., Kramer, J. H., … Jagust, W. J. (2014). Before it is too late: professional re‐ sponsibilities in late‐onset Alzheimer’s research and pre‐symptomatic prediction. Frontiers in Human Neuroscience, 20, 921. 38For example, Vanderschaeghe, G., Schaeverbeke, J., Vandenberghe, R., & Dierickx, K. (2017). Amnestic MCI patients’ perspectives toward disclosure of amyloid PET results in a research context. Neuroethics, 10, 281–297; Holt, G. R. (2011). Timely diagnosis and dis‐ closure of Alzheimer disease gives patients opportunities to make choices. Southern Medical Journal, 104, 779–780. 42Le Couteur, D. G., Doust, J., Creasey, H., & Brayne, C. (2013). Political drive to screen for pre‐dementia: not evidence based and ignores the harms of diagnosis. British Medical Journal, 347(9), f5125. 43Alzheimer Europe. (2016). Discussion paper on ethical issues linked to the changing defi‐ nitions/use of terms related to Alzheimer’s disease. Retrieved from https://www.alzhei‐ mer-europe.org/Publications/Alzheimer-Europe-Reports 44Ibid. 41Timmermans, S., & Buchbinder, M. (2010). Patients‐in‐waiting: Living between sickness and health in the genomics era. Journal of Health and Social Behavior, 51, 408. family may also change their attitude or behaviour towards those per‐ sons and this may negatively interfere with their relationships. course pose new questions. Who should use this medication and how could we prevent over‐medication? How to make sure interests of patients and not only those of the pharmaceutical industry are prioritized? Who should pay for medication, how can equal access be assured, and how much priority should AD prevention get? Would it, at some point, be a good idea to initiate screening programmes and, if yes, in which population?42 On the other hand, it has been argued that people do have a right to know their risks, if they want, and that this would enable one to plan and prepare for the future – financially, mentally, with regard to life plans or lifestyle. Even though a risk prediction is not ‘actionable’ in the sense of available preventive or treatment options, it is often claimed it can still be used for such non‐medical purposes.38 However, as we have argued elsewhere in more detail, as long as the prognostic value of the diagnosis ‘preclinical AD’ is so unclear and hence risk pre‐ diction is inaccurate, this argument has a limited validity.39 5.4 \| Taking stock So, from an individual as well as societal perspective, it is question‐ able whether the reconceptualization of AD is desirable. Is it a good idea to distinguish, to name and to detect a condition of ‘being at risk for developing AD dementia’ in the first place? The answer is that it might be, in the long run, if the hypothesis on which current research efforts are based proves to be right, and if effective, safe and affordable preventive medication is developed. However, this is by no means a certainty. At present, there is insufficient knowledge about the actual fre‐ quency of these potential harms and burdens and about their sever‐ ity. Little is known about the psychological impact of learning one’s risk status, or learning that one has ‘preclinical’ or ‘prodromal’ AD. 42Le Couteur, D. G., Doust, J., Creasey, H., & Brayne, C. (2013). Political drive to screen for pre‐dementia: not evidence based and ignores the harms of diagnosis. British Medical Journal, 347(9), f5125. 34Ibid. 144 SCHERMER and RICHARD 43Alzheimer Europe. (2016). Discussion paper on ethical issues linked to the changing defi‐ nitions/use of terms related to Alzheimer’s disease. Retrieved from https://www.alzhei‐ mer-europe.org/Publications/Alzheimer-Europe-Reports 44Ibid The authors declare no conflict of interest. The authors declare no conflict of interest. While the reconceptualization of AD, and especially the introduction of the notion of preclinical or asymptomatic AD, might seem attrac‐ tive for research into preventive strategies, and may have the poten‐ tial to benefit future patients, it will not benefit individuals in the short term. It may lead to diagnosing a pre‐symptomatic condition that in a considerable proportion of cases will never become symptomatic. This can be harmful for individuals and their caregivers. It is impor‐ tant to consider the possible harmful effects of creating these new, uncertain and unclear conditions of pre‐dementia AD in evaluating the defensibility of the proposed reconceptualization. A fundamental shortcoming in the current scientific AD debate is that illness is over‐ looked and that the disease is being oversimplified by characterizing it as the presence of biological abnormalities which in themselves cor‐ relate poorly with the clinical symptoms of cognitive impairment. entities. The AD research community should take responsibility to pre‐ vent terms like ‘preclinical AD’, ‘asymptomatic AD’ or ‘at risk for AD’ from prematurely entering the consulting room and the public domain.45 reconceptualization of AD turn out to be unequivocally for the better. However, if the attempts to develop such medication continue to fail, the reconceptualization may do more harm than good. ACKNOWLEDGEMENTS This work is part of the research programme ‘Early diagnosis of Alzheimer’s disease – conceptual and ethical issues’ with pro‐ ject number 731010012, which is financed by the Netherlands Organisation for Health Research and Development (ZonMW). 46A telling sign is the way in which some publications simply replace the term ‘research criteria’ with ‘clinical criteria’, thereby completely bypassing the proclaimed intentions of the working groups as well as the differences between research and clinical practice. See for example: Morris, J. C., Blennow, K., Froelich, L., Nordberg, A., Soininen, H., Waldemar, G., … Dubois, B. (2014). Harmonized diagnostic criteria for Alzheimer’s disease: Recommendations. Journal of Internal Medicine, 275, 204–213. In another consensus paper, several references to the adoption in clinical practice are made, such as ‘will be available in the near future for people diagnosed with prodromal dementia’ and ‘health‐ care systems will need to identify and engage with prodromal and preclinical populations’. Ritchie C. W., Russ, T. C., Banerjee, S., Barber, B., Boaden, A., Fox, N. C., … Burns, A. (2017). The Edinburgh Consensus: Preparing for the advent of disease‐modifying therapies for Alzheimer’s disease. Alzheimer’s Research and Therapy, 1, 85. 45The IWG actually agrees with this: ‘because of the uncertainty [about how big the risk for developing AD dementia is] and for ethical reasons, we emphasise that reference to preclinical AD should be avoided. At the diagnostic or clinical level, any (clinically asymp‐ tomatic) individual should be described as being “at risk for AD” … but should not be de‐ fined as having preclinical AD.’ Dubois et al. (2010), op. cit. note 3, p. 1122. 5.3 \| Societal effects This might also reinforce prejudice about elderly people, stigmatize them and increase age discrimination. If medication becomes available in the longer run, depending on its effectiveness and risk‐ and side‐effect profiles, this would of Moreover, we believe clinicians who are also involved in research should be acutely aware of these issues and should take utmost care not to conflate research language with clinical language when speaking to patients. We should consider the reconceptualization and the proposed terminology and classification as research tools, not as referring to clinical SCHERMER and RICHARD 145 45The IWG actually agrees with this: ‘because of the uncertainty [about how big the risk for developing AD dementia is] and for ethical reasons, we emphasise that reference to preclinical AD should be avoided. At the diagnostic or clinical level, any (clinically asymp‐ tomatic) individual should be described as being “at risk for AD” … but should not be de‐ fined as having preclinical AD.’ Dubois et al. (2010), op. cit. note 3, p. 1122. 46A telling sign is the way in which some publications simply replace the term ‘research criteria’ with ‘clinical criteria’, thereby completely bypassing the proclaimed intentions of the working groups as well as the differences between research and clinical practice. See for example: Morris, J. C., Blennow, K., Froelich, L., Nordberg, A., Soininen, H., Waldemar, G., … Dubois, B. (2014). Harmonized diagnostic criteria for Alzheimer’s disease: Recommendations. Journal of Internal Medicine, 275, 204–213. In another consensus paper, several references to the adoption in clinical practice are made, such as ‘will be available in the near future for people diagnosed with prodromal dementia’ and ‘health‐ care systems will need to identify and engage with prodromal and preclinical populations’. Ritchie C. W., Russ, T. C., Banerjee, S., Barber, B., Boaden, A., Fox, N. C., … Burns, A. (2017). The Edinburgh Consensus: Preparing for the advent of disease‐modifying therapies for Alzheimer’s disease. Alzheimer’s Research and Therapy, 1, 85. Maartje H. N. Schermer http://orcid.org/0000-0003-4283-9659 Edo Richard http://orcid.org/0000-0002-7250-3390 Maartje H. N. Schermer http://orcid.org/0000-0003-4283-9659 Edo Richard http://orcid.org/0000-0002-7250-3390 Maartje H. N. Schermer http://orcid.org/0000-0003-4283-9659 Edo Richard http://orcid.org/0000-0002-7250-3390 Maartje H. N. Schermer is Civis Mundi Professor of Philosophy of Medicine at Erasmus MC, Rotterdam University Medical Center. Some of her research interests are in the ethics and philosophy of preventive medicine, aging and concepts of health and disease. We conclude that the reconceptualization of AD is legitimate and meaningful for usage within a narrowly defined research community studying a clearly defined biological condition, namely the presence of specific measurable biomarkers, but that translation to clinical practice poses various ethical and communication problems. It is too early to move those concepts from research into the clinical setting, since they are based on a disputed hypothesis and since attempts to do so may actually be harmful to the people concerned. The distinc‐ tion between research and clinical practice may be difficult to main‐ tain, however, and it appears as if the use of biomarkers is slowly creeping into clinical practice, without proper scientific underpinning.46 Edo Richard is a neurologist at Radboud University Medical Centre, Nijmegen, the Netherlands. He combines patient care with re‐ search on cognitive disorders and dementia. His main research in‐ tererests are in dementia prevention research using large investiga‐ tor‐initiated clinical trials. He has a specific interest in the ethics of research into dementia and the impact of biomarker development on clinical practice in everyday memory clinics. Whether it is a good idea to move toward ever‐earlier diagnosis of AD, or of detecting at‐risk states for AD dementia, is a complex ques‐ tion. A good predictive value and actionability are important precondi‐ tions for the ethical implementation of predictive testing. With regard to the first condition, biomarker tests for AD currently fall short, while, for the time being, the second is limited to ‘preparing for one’s future’. Only if the promise of preventive medication materializes, will the How to cite this article: Schermer MHN, Richard E. On the reconceptualization of Alzheimer’s disease. Bioethics. 2019;33:138–145. https://doi.org/10.1111/bioe.12516
https://openalex.org/W2069077889	https://europepmc.org/articles/pmc4359867?pdf=render	English	null	Hybrid Tumor of the Parotid Gland: A Case Report and Review of the Literature	Case reports in otolaryngology	2,015	cc-by	2,945	Correspondence should be addressed to Elie Alam; elie.elalam@gmail.com Correspondence should be addressed to Elie Alam; elie.elalam@gmail.com Received 23 September 2014; Revised 29 December 2014; Accepted 8 January 2015 Academic Editor: Jeffrey P. Pearson Copyright © 2015 Alain Sabri et al. 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. The parotid gland is the most common location of benign neoplasms affecting major salivary glands. Hybrid tumors are very rare tumor entities which are composed of two different tumor types, each of which conforms to an exactly defined tumor category. The tumor entities of a hybrid tumor are not separated but have an identical origin within the same topographical area. This report describes a 51-year-old male with three neoplasms occurring within a single parotid gland tumor. The clinical, radiological, and histologic features are described in addition to a review of the literature. Hindawi Publishing Corporation Case Reports in Otolaryngology Volume 2015, Article ID 192453, 5 pages http://dx.doi.org/10.1155/2015/192453 Hindawi Publishing Corporation Case Reports in Otolaryngology Volume 2015, Article ID 192453, 5 pages http://dx.doi.org/10.1155/2015/192453 Hindawi Publishing Corporation Case Reports in Otolaryngology Volume 2015, Article ID 192453, 5 pages http://dx.doi.org/10.1155/2015/192453 Alain Sabri,1 Ibrahim Bawab,2 Ibrahim Khalifeh,3 and Elie Alam2 1Chair, Otolaryngology Head and Neck Surgery Cleveland Clinic Abu Dhabi, P.O. Box 112412, Abu Dhabi, UAE 2Department of Otolaryngology Head and Neck Surgery, American University of Beirut Medical Center, P.O. Box 11-0236, Riad El Solh, Beirut 1107-2020, Lebanon 3Department of Pathology and Laboratory Medicine, American University of Beirut Medical Center, P.O. Box 11-0236, Riad El Solh, Beirut 1107-2020, Lebanon 1Chair, Otolaryngology Head and Neck Surgery Cleveland Clinic Abu Dhabi, P.O. Box 112412, Abu Dhabi, UAE 2Department of Otolaryngology Head and Neck Surgery, American University of Beirut Medical Center, P.O. Box 11-0236, Riad El Solh, Beirut 1107-2020, Lebanon 3Department of Pathology and Laboratory Medicine, American University of Beirut Medical Center, P.O. Box 11-0236, Ri d El S lh B i t 1107 2020 L b 1Chair, Otolaryngology Head and Neck Surgery Cleveland Clinic Abu Dhabi, P.O. Box 112412, Abu Dhabi, UAE 2Department of Otolaryngology Head and Neck Surgery, American University of Beirut Medical Center, P.O. Box Riad El Solh, Beirut 1107-2020, Lebanon 3Department of Pathology and Laboratory Medicine, American University of Beirut Medical Center, P.O. Box 11-0 Riad El Solh, Beirut 1107-2020, Lebanon 2. Case Report A 51-year-old male patient, smoker, known to have diabetes insipidus type II, presented with a large deforming right hemifacial mass growing progressively over a period of 10 years, measuring 11 × 14 × 6 cm extending from the level of the right temporalis muscle above the zygomatic arch superiorly, down to the level of the mandible inferiorly, medially extending to the right lateral epicanthal fold, and laterally abutting the right external auditory meatus. The mass was firm, fixed but not tender, rubbery consistent with no overlying skin changes. Weakness was noted over the right eyebrow and right lower lip. No trismus or respiratory distress was present. Oral exam was unremarkable and a fiberoptic flexible laryngoscopy showed no narrowing of the nasopharyngeal, oropharyngeal, or hypopharyngeal walls. Narrowing of the right external auditory canal was evident on otoscopic exam. To note that the patient underwent a previous surgery in an outside hospital in attempt of removing the tumor bulk but the resection was minimal and a small portion of the tumor was removed, no pathological studies were taken at the time. The parotid gland is the most usual location of benign neo- plasms affecting major salivary glands and quite often the recurrence of these tumors is noticed, especially in the case of pleomorphic adenoma. When synchronous tumors of the parotid gland are encountered, the most common histology is that of multiple Warthin’s tumors [1]. Multifocal primary tumor (MPT) in the parotid gland is a rare phenomenon [2, 3]; when it occurs, the most common combination is Warthin tumor and a pleomorphic adenoma [4, 5]. Hybrid carcinomas of the salivary gland are a recently defined and rare tumor entity, consisting of two histologically distinct types of carcinoma within the same topographic location [6]. Hybrid tumors must also be distinguished from the mul- tiple occurrences of salivary gland tumors which can develop syn- or metachronously. To our knowledge, no cases in the literature mentioned the occurrence of a hybrid tumor consisting of three different carcinomas in a single parotid gland. 2 Case Reports in Otolaryngology (a) (b) (c) (d) (e) (f) (g) (h) (i) Figure 1: (a) Tumor involving the right cheek area extending from the temporal area to the mandibular area (frontal view). (b) Semilateral view. (c) Lateral view. (d) CT scan: axial cut of the upper neck with IV contrast showing narrowing of the external auditory canal by the tumor. 2.2. Postoperatively. Patient was scheduled for postoperative radiation. 2. Case Report The central epithelial cells are smaller and have eosinophilic cytoplasm, round to oval nuclei, and a high nuclear to cytoplasmic ratio. (b) Focally, the epithelial-myoepithelial carcinoma component exhibits myxochondroid rich background with anastomosing stands of epithelial cells reminiscent of pleomorphic adenoma morphologically. (c) Basal cell adenocarcinoma: prominent eosinophilic hyaline material is seen around nodules of tumor composed of small cells with more chromatic nuclei around larger cells with paler nuclei. (d) Adenoid cystic carcinoma: tubular and cribriform and solid tumor nests. Basal lamina-containing cyst-like spaces are noted. The tumor cells show high grade cytology with markedly increased nuclear to cytoplasmic ratio. (e) Nests of tumor invading the peripheral portion of the nerve bundle in the areas dominated by adenoid cystic carcinoma. (f) The proliferative index by Ki-67 stain labeling showed significantly higher labeling index in the adenoid cystic component (lower left corner) in comparison to the epithelial-myoepithelial component (upper right corner). (c) (b) (a) (a) (c) (b) (f) (d) (e) (d) (e) (f) Figure 2: (a) Epithelial-myoepithelial carcinoma. The ductal structures are composed of outer layer of polygonal, clear myoepithelial cells with variable sized and shaped nuclei. The central epithelial cells are smaller and have eosinophilic cytoplasm, round to oval nuclei, and a high nuclear to cytoplasmic ratio. (b) Focally, the epithelial-myoepithelial carcinoma component exhibits myxochondroid rich background with anastomosing stands of epithelial cells reminiscent of pleomorphic adenoma morphologically. (c) Basal cell adenocarcinoma: prominent eosinophilic hyaline material is seen around nodules of tumor composed of small cells with more chromatic nuclei around larger cells with paler nuclei. (d) Adenoid cystic carcinoma: tubular and cribriform and solid tumor nests. Basal lamina-containing cyst-like spaces are noted. The tumor cells show high grade cytology with markedly increased nuclear to cytoplasmic ratio. (e) Nests of tumor invading the peripheral portion of the nerve bundle in the areas dominated by adenoid cystic carcinoma. (f) The proliferative index by Ki-67 stain labeling showed significantly higher labeling index in the adenoid cystic component (lower left corner) in comparison to the epithelial-myoepithelial component (upper right corner). 2.3. Pathology. Mass is composed of multiple components, with epithelial-myoepithelial carcinoma being dominant (80% of the tumor mass) with a minor component of solid variant of adenoid cystic carcinoma and basal cell adenocar- cinoma (Figure 2). The diagnosis of a hybrid carcinoma was made, in the presence of perineural invasion and absence of lymphovascular invasion. The surgical margins were negative for malignancy. 2. Case Report (e) CT scan: coronal cut of the upper neck with IV contrast showing erosion of the right zygomatic arch. (f) Intraoperative image showing an extended modified Blair incision. (g) Intraoperative image showing the parotid mass well circumscribed after elevation of the skin flap. (h) Tumor after “en bloc” excision. (i) Postoperative image of the patient. (c) (a) (b) (a) (b) (c) (f) (d) (e) (d) (f) (e) (i) (h) (g) (g) (h) (i) Figure 1: (a) Tumor involving the right cheek area extending from the temporal area to the mandibular area (frontal view). (b) Semilateral view. (c) Lateral view. (d) CT scan: axial cut of the upper neck with IV contrast showing narrowing of the external auditory canal by the tumor. (e) CT scan: coronal cut of the upper neck with IV contrast showing erosion of the right zygomatic arch. (f) Intraoperative image showing an extended modified Blair incision. (g) Intraoperative image showing the parotid mass well circumscribed after elevation of the skin flap. (h) Tumor after “en bloc” excision. (i) Postoperative image of the patient. The decision was made to surgically excise the mass. 2.1. At Our Center. CT scan of the brain and neck showed 11 × 6.2 × 14.5 cm heterogeneously enhancing right parotid mass with areas of necrosis, involving the deep lobe of the parotid gland. It is invading the lateral aspect of the masticator space, right temporomandibular joint, and external auditory canal causing narrowing of the latter and extending superiorly into the temporalis muscle and involving the masseter muscle (Figure 1). The parapharyngeal space appears normal. There is erosion of the right zygomatic arch and a few subcentimetric lymph nodes surrounding the parotid mass posteriorly.i An extended right radical parotidectomy with excision of a portion of temporalis muscle, part of the masseter mus- cle, and zygomatic arch was made. Intraoperatively, the facial nerve was disappearing into tumor and was completely encased and invaded by tumor; a frozen section biopsy taken intraoperatively demonstrated high suspicion of malignancy, so the decision was made to sacrifice the facial nerve. 2.2. Postoperatively. Patient was scheduled for postoperative radiation. A fine needle aspirate was done showing a picture sug- gestive of pleomorphic adenoma. Case Reports in Otolaryngology 3 (a) (b) (c) (d) (e) (f) Figure 2: (a) Epithelial-myoepithelial carcinoma. The ductal structures are composed of outer layer of polygonal, clear myoepithelial cells with variable sized and shaped nuclei. 2. Case Report In most cases adenoid cystic carcinoma has been the predominant component in hybrid carcinomas [11]. In con- trast, biphasically differentiated tumors are a mixture of two cellular patterns with a corresponding term in the tumor clas- sification. Examples of a biphasic differentiation are basaloid- squamous carcinoma, adenosquamous carcinoma or sarco- matoid carcinoma, and epithelial-myoepithelial carcinoma, mucoepidermoid carcinoma, or adenoid cystic carcinoma. Hybrid tumors must also be distinguished from the multiple occurrences of salivary gland tumors which can develop syn- or metachronously [10]. References Sternschein, “Rare syn- chronous parotid tumors of different histologic types,” Plastic and Reconstructive Surgery, vol. 72, no. 6, pp. 798–802, 1983. Epithelial-myoepithelial carcinoma (EMC) is an uncom- mon epithelial neoplasm, comprising approximately 1% of all salivary gland tumors. The tumor is mainly composed of variable portions of ductal and clear staining myoepithelial cells. EMC is predominantly a tumor of the major salivary glands, especially the parotid gland, but they may also arise in minor salivary glands [14–16]. [8] J. A. Schilling, B. L. Block, and J. C. Speigel, “Synchronous unilateral parotid neoplasms of different histologic types,” Head and Neck, vol. 11, no. 2, pp. 179–183, 1989. [9] M. Ethunandan, C. A. Pratt, A. Morrison, R. Anand, D. W. Macpherson, and A. W. Wilson, “Multiple synchronous and metachronous neoplasms of the parotid gland: the Chichester experience,” British Journal of Oral and Maxillofacial Surgery, vol. 44, no. 5, pp. 397–401, 2006. The aspirates of epithelial-myoepithelial carcinomas have been frequently misread as pleomorphic adenoma [17]. A dual cell population representing duct epithelial and myoep- ithelial cells with stromal substance is a feature common to both epithelial-myoepithelial carcinoma and pleomor- phic adenoma. Although the presence of the double-layered tubules consisting of duct epithelial cells surrounded by myoepithelial cells is diagnostic of epithelial-myoepithelial carcinoma, this pattern is not consistently observed in aspi- rates of epithelial-myoepithelial carcinomas. [10] G. Seifert and K. Donath, “Hybrid tumours of salivary glands. Definition and classification of five rare cases,” European Journal of Cancer Part B: Oral Oncology, vol. 32, no. 4, pp. 251–259, 1996. [11] L. M. Ru´ız-Godoy R., A. Mosqueda-Taylor, L. Su´arez-Roa, A. Poitevin, E. Bandala-S´anchez, and A. Meneses-Garc´ıa, “Hybrid tumours of the salivary glands. A report of two cases involving the palate and a review of the literature,” European Archives of Oto-Rhino-Laryngology, vol. 260, no. 6, pp. 312–315, 2003. [12] K. Kainuma, A. Oshima, H. Suzuki, M. Fukushima, H. Shimojo, and S.-I. Usami, “Hybrid carcinoma of the parotid gland: report of a case (epithelial-myoepithelial carcinoma and sali- vary duct carcinoma) and review of the literature,” Acta Oto- Laryngologica, vol. 130, no. 1, pp. 185–189, 2010. Because of the tendency of local recurrence and the low metastatic potential, the tumor is now recognized to be a low-grade malignant tumor in the WHO salivary gland classification. References cystic carcinoma in two cases, myoepithelial carcinoma and salivary duct carcinoma in one case, acinic cell carcinoma and salivary duct carcinoma in one, and squamous cell carcinoma and salivary duct carcinoma in two cases. The prevalence of hybrid carcinomas was 0.4% among the parotid gland tumors in their series [6]. [1] S. D. Stavrianos, N. R. McLean, and J. V. Soames, “Synchronous unilateral parotid neoplasms of different histological types,” European Journal of Surgical Oncology, vol. 25, no. 3, pp. 331– 332, 1999. [2] C. J. Zeebregts, W. J. B. Mastboom, G. van Noort, and R. J. van Det, “Synchronous tumours of the unilateral parotid gland: rare or undetected?” Journal of Cranio-Maxillofacial Surgery, vol. 31, no. 1, pp. 62–66, 2003. Additionally, in 2003, Ru´ız-Godoy et al. reported two patients with hybrid tumours in minor salivary glands of the palate. The first case involved adenoid cystic carcinoma and mucoepidermoid carcinoma, and the second case exhibited adenoid cystic carcinoma and epithelial-myoepithelial carci- noma [11]. [3] S. Tanaka, K. Tabuchi, K. Oikawa et al., “Synchronous unilateral parotid gland neoplasms of three different histological types,” Auris Nasus Larynx, vol. 34, no. 2, pp. 263–266, 2007. In 2010, Kainuma et al. described a case of a 74-year- old male with a hybrid carcinoma composed of epithelial- myoepithelial and salivary duct carcinomas of the right parotid gland [12].i [4] P. M. Y. Goh and E. Cheah, “Synchronous tumours of the parotid gland with different histology,” British Journal of Oral and Maxillofacial Surgery, vol. 27, no. 3, pp. 198–202, 1989. [5] G. Seifert and K. Donath, “Multiple tumours of the salivary glands—terminology and nomenclature,” European Journal of Cancer Part B: Oral Oncology, vol. 32, no. 1, pp. 3–7, 1996. Basal cell adenocarcinoma is a rare entity that was first defined as a malignant salivary gland tumor in 1991; although histomorphologic features of the tumors are similar to basal cell adenomas, proof of an infiltrative and destructive growth is essential for diagnosis. Due to their biologic behavior and prognosis, basal cell adenocarcinomas should be classified as low-grade carcinomas [13]. [6] T. Nagao, I. Sugano, Y. Ishida et al., “Hybrid carcinomas of the salivary glands: report of nine cases with a clinicopathologic, immunohistochemical, and p53 gene alteration analysis,” Mod- ern Pathology, vol. 15, no. 7, pp. 724–733, 2002. [7] I. P. Janecka, K. H. Perzin, and M. J. References The importance of this case lies in the rarity to see simultaneous different cancers within the parotid gland and to educate physicians that treatment for such cases is towards the tumor with the most aggressive type. Moreover, this case introduces epithelial-myoepithelial carcinoma and stresses that high clinical suspicion should be present for such an entity during patient history and disease progression since this type can often be mistaken for pleomorphic adenoma by FNA. [13] A. Franzen, K. Koegel, H.-J. Knieriem, and M. Pfaltz, “Basal cell adenocarcinoma of the parotid gland: a rare tumor entity. Case report and review of the literature,” HNO, vol. 46, no. 9, pp. 821– 825, 1998. [14] J. G. Batsakis, A. K. El-Naggar, and M. A. Luna, “Epithelial- myoepithelial carcinoma of salivary glands,” Annals of Otology, Rhinology & Laryngology, vol. 101, no. 6, pp. 540–542, 1992. [15] K. J. Cho, A. K. El-Naggar, N. G. Ordonez, M. A. Luna, J. Austin, and J. G. Batsakis, “Epithelial-myoepithelial carcinoma of salivary glands. A clinicopathologic, DNA flow cytometric, and immunohistochemical study of Ki-67 and HER-2/neu oncogene,” The American Journal of Clinical Pathology, vol. 103, no. 4, pp. 432–437, 1995. 3. Discussion y In 1996, Seifert and Donath reported 5 cases of hybrid tumors between 1965 and 1994 in the tissue samples of more than 6600 salivary gland tumors covered by the Salivary Gland Register (Institute of Pathology, University of Ham- burg, Germany). This means less than 0.1% of all registered tumours [10]. Very few articles have described the presence of synchronous parotid tumor. Unilateral synchronous neoplasms of the parotid gland are rare. The incidence ranges from 0.2% to 0.7% of parotid gland tumors [1–8].h The combination most commonly seen is pleomorphic adenoma and a Warthin’s tumor [9]. Hybrid tumors are very rare tumor entities which are composed of two or more different tumor types, each of which conforms to an exactly defined tumor category. The tumor entities of a hybrid tumor are not separated but have an identical origin within the same topographical area [10]. Moreover, in 2002, Nagao et al. described nine cases of hybrid carcinomas. The combinations of carcinoma compo- nents in their report were as follows: epithelial-myoepithelial carcinoma and basal cell adenocarcinoma in two cases, epithelial-myoepithelial carcinoma and squamous cell car- cinoma in one case, salivary duct carcinoma and adenoid 4 Conflict of Interests The authors declare that there is no conflict of interests regarding the publication of this paper. [16] I. Fonseca and J. Soares, “Epithelial-myoepithelial carcinoma of the salivary glands. A study of 22 cases,” Virchows Archiv A: Case Reports in Otolaryngology 5 Case Reports in Otolaryngology Pathological Anatomy and Histopathology, vol. 422, no. 5, pp. 389–396, 1993. [17] C. J. R. Stewart, S. Hamilton, I. L. Brown, and K. Mackenzie, “Salivary epithelial-myoepithelial carcinoma: report of a case misinterpreted as pleomorphic adenoma on fine needle aspira- tion (FNA),” Cytopathology, vol. 8, no. 3, pp. 203–209, 1997.
https://openalex.org/W3195136364	https://escholarship.org/content/qt54c45555/qt54c45555.pdf?t=rx8prf	English	null	FluB-RAM and FluB-RANS: Genome re-arrangement as safe and efficacious live attenuated influenza B virus vaccines.	null	2,021	cc-by	19,807	UC Irvine UC Irvine Previously Published Works Title FluB-RAM and FluB-RANS: Genome Rearrangement as Safe and Efficacious Live Attenuated Influenza B Virus Vaccines FluB-RAM and FluB-RANS: Genome Rearrangement as Safe and Efficacious Live Attenuated Influenza B Virus Vaccines https://escholarship.org/uc/item/54c45555 Journal Vaccines, 9(8) ISSN 2076-393X Authors Cardenas-Garcia, Stivalis Cáceres, C Joaquín Jain, Aarti et al. Publication Date 2021 DOI 10.3390/vaccines9080897 Peer reviewed Permalink https://escholarship.org/uc/item/54c45555 FluB-RAM and FluB-RANS: Genome Rearrangement as Safe and Efﬁcacious Live Attenuated Inﬂuenza B Virus Vaccines ardenas-Garcia 1 , C. Joaquín Cáceres 1 , Aarti Jain 2, Ginger Geiger 1 , Jong-Suk Mo 1, as Jasinskas 2, Rie Nakajima 2, Daniela S. Rajao 1 , D. Huw Davies 2 and Daniel R. Perez 1,* Stivalis Cardenas-Garcia 1 , C. Joaquín Cáceres 1 , Aarti Jain 2, Ginger Geiger 1 , Jong-Suk Mo 1, Algimantas Jasinskas 2, Rie Nakajima 2, Daniela S. Rajao 1 , D. Huw Davies 2 and Daniel R. Perez 1,* 1 Department of Population Health, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USA; stivalis@uga.edu (S.C.-G.); cjoaquincaceres@uga.edu (C.J.C.); imginger@uga.edu (G.G.); jm45001@uga.edu (J.-S.M.); daniela.rajao@uga.edu (D.S.R.) 2 f h l d h S h l f d f C l f 1 Department of Population Health, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USA; stivalis@uga.edu (S.C.-G.); cjoaquincaceres@uga.edu (C.J.C.); imginger@uga.edu (G.G.); jm45001@uga.edu (J.-S.M.); daniela.rajao@uga.edu (D.S.R.) p p , g y , y g , Athens, GA 30602, USA; stivalis@uga.edu (S.C.-G.); cjoaquincaceres@uga.edu (C.J.C.); imginger@uga.edu (G.G.); jm45001@uga.edu (J.-S.M.); daniela.rajao@uga.edu (D.S.R.) 2 Department of Physiology and Biophysics, School of Medicine, University of California Irvine, Irvine, CA 92697, USA; aartij@hs.uci.edu (A.J.); ajasinsk@hs.uci.edu (A.J.); rie3@hs.uci.edu (R.N.); ddavies@uci.edu (D.H.D.) g g g j g j g 2 Department of Physiology and Biophysics, School of Medicine, University of California Irvine, Irvine, CA 92697, USA; aartij@hs.uci.edu (A.J.); ajasinsk@hs.uci.edu (A.J.); rie3@hs.uci.edu (R.N.); ddavies@uci.edu (D.H.D.) * Correspondence: dperez1@uga.edu; Tel.: +1-(706)-542-5506 Abstract: Inﬂuenza B virus (IBV) is considered a major respiratory pathogen responsible for seasonal respiratory disease in humans, particularly severe in children and the elderly. Seasonal inﬂuenza vaccination is considered the most efﬁcient strategy to prevent and control IBV infections. Live attenuated inﬂuenza virus vaccines (LAIVs) are thought to induce both humoral and cellular immune responses by mimicking a natural infection, but their effectiveness has recently come into question. Thus, the opportunity exists to ﬁnd alternative approaches to improve overall inﬂuenza vaccine effectiveness. Two alternative IBV backbones were developed with rearranged genomes, rearranged M (FluB-RAM) and a rearranged NS (FluB-RANS). Both rearranged viruses showed temperature sensitivity in vitro compared with the WT type B/Bris strain, were genetically stable over multiple passages in embryonated chicken eggs and were attenuated in vivo in mice. In a prime-boost regime in naïve mice, both rearranged viruses induced antibodies against HA with hemagglutination inhibi- tion titers considered of protective value. In addition, antibodies against NA and NP were readily detected with potential protective value. Citation: Cardenas-Garcia, S.; Cáceres, C.J.; Jain, A.; Geiger, G.; Mo, J.-S.; Jasinskas, A.; Nakajima, R.; Rajao, D.S.; Davies, D.H.; Perez, D.R. FluB-RAM and FluB-RANS: Genome Rearrangement as Safe and Efﬁcacious Live Attenuated Inﬂuenza B Virus Vaccines. Vaccines 2021, 9, 897. https://doi.org/10.3390/ vaccines9080897 Keywords: LAIV; inﬂuenza; HA; IgA; IgG; vaccine; genome rearrangement Keywords: LAIV; inﬂuenza; HA; IgA; IgG; vaccine; genome rearrangement Academic Editors: Amit Gaba and Laurent Verkoczy Academic Editors: Amit Gaba and Laurent Verkoczy Received: 31 May 2021 Accepted: 5 August 2021 Published: 12 August 2021 FluB-RAM and FluB-RANS: Genome Rearrangement as Safe and Efﬁcacious Live Attenuated Inﬂuenza B Virus Vaccines Upon lethal IBV challenge, mice previously vaccinated with either FluB-RAM or FluB-RANS were completely protected against clinical disease and mortality. In conclusion, genome re-arrangement renders efﬁcacious LAIV candidates to protect mice against IBV. Powered by the California Digital Library University of California Powered by the California Digital Library University of California eScholarship.org eScholarship.org 1. Introduction Inﬂuenza B viruses (IBVs) in the Orthomyxoviridae family were ﬁrst isolated in 1940 in Irvington, NY [1]. IBVs are enveloped by a host-derived lipid bilayer and contain eight segments of single-stranded, negative-sense RNA [2] that encode for at least 11 proteins: polymerase basic 1 (PB1), polymerase basic 2 (PB2), polymerase acidic (PA), hemagglutinin (HA, surface glycoprotein), nucleoprotein (NP), neuraminidase (NA, surface glycoprotein), NB (surface glycoprotein), matrix protein 1 (M1), matrix protein 2 (BM2), non-structural protein 1 (NS1), and non-structural protein 2 (NS2) [3–7]. IBVs are of public health rele- vance thanks to their association with severe respiratory disease in humans, particularly in pediatric and elderly populations. Two antigenically distinct lineages co-circulate world- wide identiﬁed as Victoria and Yamagata lineages that show no serological cross reactivity, providing limited cross protection against each other [8–10]. The incidence of IBV infec- tions varies from season to season, linked to 0.69–61% of the inﬂuenza-induced pediatric mortalities registered in the United States from 2004 to 2020 [11]. During the 2019–2020 inﬂuenza season, IBV showed an early onset and the incidence of IBV infections in the United States increased compared with previous seasons. Compared with the 2018–2019 inﬂuenza season in which about 7% of inﬂuenza-positive samples corresponded to IBV [12], Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/vaccines Vaccines 2021, 9, 897. https://doi.org/10.3390/vaccines9080897 2 of 21 Vaccines 2021, 9, 897 2 of 21 >45% of the inﬂuenza-positive samples were positive for IBV during the 2019–2020 season. The 2019–2020 IBV season was associated with 122 (61.3%) of pediatric deaths [11] and 5174 (26.8%) hospitalizations, with the highest rate among adults ≥65 years old [13]. >45% of the inﬂuenza-positive samples were positive for IBV during the 2019–2020 season. The 2019–2020 IBV season was associated with 122 (61.3%) of pediatric deaths [11] and 5174 (26.8%) hospitalizations, with the highest rate among adults ≥65 years old [13]. ( ) p , g g ≥ y [ ] Although vaccination is the most effective strategy to ameliorate the impact of in- ﬂuenza infections, the incidence of IBV shows an increasing trend. 1. Introduction This is in part due to vaccine mismatch in trivalent vaccine formulations that contain only one IBV strain from one of the lineages [14–21]. These observations underscore the importance of includ- ing both IBV lineages in seasonal vaccine formulations, as is the case in several of the most recently FDA-approved quadrivalent vaccines [22]. However, additional efforts are warranted in order to improve vaccine protection against IBV. Live attenuated vaccine platforms have been among the most explored over the years (reviewed in [23]). In addi- tion to the cold-adapted LAIVs developed in the 1960s that form the basis of the current LAIVs approved for human use, alternative LAIV approaches have been developed that include modiﬁcations and deletions to the NS1 gene segment, generation of M2 deﬁcient viruses, and alternative virus backbones with temperature sensitive phenotypes, among others [24–30]. We have previously shown that genome re-arrangement is a suitable strat- egy for the development of inﬂuenza A virus LAIVs [31]. In the present study, we expanded these studies into IBV and produced two distinct genome re-arrangements in the back- bone of the B/Brisbane/60/2008 strain (Victoria lineage). The FluB-RAM re-arrangement involved producing a chimeric segment 1 that encodes PB1 and BM2, and a series of mutations in segment 7 to completely abrogate expression of BM2 from the latter. The FluB-RANS re-arrangement used a similar strategy, whereby NS2 was cloned downstream of PB1 and segment 8 contains multiple mutations that precludes NS2 expression. The safety and efﬁcacy of the FluB-RAM and FluB-RANS viruses were evaluated in DBA/2J mice [26,30,32,33]. Both vaccine candidates were immunogenic and effectively protected mice against homologous lethal IBV challenge. 2.1. Cells and Eggs Madin Darby canine kidney (MDCK) cells and 293T cells (ATCC CRL-3216) were used for reverse genetics of virus strains. Speciﬁc pathogen-free embryonated chicken eggs (ECEs) used for virus propagation and stock titration were obtained from Charles Rivers (Wilmington, MA, USA). 2.2. Recombinant Plasmids Infected cells were incubated at 33 °C, 35 °C, and 37 °C to assess viral growth at 0 12 24 48 72 96 2 4 6 8 0 Hours post infection TCID50/ml (Log10) 33℃ * * * * 0 12 24 48 72 96 2 4 6 8 0 Hours post infection TCID50/ml (Log10) 35℃ * * * ** * 0 12 24 48 72 96 2 4 6 8 0 Hours post infection TCID50/ml (Log10) 37℃ * ** ** A B C FluB-RAM FluB-RANS pSCG-PB1NS2 pSCG-PB1BM2 pDPPB1-B/Bris pDPPB1-B/Bris 500 bp - 1000 bp - FluB-RAM FluB-RANS B/Bris wt Segment 7 M1-∆BM2 Segment 1 PB1-BM2 Membrane Interaction Domain 1 248 PA vRNA cRNA cRNA vRNA vRNA A B C D 1 PB2 G4S2A BM2 PB1 1 752 886 1 109 FluB-RAM Stop/M1V/L2I/E3Stop/P4Stop/M21Stop/M37Stop Segment 8 NS1-∆NEP Segment 2 PB1-NEP RBD Effector Domain 1 282 PA vRNA cRNA cRNA vRNA vRNA A B C D 1 PB2 G4S2A NEP PB1 752 1 123 1 900 FluB-RANS W13Stop/M15T/M18T/M31T/F38Stop ure 1. (A) Schematic representation of the modiﬁed PB1 and M or NS segments carried by the FluB-RAM (top) and B-RANS (bottom) viruses. (B) RT-PCR from newly rescued FluB-RAM and FluB-RANS carrying the rearranged PB1 e segment. RT-PCR was performed from RNA extracted from FluB-RAM and FluB-RANS to amplify their rearranged gene segments. The plasmid carrying the PB1 WT and the rearranged PB1 plasmids were included in the reactions as trols. The agarose gel image showing the RT-PCR products is a demonstration that both rearranged PB1 gene segments pliﬁed from FluB-RAM and FluB-RANS RNA carry either the BM2 or the BNS2, as conﬁrmed by the corresponding trols. (C) Comparative growth kinetics of B/Bris WT, FluB-RAM, and FluB-RANS. 2.2. Recombinant Plasmids The modifications on the plasmids were confirmed by Sanger sequencing using Psomagen services (Rockville, MD, USA). were propagated in the top 10 chemically competent E. coli cells (ThermoFisher Scientiﬁc, Waltham, MA, USA). Plasmid puriﬁcations were carried out using QIAGEN Plasmid Maxi Kit (Qiagen, Gaithersburg, MD, USA). The modiﬁcations on the plasmids were conﬁrmed by Sanger sequencing using Psomagen services (Rockville, MD, USA). W13Stop, M15T, M18T, M31T, and F38Stop (Figure 1A). Plasmids were propagated in the top 10 chemically competent E. coli cells (ThermoFisher Scientific, Waltham, MA, USA). Plasmid purifications were carried out using QIAGEN Plasmid Maxi Kit (Qiagen, Gaithersburg, MD, USA). The modifications on the plasmids were confirmed by Sanger sequencing using Psomagen services (Rockville, MD, USA). gure 1. (A) Schematic representation of the modified PB1 and M or NS segments carried by the FluB-RAM (top) and uB-RANS (bottom) viruses. (B) RT-PCR from newly rescued FluB-RAM and FluB-RANS carrying the rearranged PB1 ne segment. RT-PCR was performed from RNA extracted from FluB-RAM and FluB-RANS to amplify their rearranged B1 gene segments. The plasmid carrying the PB1 WT and the rearranged PB1 plasmids were included in the reactions as ntrols. The agarose gel image showing the RT-PCR products is a demonstration that both rearranged PB1 gene segments mplified from FluB-RAM and FluB-RANS RNA carry either the BM2 or the BNS2, as confirmed by the corresponding ntrols. (C) Comparative growth kinetics of B/Bris WT, FluB-RAM, and FluB-RANS. MDCK cells were inoculated with e three viruses at an MOI of 0.01. 2.2. Recombinant Plasmids The plasmid carrying the PB1 WT and the rearranged PB1 plasmids were included in the reactions as controls. The agarose gel image showing the RT-PCR products is a demonstration that both rearranged PB1 gene segments ampliﬁed from FluB-RAM and FluB-RANS RNA carry either the BM2 or the BNS2, as conﬁrmed by the corresponding controls. (C) Comparative growth kinetics of B/Bris WT, FluB-RAM, and FluB-RANS. MDCK cells were inoculated with the three viruses at an MOI of 0.01. Infected cells were incubated at 33 ◦C, 35 ◦C, and 37 ◦C to assess viral growth at different temperatures over time. Samples were collected at 0, 12, 24, 48, and 96 h post-infection (hpi) and titrated by TCID50 in MDCK cells. Virus titers are graphed as the mean TCID50/mL ± SD. Samples with undetected virus titers were assigned the limit of detection value (0.699 TCID50/mL). Data analysis and graphs were prepared using Prism v9. Curves were analyzed using multiple t-tests followed by the Holm–Sidak method to correct for multiple comparisons. Signiﬁcant differences from the WT B/Bris are denotated by stars (). * = p < 0.01, * = p < 0.001, and = p < 0.0001. Figure 1. (A) Schematic representation of the modified PB1 and M or NS segments carried by the FluB-RAM (top) and FluB-RANS (bottom) viruses. (B) RT-PCR from newly rescued FluB-RAM and FluB-RANS carrying the rearranged PB1 gene segment. RT-PCR was performed from RNA extracted from FluB-RAM and FluB-RANS to amplify their rearranged PB1 gene segments. The plasmid carrying the PB1 WT and the rearranged PB1 plasmids were included in the reactions as controls. The agarose gel image showing the RT-PCR products is a demonstration that both rearranged PB1 gene segments amplified from FluB-RAM and FluB-RANS RNA carry either the BM2 or the BNS2, as confirmed by the corresponding controls. (C) Comparative growth kinetics of B/Bris WT, FluB-RAM, and FluB-RANS. MDCK cells were inoculated with the three viruses at an MOI of 0.01. Infected cells were incubated at 33 °C, 35 °C, and 37 °C to assess viral growth at different temperatures over time. Samples were collected at 0, 12, 24, 48, and 96 h post-infection (hpi) and titrated by Figure 1. (A) Schematic representation of the modiﬁed PB1 and M or NS segments carried by the FluB-RAM (top) and FluB-RANS (bottom) viruses. (B) RT-PCR from newly rescued FluB-RAM and FluB-RANS carrying the rearranged PB1 gene segment. 2.2. Recombinant Plasmids MDCK cells were inoculated with the A Segment 7 M1-∆BM2 Segment 1 PB1-BM2 Membrane Interaction Domain 1 248 PA vRNA cRNA cRNA vRNA vRNA A B C D 1 PB2 G4S2A BM2 PB1 1 752 886 1 109 FluB-RAM Stop/M1V/L2I/E3Stop/P4Stop/M21Stop/M37Stop Segment 8 NS1-∆NEP Segment 2 PB1-NEP RBD Effector Domain 1 282 PA vRNA cRNA cRNA vRNA vRNA A B C D 1 PB2 G4S2A NEP PB1 752 1 123 1 900 FluB-RANS W13Stop/M15T/M18T/M31T/F38Stop A B B FluB-RAM FluB-RANS pSCG-PB1NS2 pSCG-PB1BM2 pDPPB1-B/Bris pDPPB1-B/Bris 500 bp - 1000 bp - B 0 12 24 48 72 96 2 4 6 8 0 Hours post infection TCID50/ml (Log10) 33℃ * * * ** * 0 12 24 48 72 96 2 4 6 8 0 Hours post infection TCID50/ml (Log10) 35℃ * * * ** * 0 12 24 48 72 96 2 4 6 8 0 Hours post infection TCID50/ml (Log10) 37℃ * ** ** C C Figure 1. (A) Schematic representation of the modified PB1 and M or NS segments carried by the FluB-RAM (top) and FluB-RANS (bottom) viruses. (B) RT-PCR from newly rescued FluB-RAM and FluB-RANS carrying the rearranged PB1 gene segment. RT-PCR was performed from RNA extracted from FluB-RAM and FluB-RANS to amplify their rearranged PB1 gene segments. The plasmid carrying the PB1 WT and the rearranged PB1 plasmids were included in the reactions as controls. The agarose gel image showing the RT-PCR products is a demonstration that both rearranged PB1 gene segments amplified from FluB-RAM and FluB-RANS RNA carry either the BM2 or the BNS2, as confirmed by the corresponding controls. (C) Comparative growth kinetics of B/Bris WT, FluB-RAM, and FluB-RANS. MDCK cells were inoculated with the three viruses at an MOI of 0.01. Infected cells were incubated at 33 °C, 35 °C, and 37 °C to assess viral growth at different temperatures over time. Samples were collected at 0, 12, 24, 48, and 96 h post-infection (hpi) and titrated by Figure 1. (A) Schematic representation of the modiﬁed PB1 and M or NS segments carried by the FluB-RAM (top) and FluB-RANS (bottom) viruses. (B) RT-PCR from newly rescued FluB-RAM and FluB-RANS carrying the rearranged PB1 gene segment. RT-PCR was performed from RNA extracted from FluB-RAM and FluB-RANS to amplify their rearranged PB1 gene segments. 2.2. Recombinant Plasmids DNA fragments ﬂanked by AarI sites and encoding, in the 5′-3′ direction, the 82 codons of the C-terminus of B/Bris PB1, followed by codons encoding the sequence Gly-Gly-Gly- Gly-Ser (G4S), the 2A protease from Thosea asigna virus ORF (Tav 2A), either the BM2 ORF or BNS2 ORF of B/Bris, followed by the untranslated region of B/Bris PB1, were synthe- sized and cloned into pUC57 using GenScript services (Piscataway, NJ, USA). The synthetic fragments were subcloned using appropriate restriction sites into the reverse genetics pDP2002 vector encoding the wild type B/Bris PB1 gene segment [26] to generate the plas- mids pSCG_PB1G4S2ATavBM2_FluB (pSCG-PB1BM2) and pSCG_PB1G4S2ATavBNS2_FluB (pSCG-PB1BNS2), respectively. The reverse genetics plasmids that encode the B/Bris M seg- ment and B/Bris NS segment were mutagenized to obliterate expression of BM2 and BNS2, respectively. In plasmid pSCG_M_FLuB_stops_at_BM2 (pSCG-BM1-∆M2), the nucleotide sequence 771-AGTGATCTAATGATTTCAGATTCTTACAATTTGTTCTTTTATCTTATCAG- CTCTCCATTTCTAGGCTTGGACAATAGGGCATTTGAATCAAATAAAAAGAGGAATA- AACTAG-881 leads to the following aa mutations: an extra stop codon at the end of the M1 ORF; and M1V, L2I, E3Stop, P4Stop, M21Stop, and M37Stop for BM2 ORF (Figure 1A). In pSCG_NS_FLuB_stops_at_NS2 (pSCG-BNS1-∆NEP), the sequence 733-CTGTAGAGGAC- GAAGAAGACGGCCATCGGATCCTCAACTCACTCTTCGAGCGTCTTAACGAAGGAC- ATTCAAAGCCAATAA-813 leads to the following aa mutations: Q to L at the acceptor splicing boundary, W13Stop, M15T, M18T, M31T, and F38Stop (Figure 1A). Plasmids 3 of 21 TCG- A- Vaccines 2021, 9, 897 were propagated in the top 10 chemically competent E. coli cells (ThermoFisher Scientiﬁc, Waltham, MA, USA). Plasmid puriﬁcations were carried out using QIAGEN Plasmid Maxi Kit (Qiagen, Gaithersburg, MD, USA). The modiﬁcations on the plasmids were conﬁrmed by Sanger sequencing using Psomagen services (Rockville, MD, USA). W13Stop, M15T, M18T, M31T, and F38Stop (Figure 1A). Plasmids were propagated in the top 10 chemically competent E. coli cells (ThermoFisher Scientific, Waltham, MA, USA). Plasmid purifications were carried out using QIAGEN Plasmid Maxi Kit (Qiagen, Gaithersburg, MD, USA). The modifications on the plasmids were confirmed by Sanger sequencing using Psomagen services (Rockville, MD, USA). were propagated in the top 10 chemically competent E. coli cells (ThermoFisher Scientiﬁc, Waltham, MA, USA). Plasmid puriﬁcations were carried out using QIAGEN Plasmid Maxi Kit (Qiagen, Gaithersburg, MD, USA). The modiﬁcations on the plasmids were conﬁrmed by Sanger sequencing using Psomagen services (Rockville, MD, USA). W13Stop, M15T, M18T, M31T, and F38Stop (Figure 1A). Plasmids were propagated in the top 10 chemically competent E. coli cells (ThermoFisher Scientific, Waltham, MA, USA). Plasmid purifications were carried out using QIAGEN Plasmid Maxi Kit (Qiagen, Gaithersburg, MD, USA). 2.4. Stability of FluB-RAM and FluB-RANS Viruses through Serial Passages in ECEs Serial passages were performed in 11-day-old SPF embryonated chicken eggs as follows: serial 10-fold dilutions from FluB-RAM and FluB-RANS E1 viruses were prepared in 1X phosphate buffered saline (PBS) and 100 µL from each dilution was inoculated into each of ﬁve ECEs through the allantoic cavity to generate E2. The inoculated ECEs were incubated at 33 ◦C for 48 h. Allantoic ﬂuids were then tested for hemagglutination activity by the hemagglutination assay (HA). Fluids collected from the previous to the last dilution with 5/5 embryos positive for HA activity were pooled together and used to prepare 10-fold dilutions to inoculate the next set of embryos. The same procedure was repeated until ﬁve passages had been completed, generating passage E6. Aliquots from each passage were stored at −80 ◦C until needed. RNA was extracted from ﬂuids collected at each passage and from the original virus stock using the MagNA Pure LC Total Nucleic Acid Isolation Kit (Roche, San Francisco, CA, USA). The PB1, M, and/or NS gene segments were ampliﬁed by RT-PCR using SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase (ThermoFisher Scientiﬁc). Sanger sequencing (Psomagen) was then performed from the resulting RT-PCR products to conﬁrm the re-arrangement at the PB1 gene segments and the presence of the introduced mutations within the M and NS gene segments, respectively. Multi-segment RT-PCR (using the same RT-PCR system) was performed as previously described [35] for full genome sequencing using next generation sequencing (NGS) as follows: amplicon libraries were prepared using the Nextera XT DNA library preparation kit (Illumina, San Diego, CA, USA) following the manufacturer’s protocol. Barcoded libraries were multiplexed and sequenced on the high-throughput Illumina MiSeq NGS platform (Illumina) in a paired-end 150-nucleotide run format. De novo genome assembly was performed as described previously [36]. 2.3. Rescue of FluB-RAM and FluB-RANS Viruses with Rearranged Genomes 2.3. Rescue of FluB-RAM and FluB-RANS Viruses with Rearranged Genomes Recombinant viruses were rescued by reverse genetics as previously described [34]. We employed a 6 + 2 method whereby six plasmids, each containing a single cDNA copy of the wild type gene segments from the B/Brisbane/60/2008, were mixed with the corre- sponding pair of plasmids (either pSCG-PB1BM2 and pSCG-BM1-∆M2, or pSCG-PB1BNS2 and pSCG-BNS1-∆NEP) to produce the B/Bris rearranged M (FluB-RAM) and B/Bris rearranged NS (FluB-RANS) viruses, respectively. The identity of the rescued viruses was conﬁrmed by Sanger sequencing (Psomagen). The recombinant viruses were propagated and titrated in 11-day-old SPF ECEs incubated at 33 ◦C for 48 h. Virus stocks were stored at −80◦C until further use. These stocks constitute the ﬁrst passage in ECEs (E1). 2.2. Recombinant Plasmids RT-PCR was performed from RNA extracted from FluB-RAM and FluB-RANS to amplify their rearranged PB1 gene segments. The plasmid carrying the PB1 WT and the rearranged PB1 plasmids were included in the reactions as controls. The agarose gel image showing the RT-PCR products is a demonstration that both rearranged PB1 gene segments ampliﬁed from FluB-RAM and FluB-RANS RNA carry either the BM2 or the BNS2, as conﬁrmed by the corresponding controls. (C) Comparative growth kinetics of B/Bris WT, FluB-RAM, and FluB-RANS. MDCK cells were inoculated with the three viruses at an MOI of 0.01. Infected cells were incubated at 33 ◦C, 35 ◦C, and 37 ◦C to assess viral growth at different temperatures over time. Samples were collected at 0, 12, 24, 48, and 96 h post-infection (hpi) and titrated by TCID50 in MDCK cells. Virus titers are graphed as the mean TCID50/mL ± SD. Samples with undetected virus titers were assigned the limit of detection value (0.699 TCID50/mL). Data analysis and graphs were prepared using Prism v9. Curves were analyzed using multiple t-tests followed by the Holm–Sidak method to correct for multiple comparisons. Signiﬁcant differences from the WT B/Bris are denotated by stars (). * = p < 0.01, * = p < 0.001, and = p < 0.0001. 4 of 21 Vaccines 2021, 9, 897 2.6. Mouse Studies Male and female DBA/2J mice (5 weeks old) were purchased from Jackson’s Labo- ratories (Bar Harbor, ME) and raised until 7 weeks of age. Mice were housed in negative pressure caging in the Davison Life Sciences Complex, University of Georgia and were provided food and water ad libitum for the duration of the experiment. 2.5. Virus Growth Kinetics MDCK cells were seeded in six-well plates and incubated overnight at 37 ◦C, under 5% CO2. The next day, cells were inoculated with 0.01 MOI of either the B/Bris WT, FluB-RAM, or FluB-RANS virus contained in 500 µL, each in triplicate wells. Three set of plates were prepared for each virus. Inoculated cells were incubated for 1 h at 35 ◦C/5% CO2 with gentle rocking of the plates every 15 min. Subsequently, the virus inoculum was removed, and the cells were washed twice with 1× PBS and replenished with 2 mL of fresh Opti-MEM (Gibco, ThermoFisher Scientiﬁc) supplemented with 1× antibiotic/antimycotic solution (Gibco, ThermoFisher Scientiﬁc) and 1 µg/mL of L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK)-treated Trypsin. Plates were set to incubate at either 33, 35, or 37 ◦C at 5% CO2. Supernatants (200 µL) were collected at 0, 12, 24, 48, 72, and 96 h post-inoculation (hpi) and stored at −80 ◦C until processed. The amount of virus present in the collected samples was titrated by TCID50 in MDCK cells, determining virus presence by HA assay. Virus titers were calculated using the Reed and Muench protocol [37] and plotted as the mean TCID50/mL ± SD. Vaccines 2021, 9, 897 5 of 21 2.8. Vaccine Efﬁcacy Mice from the vaccine safety study (n = 8/group, 1 2 females) were challenged i.n. with a lethal dose (107 EID50/mouse) of the B/Brisbane/60/2008 PB2-F406Y (B/Bris/ F406Y) strain [26] contained in 50 µL. A subset of mice in the mock group (n = 8, 1 2 female) remained unchallenged and served as negative controls. Mice were monitored twice daily to record clinical signs and mortality for up to 14 days post-challenge (dpc). Body weight was recorded for up to 12 dpc. At 14 dpc, survivors were anesthetized, terminally bled to collect sera, and subsequently humanely euthanized (Figure 2A). 2.7. Vaccine Safety A prime-boost strategy using the same administration route and inoculum was imple- mented 20 days apart. Seven-week-old mice were vaccinated intranasally (i.n.) with 50 µL of inoculum distributed equally between nares. Male and female mice, housed separately, were allocated into four groups ( 1 2 females/group) as follows: G1. FluB-RAM (n = 12); G2. FluB-RANS (n = 12); G3. 1× PBS (mock, n = 24); and G4. B/Bris WT (n = 12, positive control). The FluB-RAM, FluB-RANS, and control B/Bris WT viruses were administered at a target dose of 106 EID50/mouse. Mice were monitored daily to record clinicals signs and mortality. Body weight was recorded daily for up to 12 days following vaccination (dpv) and boost (dpb). At 19 dpb, a subset of mice from each group (n = 4/group, 1 2 females) was anesthetized with isoﬂurane, terminally bled to collect sera, and subsequently humanely euthanized (Figure 2A). 2.9. Hemagglutination Inhibition (HI) Assay Sera were prepared from whole blood collected at 19 dpb (n = 4/group, except for FluB-RAM) and 14 dpc (n = 8/group) by centrifugation at 1000× g for 15 min at room temperature. The sera were treated with receptor destroying enzyme (RDE) and the HI assay was performed in V-bottomed microtiter plates, using four hemagglutination units (HAU) of viral antigen (B/Bris WT) per 25 µL, as recommended by the OIE [1], using a suspension of turkey red blood cells (0.5%). HI titers were plotted using Prism v9 (Graph- Pad, San Diego, CA, USA). The limit of detection was at dilution of 1/10, and samples with undetectable titers were assigned a dilution value of 1/8 for statistical purposes. 2.10. Virus Neutralization (VN) Assay Sera collected at 19 dpb were treated with RDE. In 96-well plates, twofold dilu- tions (50 µL) were prepared from treated sera using 1X PBS supplemented with antibi- otic/antimycotic solution. Next, 100 TCID50 (in 50 µL) of either B/Bris (homologous) or B/Wisconsin/01/2010 (B/Wis, heterologous) were added to the corresponding wells containing serum dilutions. Serum/virus mixes were incubated at 37 ◦C for 1 h. Thereafter, the serum/virus mixes were added to MDCK cell monolayers and set to incubate at 4 ◦C for 15 min and then at 35 ◦C for 45 min. After incubation, the serum/virus mixes were removed from the cell monolayers and 200 µL of Opti-MEM (Gibco) supplemented with 1X antibiotic/antimycotic solution (Gibco) and 1 µg/mL of TPCK-Trypsin was added to each well. Plates were set to incubate for 72 h at 35 ◦C, under 5% CO2. Virus neutralization titers were determined by HA assay. The limit of detection was at dilution of 1/10. Samples with undetectable VN titers were assigned a dilution value of 1/8 for statistical purposes. 6 of 21 ubseque Vaccines 2021, 9, 897 e 2. (A) Experimental timeline. Seven-week-old mice (n = 12/ group, ½ females) were vaccinated or mock vaccinat asally with 1× PBs, B/Bris WT, FluB-RAM, or FluB-RANS at day 0, monitoring body weight for up to 12 dpv an al signs for up to 20 dpv. Twenty days after vaccination (20 dpv), all mice were boosted with the same mock ne treatment as before and their body weight was monitored for up to 12 dpb (day 32) and clinical signs were mon for up to 21 dpb (day 41). At day 39 (19 dpb), a subset of mice was bled and humanely euthanized. 2.10. Virus Neutralization (VN) Assay The remini 0 1 2 3 4 5 6 7 8 9 10 11 12 60 70 80 90 100 110 120 Days post-prime Body Weight (% change) Male mice - body weight changes post-prime n = 1 † n = 4 † 0 1 2 3 4 5 6 7 8 9 10 11 12 60 70 80 90 100 110 120 Days post-prime Body Weight (% change) Female mice - body weight changes post-prime n = 1 † A B Males Females 10 20 40 80 160 320 640 HI titer 19 days post-boost C FluB-RAM FluB-RANS PBS B/Bris wt B/Bris/PB2 F406Y B/Wis/PB2 F406Y 10 20 40 80 160 320 640 Neutralization titer PBS FluB-RAM FluB-RANS FluB att 19 days post-boost FluB att 7-week-old DBA/2J (n=12/group, ½ females) 0 20 39 41 55 (14 dpc) Boost Blood collection Challenge 107 EID50/mouse Termination and Bleeding 12 32 Body Weight recording Body Weight recording Body Weight recording Recording of Clinical signs Recording of Clinical signs Prime 106 EID50/mouse Figure 2. (A) Experimental timeline. Seven-week-old mice (n = 12/ group, 1 2 females) were vaccinated or mock vaccinated ntranasally with 1× PBs, B/Bris WT, FluB-RAM, or FluB-RANS at day 0, monitoring body weight for up to 12 dpv and linical signs for up to 20 dpv. Twenty days after vaccination (20 dpv), all mice were boosted with the same mock or vaccine reatment as before and their body weight was monitored for up to 12 dpb (day 32) and clinical signs were monitored or up to 21 dpb (day 41). At day 39 (19 dpb), a subset of mice was bled and humanely euthanized. The remining mice n = 8/group, 1 2 females) were challenged at day 41 (21 dpb). Mice were observed for up to 14 dpc (day 55) for clinical igns and mortality, and body weight was recorded for up to 12 dpc (day 52). At 14 dpc, all remaining mice were bled nd humanely euthanized; nasal washes (NW) were collected as well. (B) Monitoring of body weight and survival in male and female mice. After prime vaccination, body weight was monitored for up to 12 dpv; survival was recorded until he day before the boost. (C) HI antibody titers and VN titers after boost. Blood samples for serology were collected at 9 dpb. 2.10. Virus Neutralization (VN) Assay † = number of dead mice. ure 2. (A) Experimental timeline. Seven-week-old mice (n = 12/ group, ½ females) were vaccinated or mock vaccin anasally with 1× PBs, B/Bris WT, FluB-RAM, or FluB-RANS at day 0, monitoring body weight for up to 12 dpv cal signs for up to 20 dpv. Twenty days after vaccination (20 dpv), all mice were boosted with the same moc cine treatment as before and their body weight was monitored for up to 12 dpb (day 32) and clinical signs were m d for up to 21 dpb (day 41). At day 39 (19 dpb), a subset of mice was bled and humanely euthanized. The remi Figure 2. (A) Experimental timeline. Seven-week-old mice (n = 12/ group, 1 2 females) were vaccinated or mock vaccinated intranasally with 1× PBs, B/Bris WT, FluB-RAM, or FluB-RANS at day 0, monitoring body weight for up to 12 dpv and clinical signs for up to 20 dpv. Twenty days after vaccination (20 dpv), all mice were boosted with the same mock or vaccine treatment as before and their body weight was monitored for up to 12 dpb (day 32) and clinical signs were monitored for up to 21 dpb (day 41). At day 39 (19 dpb), a subset of mice was bled and humanely euthanized. The remining mice (n = 8/group, 1 2 females) were challenged at day 41 (21 dpb). Mice were observed for up to 14 dpc (day 55) for clinical signs and mortality, and body weight was recorded for up to 12 dpc (day 52). At 14 dpc, all remaining mice were bled and humanely euthanized; nasal washes (NW) were collected as well. (B) Monitoring of body weight and survival in male and female mice. After prime vaccination, body weight was monitored for up to 12 dpv; survival was recorded until the day before the boost. (C) HI antibody titers and VN titers after boost. Blood samples for serology were collected at 19 dpb. Sera were separated and used to perform HI assays comparing males and females, as well as to perform VN assays against B/Bris or B/Wis. Body weight values, HI titers, and VN titers were graphed as the group mean ± SD. Data analysis and graphs were prepared using Prism v9. Signiﬁcant differences between groups are denotated by stars (). * = p < 0.01. † = number of dead mice. 2.10. Virus Neutralization (VN) Assay Seven-week-old mice (n = 12/ group, 1 2 females) were vaccinated or mock vaccinate ure 2. (A) Experimental timeline. Seven-week-old mice (n = 12/ group, ½ females) were vaccinated or mock vaccin anasally with 1× PBs, B/Bris WT, FluB-RAM, or FluB-RANS at day 0, monitoring body weight for up to 12 dpv cal signs for up to 20 dpv. Twenty days after vaccination (20 dpv), all mice were boosted with the same moc cine treatment as before and their body weight was monitored for up to 12 dpb (day 32) and clinical signs were m d for up to 21 dpb (day 41). At day 39 (19 dpb), a subset of mice was bled and humanely euthanized. The remin Figure 2. (A) Experimental timeline. Seven-week-old mice (n = 12/ group, 1 2 females) were vaccinated or mock vaccinated intranasally with 1× PBs, B/Bris WT, FluB-RAM, or FluB-RANS at day 0, monitoring body weight for up to 12 dpv and clinical signs for up to 20 dpv. Twenty days after vaccination (20 dpv), all mice were boosted with the same mock or vaccine treatment as before and their body weight was monitored for up to 12 dpb (day 32) and clinical signs were monitored for up to 21 dpb (day 41). At day 39 (19 dpb), a subset of mice was bled and humanely euthanized. The remining mice (n = 8/group, 1 2 females) were challenged at day 41 (21 dpb). Mice were observed for up to 14 dpc (day 55) for clinical signs and mortality, and body weight was recorded for up to 12 dpc (day 52). At 14 dpc, all remaining mice were bled and humanely euthanized; nasal washes (NW) were collected as well. (B) Monitoring of body weight and survival in male and female mice. After prime vaccination, body weight was monitored for up to 12 dpv; survival was recorded until the day before the boost. (C) HI antibody titers and VN titers after boost. Blood samples for serology were collected at 19 dpb. Sera were separated and used to perform HI assays comparing males and females, as well as to perform VN assays against B/Bris or B/Wis. Body weight values, HI titers, and VN titers were graphed as the group mean ± SD. Data analysis and graphs were prepared using Prism v9. Signiﬁcant differences between groups are denotated by stars (). * = p < 0.01. 2.10. Virus Neutralization (VN) Assay Sera were separated and used to perform HI assays comparing males and females, as well as to perform VN assays gainst B/Bris or B/Wis. Body weight values, HI titers, and VN titers were graphed as the group mean ± SD. Data analysis nd graphs were prepared using Prism v9. Signiﬁcant differences between groups are denotated by stars (). * = p < 0.01. A 7-week-old DBA/2J (n=12/group, ½ females) 0 20 39 41 55 (14 dpc) Boost Blood collection Challenge 107 EID50/mouse Termination and Bleeding 12 32 Body Weight recording Body Weight recording Body Weight recording Recording of Clinical signs Recording of Clinical signs Prime 106 EID50/mouse A Termination and Bleeding 0 1 2 3 4 5 6 7 8 9 10 11 12 60 70 80 90 100 110 120 Days post-prime Body Weight (% change) Male mice - body weight changes post-prime n = 1 † n = 4 † 0 1 2 3 4 5 6 7 8 9 10 11 12 60 70 80 90 100 110 120 Days post-prime Body Weight (% change) Female mice - body weight changes post-prime n = 1 † B FluB-RAM FluB-RANS PBS B/Bris wt FluB att 0 1 2 3 4 5 6 7 8 9 10 11 12 60 70 80 90 100 110 120 Days post-prime Body Weight (% change) Male mice - body weight changes post-prime n = n = 4 † B B B † 0 1 2 3 4 5 6 7 8 9 10 11 12 60 70 80 90 100 110 120 Days post-prime Body Weight (% change) Female mice - body weight changes post-prime n = 1 † FluB-RAM FluB-RANS PBS B/Bris wt FluB att Female mice - body weight changes post-prime Days post-prime Males Females 10 20 40 80 160 320 640 HI titer 19 days post-boost C B/Bris/PB2 F406Y B/Wis/PB2 F406Y 10 20 40 80 160 320 640 Neutralization titer PBS FluB-RAM FluB-RANS FluB att 19 days post-boost ** Males Females 10 20 40 80 160 320 640 HI titer 19 days post-boost C C erimental timeline. Seven-week-old mice (n = 12/ group, ½ females) were vaccinated or mock vac 1 PB B/B i WT Fl B RAM Fl B RANS t d 0 it i b d i ht f t 12 d Experimental timeline. 2.11. Microarray for IgG and IgA Determination Sera collected at 19 dpb and 14 dpc, and nasal washed collected at 14 dpc were analyzed through protein microarrays to determine anti-HA, -NA, and -NP IgG and IgA levels from multiple Victoria- and Yamagata-like IBVs (Table 1). Puriﬁed IBV protein antigens were purchased from Sino Biological (Wayne, PA) (Table 1). Microarrays were carried out as described elsewhere [38]. The results are expressed as the group mean ﬂuorescence intensity (MFI) ± SD. The higher the MFI, the more Abs bound to a particular antigen. MFIs were plotted using Prism v9 (GraphPad). Vaccines 2021, 9, 897 7 of 21 Table 1. Protein antigens used in protein microarray analysis. Protein Region IBV Strain Lineage Expression System Catalog No. HA HA1 B/Victoria/02/1987 Victoria HEK293 40163-V08H HA HA1 B/Wisconsin/01/2012 Victoria HEK293 40462-V08H1 HA HA1 B/Brisbane/60/2008 Victoria HEK293 40016-V08H1 HA HA1 B/Ohio/01/2005 Victoria HEK293 40460-V08H1 HA HA1 B/Massachusetts/03/2010 Victoria HEK293 40191-V08H1 HA HA1 B/Malaysia/2506/2004 Victoria HEK293 11716-V08H1 HA HA1 B/HongKong/05/1972 Victoria HEK293 40461-V08H1 HA HA1 B/Yamagata/16/1988 Yamagata HEK293 40157-V08H1 HA HA1 B/Victoria/504/2000 Yamagata HEK293 40391-V08H HA HA1 B/Brisbane/3/2007 Yamagata HEK293 40431-V08H1 HA HA1 B/Phuket/3073/2013 Yamagata HEK293 40498-V08H1 HA HA1 B/Florida/07/2004 Yamagata HEK293 40432-V08H1 HA HA1 B/Utah/02/2012 Yamagata HEK293 40463-V08H1 HA HA1 B/Florida/4/2006 Yamagata HEK293 11053-V08H1 HA HA1+HA2 B/Brisbane/60/2008 Victoria E. coli 40016-V08B HA HA1+HA2 B/Malaysia/2506/2004 Victoria HEK293 11716-V08H HA HA1+HA2 B/Massachusetts/03/2010 Victoria Baculovirus 40191-V08B HA HA1+HA2 B/Florida/4/2006 Yamagata HEK293 11053-V08H HA HA1+HA2 B/Phuket/3073/2013 Yamagata Baculovirus 40498-V08B HA HA1+HA2 B/Yamagata/16/1988 Yamagata Baculovirus 40157-V08B HA HA1+HA2 B/Utah/02/2012 Yamagata Baculovirus 40463-V08B HA HA1+HA2 B/Brisbane/60/2008 Victoria HEK293 40016-V08H NA NA B/Brisbane/60/2008 Victoria HEK293 40203-VNAHC NA NA B/Phuket/3073/2013 Yamagata Baculovirus 40502-V07B NP NP B/Florida/4/2006 Yamagata Baculovirus 40438-V08B Table 1. Protein antigens used in protein microarray analysis. 3. Results 3.1. FluB-RAM and FluB-RANS Viruses with Rearranged Genomes 3.1. FluB-RAM and FluB-RANS Viruses with Rearranged Genomes The currently available inﬂuenza B virus LAIVs approved for human use are based on cold-adapted/temperature sensitive mutations. More recently, we developed an alternative inﬂuenza B virus LAIV based on amino acid mutations on the PB1 segment with or without a C-terminal HA tag. To further expand the choice of potential alternative LAIV candidates against the inﬂuenza B virus and to test the hypothesis that different LAIV backbones have an impact on adaptive immunity, we developed two strategies of genome re-arrangement within the backbone of the B/Brisbane/60/2008 strain (Victoria lineage). The ﬁrst strategy, FluB-RAM, consists of moving the BM2 ORF from segment 7 into the C-terminal end of the PB1 ORF in segment 1 (Figure 1A). Segment 1 is further modiﬁed with the inclusion of a linker peptide sequence (G4S) and the Tav 2A protease sequence between the PB1 and BM2 ORFs. The strategy leads to a chimeric polymerase PB1 subunit protein carrying the G4S linker and the Tav 2A protein sequences and the BM2 protein, but with N-terminal proline. Segment 7 is mutagenized to eliminate the codon for the ﬁrst methionine in the BM2 ORF, the inclusion of an additional stop codon in the BM1 ORF, and two early stop codons in the BM2 ORF, resulting in complete obliteration of BM2 expression from its cognate segment. In the second strategy, FluB-RANS (Figure 1A), the NS2 ORF from segment 8, instead of BM2 ORF is cloned downstream of PB1. Segment 8 is further modiﬁed to produce an amino acid mutation and a stop codon at the splicing acceptor boundary and an additional stop codon in BNS2 (F38Stop). In addition, codons 15, 18, and 31 were mutated (M15T, M18T, and M31T), to prevent leaky expression of a truncated BNS2 protein. The FluB-RAM and FluB-RANS viruses were successfully rescued and propagated in ECEs. To quickly visualize whether the viruses contained the corresponding rearranged PB1 gene segments, RT-PCR targeting the region containing the BM2 or BNS2 insertions was performed (Figure 1B). The RT-PCR showed that the FluB-RAM and FluB-RANS viruses carry PB1 segments with the expected size changes (402 and 444 base pairs, respectively). Vaccines 2021, 9, 897 8 of 21 The sizes of the ampliﬁed fragments were consistent with those of the positive control reverse genetics plasmids used to generate the corresponding viruses (Figure 1B). 3.1. FluB-RAM and FluB-RANS Viruses with Rearranged Genomes Further, the sequencing results conﬁrmed the presence of the BM2 and BNS2 inserts downstream of the PB1 ORF in the corresponding viruses, as well as the mutations introduced in segments 7 and 8 that prevent the expression of BM2 or BNS2, respectively (Table 2). To further evaluate genome re-arrangement stability and the mutations introduced in the M and NS gene segments, ﬁve serial passages from an E1 stock were performed in ECEs, as described above. Segments 1 and 7 from the FluB-RAM virus and 1 and 8 from the FluB-RANS virus from each passage were ampliﬁed by RT-PCR and sequenced by Sanger (Table 2). In addition, NGS was performed on the last passage virus and compared with the original stock virus from passage 1. Both Sanger sequencing and NGS conﬁrmed the presence of the BM2 or BNS2 downstream of the PB1 gene segment in either FluB-RAM or FluB-RANS, respectively. The sequencing results also conﬁrmed the maintenance of the mutations introduced in either the M or NS gene segment from the corresponding virus. These results highlight the stability of the two genome re-arrangement strategies introduced in the B/Bris genome. Table 2. Whole genome sequencing results after serial passages in embryonated chicken eggs. FluB-RAM FluB-RANS Segment Predicted Mutations Egg Passage #6 Predicted Mutations Egg Passage #6 PB1 +BM2 ORF +BM2 ORF +BNS2 ORF +BNS2 ORF M495I (g1485a) PB2 None None None None PA None None None S719P (t2184c) HA None None None None NP None None None A318T (g1012a) NA None None None None NB None None None None BM1 Stop (c774a) g543a s a546g s Stop (c774a) None None BM2 M1V (a771g) L2I (c774a) E3Stop (g777a) P4Stop (c780t, c791g) M21Stop (a831t, t832a) M37Stop (a879t, t880a) M1V (a771g) L2I (c774a) E3Stop (g777a) P4Stop (c780t, c791g) M21Stop (a831t, t832a) M37Stop (a879t, t880a) None None NS1 None None None None NS2/NEP None None a734t (Splicing acceptor) W13Stop (g737a) M14T (t743c) M15T (t752c) M31T (t791c) F38Stop (t812a c813a) a734t (Splicing acceptor) W13Stop (g737a) M14T (t743c) M15T (t752c) M31T (t791c) F38Stop (t812a c813a) s Synonymous mutation. Higher case letters = amino acids. Lower case letters = nucleotides. Table 2. Whole genome sequencing results after serial passages in embryonated chicken eggs. s Synonymous mutation. Higher case letters = amino acids. Lower case letters = nucleotides. 3.2. 3.3. FluB-RAM and FluB-RANS Viruses Show Differences in Attenuation 3.3. FluB-RAM and FluB-RANS Viruses Show Differences in Attenuation The safety and immunogenicity of the FluB-RAM and FluB-RANS viruses were tested in DBA/2J mice, a small animal model susceptible to inﬂuenza B viruses without further adaptation [26]. DBA/2J mice (7-week-old, male and female) were inoculated with 106 EID50/mouse i.n. following a prime/boost strategy 20 days apart with the corresponding rearranged virus (Figure 2A). As a control, a group of mice was inoculated with the B/Bris WT virus (106 EID50/mouse i.n.). Prime vaccination with the FluB-RANS resulted in neither clinical signs nor body weight changes in both male and female mice (Figure 2B). In contrast, male mice primed with the FluB-RAM virus showed an average of ~10% body weight loss between 7 and 9 dpv, but started to recover from 10 dpc onwards, whereas female mice showed a slight drop in body weight (<5%) at 7 dpv and quickly recovered. Consistent with the presentation of clinical signs, no mortality was observed in mice that received the FluB-RANS virus or female mice inoculated with the FluB-RAM virus (not shown). One out of 6 male mice primed with the FluB-RAM virus had to be euthanized by 10 dpv (not shown). These observations contrast with those in the group primed with the B/Bris WT virus, where male and female mice showed body weight drops of ~20% and where 4 out 6 males and 1 out 6 females succumbed to the infection between 8 and 10 dpv (not shown). These results show that the FluB-RANS vaccine candidate is the safest between the two rearranged viruses and both viruses are attenuated in vivo compared with the B/Bris WT strain. As expected, boost vaccination resulted in neither clinical signs nor mortality in any of the groups (data not shown). 3.1. FluB-RAM and FluB-RANS Viruses with Rearranged Genomes FluB-RAM and FluB-RANS Viruses Are Attenuated In Vitro To determine the growth of the rearranged viruses at different temperatures, MDCK cells were infected with either B/Bris WT, FluB-RAM, or FluB-RANS at 0.01 MOI. The growth kinetics for each virus was assessed at 33 ◦C, 35 ◦C, and 37 ◦C for up to 96 hpi (Figure 1C). Compared with the B/Bris WT virus, both FluB-RAM and FluB-RANS showed signiﬁcantly lower replication at all three temperatures. Of note, the replication of the FluB-RANS virus was lower than that of the FluB-RAM virus at either 33 ◦C or 35 ◦C and was almost undetectable at 37 ◦C compared with the B/Bris WT and FluB-RAM viruses. Vaccines 2021, 9, 897 9 of 21 9 of 21 These results demonstrate that both FluB-RAM and FluB-RANS are attenuated in vitro. Rearranged virus yield in ECEs reached titers of 1 × 108 and 3.16 × 108 EID50/mL for FluB-RAM and FluB-RANS, respectively. 3.4. Qualitative Differences in Humoral Responses among Different Vaccine Groups In addition, FluB att Vaccines 2021, 9, 897 10 of 21 10 of 21 anti-HA1 IgG responses were signiﬁcantly higher than those for FluB-RAM for HA1 from B/Victoria/02/1987 (p = 0.0332), B/Ohio/01/2005 (p = 0.0257), B/Massachusetts/03/2010 (p = 0.0434), and B/Wisconsin/02/2012 (p = 0.0335) (Figure 3A top). Analysis of all the anti-Victoria HA responses combined and comparison between groups conﬁrmed that the FluB-RANS vaccine induced higher responses than the FluB-RAM vaccine (p < 0.0001) (Figure 3A bottom). When looking at the anti-Yamagata responses, FluB-RANS showed numerically higher anti-HA IgG responses that the other vaccine groups; however, none of those were statistically signiﬁcant (p > 0.05) owing to the high variability between samples within the group (Figure 3B top). When the responses against all the Yamagata lineage HAs were combined, the FluB-RANS group had a signiﬁcantly higher IgG response compared with the other vaccine groups (p < 0.0001 and p < 0.0001, respectively) (Figure 3B bottom). In contrast, anti-HA IgA responses were numerically higher for both IBV lineages in sam- ples from the FluB att group, but not signiﬁcantly different than the other groups (p > 0.05) (Figure 3C,D). Combining the responses against all the Victoria or Yamagata HA anti- gens, FluB att induced signiﬁcantly higher IgA responses than FluB-RAM (p < 0.0001 and p < 0.0001, respectively) and FluB-RANS (p = 0.0021 and p = 0.0097, respectively) against both lineages (Figure 3C,D bottom). Interestingly, and despite showing the least attenu- ation, FluB-RAM samples showed the lowest levels of anti-HA IgG and IgA responses among the three vaccine groups (Figure 3). g g p g Differences in serological responses against NA and NP were also observed (Figure 4). Anti-NA IgG and IgA showed a trend towards higher responses in samples from the FluB att group, although most of them were not statistically signiﬁcant (p > 0.05). How- ever, the FluB att vaccine induced a signiﬁcantly higher anti-B/Phuket/3073/2013 IgG response than the other two groups (p = 0.005 and p = 0.0016, respectively). It was noted that the NA antigen derived from the B/Phuket/3073/2013 provided more reliable sig- nals with low background noise (Figure 4A,B). In contrast, the NA antigen derived from B/Brisbane/60/2008 reacted poorly in the array when probing for IgG responses and provided a high background signal when probing for IgA responses. 3.4. Qualitative Differences in Humoral Responses among Different Vaccine Groups The trend of anti-NP IgG responses was also numerically higher in samples from the FluB att group (Figure 4C). Anti-NP IgA serum responses were low, except for the serum from one female in the FluB-RAM group, which clearly show reactivity well above background (Figure 4D). Inter- estingly, samples from the FluB-RANS and FluB-RAM groups had similar anti-NA and anti-NP responses, despite their differences in anti-HA responses. 3.4. Qualitative Differences in Humoral Responses among Different Vaccine Groups 3.4. Qualitative Differences in Humoral Responses among Different Vaccine Groups The humoral responses induced by the rearranged virus vaccines were analyzed utiliz- ing serum samples obtained at 19 days post-boost (19 dpb) from a subset of 4 mice/group (2 males, 2 females, except in the FluB-RAM group with 1 male and 2 female serum sam- ples). Please note that we included HI data from FluB att as it was part of the same study, although it was reported elsewhere [39]; these data were included for comparison purposes. Boost vaccination led to HI titers above 40, the predictive limit of protection (Figure 2C) with either rearranged virus. HI titers for the FluB-RAM group (80, 160, and 160 for each mouse, respectively) and for the FluB-RANS group (80, 80, 80, and 160 for each mouse, respec- tively) were lower than those obtained with the B/Bris att virus (160, 160, 320, and 320 for each mouse, respectively). In addition, we performed virus neutralizations assays against B/Bris (homologous) and B/Wis (heterologous). Sera from both FluB-RAM and FluB att showed similar mean neutralization titers. The neutralization titers induced by FluB-RANS sera were signiﬁcantly lower than those for the FluB att (p = 0.0095). As expected, no virus neutralization activity was detected against B/Wis (Figure 2C). To further understand possible differences in serological responses, IgG and IgA antibodies were analyzed using a protein microarray consisting of 22 HA proteins and 2 NA proteins derived from inﬂuenza B viruses (IBVs), corresponding to the two major lineages (Victoria and Yamagata), as well as a single NP protein from a prototypic IBV. Approximately one-third of the HA proteins are displayed as full length, whereas the rest correspond to the HA1 region. The array also contains many inﬂuenza A proteins, including group 1 and group 2 HA subtypes, NA subtypes, NP, M1, NS1, and NS2, which served as internal controls. Details of the strain of origin, source of the protein, and presence or absence of epitope tags are provided upon request. Side by side comparisons of the three vaccine groups, FluB-RAM, FluB-RANS, and FluB att, revealed qualitative differences in humoral responses for both IgG and IgA. Analysis of the serum samples post-boost showed that the FluB-RANS and FluB att groups had signiﬁcantly higher anti-B/Brisbane/60/2008-HA IgG responses than the FluB-RAM group (p = 0.0019 and p = 0.0064, respectively) (Figure 3A top). 3.5. FluB-RAM and FluB-RANS Effectively Protect Mice against Lethal IBV Challenge Protection efﬁcacy of the rearranged viruses was tested using a lethal challenge dose of 107 EID50/mouse of B/Bris/PB2 F406Y strain, administered i.n. [26] 3 weeks after boost. Mice in the three vaccine groups (FluB att data included for comparison) were fully protected as no signs of disease and no mortality were observed (Figure 5A,B). In contrast, PBS-vaccinated/challenged mice showed severe body weight loss. Only one female (out of 8) and none of the male mice survived in the PBS-vaccinated/challenged group, consistent with previous studies [26,30]. 3.6. Qualitative Differences in Humoral and Mucosal Responses among Different Vaccine Groups at 14 dpc 3.6. Qualitative Differences in Humoral and Mucosal Responses among Different Vaccine Groups at 14 dpc HI responses at 14 dpc were similar among the three vaccine groups, with a mean antibody titer increase of about 1 log2 compared with post-boost HI titers, particularly in samples from the rearranged vaccine groups (Figure 5C). No statistically signiﬁcant differences were observed between vaccine groups with trends like those observed post- boost. With respect to the rearranged vaccine groups, the data showed better responses in female mice than in male mice. Further analyses of serum and nasal wash samples collected at 14 dpc revealed recall IgG and IgA anti-HA responses against both the Victoria- and Yamagata-lineage antigens (Figure 6). As expected, the reactivity of serum samples from all vaccine groups against Victoria lineage HA antigens was 1.5–2-fold higher than to those of the Yamagata lineage (Figure 6A,B). Interestingly, the HA1 antigen derived Vaccines 2021, 9, 897 11 of 21 11 of 21 from B/Hong Kong/05/1972 (before the split of the two IBV lineages) reacted well with samples from all groups (Figure 6A), whereas the HA1 antigen from B/Florida/4/2006 and B/Utah/02/2007 (Yamagata lineage) shows the lowest reaction with the serum samples (Figure 6B). Of note, the full-length HA of B/Florida/4/2006 reacted well with samples from all three vaccine groups (Figure 6B). The pattern of anti-HA Victoria lineage serum IgA was similar among all vaccine groups, where differences in reactivity could be attributed to the different antigens in the array (Figure 6C). Post-challenge serum IgA responses against Yamagata-lineage HA antigens showed reactivity patterns attributed also to the different antigens, but trending towards better reactivity in samples from the FluB-RANS group (Figure 6D). 897 3. Lineage-specific IgG and IgA responses against the HA in serum at 19 dpb. IgG and IgA responses nalyzed using protein microarrays. Sera collected at 19 dpb from mice inoculated with either PBS, FluB-RA or FluB att (n = 4/group) were tested against a variety of purified IBV full HA or HA1 portion protein sed from Sino Biological. The results are expressed as the group mean fluorescence intensity (MFI) ± SD. 3.6. Qualitative Differences in Humoral and Mucosal Responses among Different Vaccine Groups at 14 dpc T I th Ab b d t ti l ti (A) A ti Vi t i I G (B) A ti Y t I G 0 20,000 40,000 60,000 B/HongKong/05/1972 B/Victoria/02/1987 B/Malaysia/2506/2004 B/Ohio/01/2005 B/Brisbane/60/2008 B/Massachusetts/03/2010 B/Wisconsin/02/2012 B/Malaysia/2506/2004 B/Brisbane/60/2008 B/Massachusetts/03/2010 MFI HA1 Full * * * * 0 25,000 50,000 B/HongKong/05/1972 B/Victoria/02/1987 B/Malaysia/2506/2004 B/Ohio/01/2005 B/Brisbane/60/2008 B/Massachusetts/03/2010 B/Wisconsin/02/2012 B/Malaysia/2506/2004 B/Brisbane/60/2008 B/Massachusetts/03/2010 MFI HA1 Full * 0 25,000 50,000 B/Yamagata/16/1988 B/Victoria/504/2000 B/Florida/07/2004 B/Florida/4/2006 B/Brisbane/3/2007 B/Utah/02/2012 B/Phuket/3073/2013 B/Yamagata/16/1988 B/Florida/4/2006 B/Utah/02/2012 B/Phuket/3073/2013 MFI HA1 Full 0 20,000 40,000 60,000 PBS FluB att FluB-RANS FluB-RAM ** 0 25,000 50,000 PBS FluB att FluB-RANS FluB-RAM ** * 0 25,000 50,000 PBS FluB att FluB-RANS FluB-RAM * A B C D IgG - Flu B HA - Victoria lineage - serum 19 dpb IgA - Flu B HA - Victoria lineage - serum 19 dpb IgA - Flu B HA - Yamagata lineage - serum 19 dpb 0 20,000 40,000 60,000 B/Yamagata/16/1988 B/Victoria/504/2000 B/Florida/07/2004 B/Florida/4/2006 B/Brisbane/3/2007 B/Utah/02/2012 B/Phuket/3073/2013 B/Yamagata/16/1988 B/Florida/4/2006 B/Utah/02/2012 B/Phuket/3073/2013 MFI HA1 Full 0 20,000 40,000 60,000 PBS FluB att FluB-RANS FluB-RAM ** IgG - Flu B HA - Yamagata lineage - serum 19 dpb FluB-RAM FluB-RANS FluB att PBS Figure 3. Lineage-specific IgG and IgA responses against the HA in serum at 19 dpb. IgG and Ig responses in serum were analyzed using protein microarrays. 3.6. Qualitative Differences in Humoral and Mucosal Responses among Different Vaccine Groups at 14 dpc he FluB inoculated with either PBS, FluB-RAM, FluB-RANS, or FluB att (n = 4/group) were tested against a variety of puriﬁed IBV full HA or HA1 portion protein antigens purchased from Sino Biological. The results are expressed as the group mean ﬂuorescence intensity (MFI) ± SD. The higher the MFI, the more Abs bound to a particular antigen. (A) Anti-Victoria IgG responses. (B) Anti-Yamagata IgG response. (C) Anti-Victoria IgA responses. (D) Anti-Yamagata IgA responses. MFIs were plotted and analyzed using Prism v9. Top graphs from each subﬁgure show the responses from each group against every single HA protein antigen; the bottom graphs summarize the combined IgG or IgA responses against a particular lineage. Statistical analysis to compare responses between groups was performed using two-way ANOVA followed by a Tukey’s test for multiple comparisons. Signiﬁcant differences between groups are denotated by stars (). = p < 0.05, = p < 0.01, * = p < 0.001, and **** = p < 0.0001. g p g y g (p ) FluB att vaccine induced a significantly higher anti-B/Phuket/3073/2013 IgG response than the other two groups (p = 0.005 and p = 0.0016, respectively). It was noted that the NA antigen derived from the B/Phuket/3073/2013 provided more reliable signals with low background noise (Figure 4A,B). In contrast, the NA antigen derived from B/Bris- bane/60/2008 reacted poorly in the array when probing for IgG responses and provided a high background signal when probing for IgA responses. The trend of anti-NP IgG re- sponses was also numerically higher in samples from the FluB att group (Figure 4C). Anti- NP IgA serum responses were low, except for the serum from one female in the FluB- RAM group, which clearly show reactivity well above background (Figure 4D). Interest- ingly, samples from the FluB-RANS and FluB-RAM groups had similar anti-NA and anti- NP responses, despite their differences in anti-HA responses. p p p Figure 4. Lineage-specific IgG and IgA responses against the NA and the NP in serum at 19 dpb. IgG and IgA responses in serum were analyzed using protein microarrays. Sera collected at 19 dpb from mice inoculated with either PBS, FluB-RAM, FluB-RANS, or FluB att (n = 4/group) were tested against purified IBV NA or NP protein antigens purchased from Sino Biological. The results are expressed as the group mean fluorescence intensity (MFI) ± SD. 3.6. Qualitative Differences in Humoral and Mucosal Responses among Different Vaccine Groups at 14 dpc The higher the MFI, the more Abs bound to a particular antigen. (A) Anti-NA IgG responses. (B) Anti-NA IgA responses. (C) Anti-NP IgG responses. (D) Anti-NP IgA responses. MFIs were plotted and analyzed using Prism v9. Statis- tical analysis to compare responses between groups was performed using two-way ANOVA fol- lowed by a Tukey’s test for multiple comparisons. Significant differences between groups are deno- tated by stars (). * = p < 0.01. 0 20,000 40,000 60,000 B/Phuket/3073/2013 B/Brisbane/60/2008 MFI 0 20,000 40,000 60,000 B/Florida/4/2006 MFI 0 1,000 2,000 3,000 B/Phuket/3073/2013 B/Brisbane/60/2008 MFI 0 20,000 40,000 B/Florida/4/2006 MFI FluB-RAM FluB-RANS FluB att PBS A B C D IgG - anti-NA - serum 19 dpb IgA - anti-NA - serum 19 dpb IgG - anti-NP - serum 19 dpb IgA - anti-NP - serum 19 dpb FluB-RAM FluB-RANS FluB att PBS Figure 4. Lineage-speciﬁc IgG and IgA responses against the NA and the NP in serum at 19 dpb. IgG and IgA responses in serum were analyzed using protein microarrays. Sera collected at 19 dpb from mice inoculated with either PBS, FluB-RAM, FluB-RANS, or FluB att (n = 4/group) were tested against puriﬁed IBV NA or NP protein antigens purchased from Sino Biological. The results are expressed as the group mean ﬂuorescence intensity (MFI) ± SD. The higher the MFI, the more Abs bound to a particular antigen. (A) Anti-NA IgG responses. (B) Anti-NA IgA responses. (C) Anti- NP IgG responses. (D) Anti-NP IgA responses. MFIs were plotted and analyzed using Prism v9. Statistical analysis to compare responses between groups was performed using two-way ANOVA followed by a Tukey’s test for multiple comparisons. Signiﬁcant differences between groups are denotated by stars (). * = p < 0.01. 3.6. Qualitative Differences in Humoral and Mucosal Responses among Different Vaccine Groups at 14 dpc Sera collected at 19 dpb from mic 0 20,000 40,000 60,000 B/HongKong/05/1972 B/Victoria/02/1987 B/Malaysia/2506/2004 B/Ohio/01/2005 B/Brisbane/60/2008 B/Massachusetts/03/2010 B/Wisconsin/02/2012 B/Malaysia/2506/2004 B/Brisbane/60/2008 B/Massachusetts/03/2010 MFI HA1 Full * * * * 0 20,000 40,000 60,000 PBS FluB att FluB-RANS FluB-RAM **** A IgG - Flu B HA - Victoria lineage - serum 19 dpb A B 0 20,000 40,000 60,000 B/HongKong/05/1972 B/Victoria/02/1987 B/Malaysia/2506/2004 B/Ohio/01/2005 B/Brisbane/60/2008 B/Massachusetts/03/2010 B/Wisconsin/02/2012 B/Malaysia/2506/2004 B/Brisbane/60/2008 B/Massachusetts/03/2010 MFI HA1 Full * * * * A IgG - Flu B HA - Victoria lineage - serum 19 dpb B 0 20,000 40,000 60,000 B/Yamagata/16/1988 B/Victoria/504/2000 B/Florida/07/2004 B/Florida/4/2006 B/Brisbane/3/2007 B/Utah/02/2012 B/Phuket/3073/2013 B/Yamagata/16/1988 B/Florida/4/2006 B/Utah/02/2012 B/Phuket/3073/2013 MFI HA1 Full IgG - Flu B HA - Yamagata lineage - serum 19 dpb B IgG - Flu B HA - Yamagata lineage - serum 19 dpb 0 20,000 40,000 60,000 PBS FluB att FluB-RANS FluB-RAM ** 0 20,000 40,000 60,000 PBS FluB att FluB-RANS FluB-RAM 0 25,000 50,000 B/Yamagata/16/1988 B/Victoria/504/2000 B/Florida/07/2004 B/Florida/4/2006 B/Brisbane/3/2007 B/Utah/02/2012 B/Phuket/3073/2013 B/Yamagata/16/1988 B/Florida/4/2006 B/Utah/02/2012 B/Phuket/3073/2013 MFI HA1 Full 0 25,000 50,000 PBS FluB att FluB-RANS FluB-RAM *** D IgA - Flu B HA - Yamagata lineage - serum 19 dpb NS FluB att PBS 0 25,000 50,000 B/HongKong/05/1972 B/Victoria/02/1987 B/Malaysia/2506/2004 B/Ohio/01/2005 B/Brisbane/60/2008 B/Massachusetts/03/2010 B/Wisconsin/02/2012 B/Malaysia/2506/2004 B/Brisbane/60/2008 B/Massachusetts/03/2010 MFI HA1 Full * 0 25,000 50,000 PBS FluB att FluB-RANS FluB-RAM **** * C IgA - Flu B HA - Victoria lineage - serum 19 dpb FluB-RAM FluB C D C IgA - Flu B HA - Victoria lineage - serum 19 dpb D IgA - Flu B HA - Yamagata lineage - serum 19 dpb D IgA - Flu B HA - Yamagata lineage - serum 19 dpb Lineage-specific IgG and IgA responses against the HA in serum at 19 dpb. IgG and IgA responses in s yzed using protein microarrays. Sera collected at 19 dpb from mice inoculated with either PBS, FluB-RAM, FluB att (n = 4/group) were tested against a variety of purified IBV full HA or HA1 portion protein ant Figure 3. Lineage-specific IgG and IgA responses against the HA in serum at 19 dpb. IgG and IgA responses in serum were analyzed using protein microarrays. Sera collected at 19 dpb from mice Vaccines 2021, 9, 897 12 of 21 gure 4). he FluB 12 of 21 gure 4). 3.6. Qualitative Differences in Humoral and Mucosal Responses among Different Vaccine Groups at 14 dpc 0 20,000 40,000 60,000 B/Phuket/3073/2013 B/Brisbane/60/2008 MFI 0 1,000 2,000 3,000 B/Phuket/3073/2013 B/Brisbane/60/2008 MFI FluB-RAM FluB-RANS FluB att PBS A B IgG - anti-NA - serum 19 dpb IgA - anti-NA - serum 19 dpb 0 20,000 40,000 60,000 B/Phuket/3073/2013 B/Brisbane/60/2008 MFI ** FluB-RAM Fl A IgG - anti-NA - serum 19 dpb 0 1,000 2,000 3,000 B/Phuket/3073/2013 B/Brisbane/60/2008 MFI B-RANS FluB att PBS B IgA - anti-NA - serum 19 dpb IgG - anti-NA - serum 19 dpb IgA - anti-NA - serum 19 dpb IgA - anti-NA - serum 19 dpb A B B/Brisbane/60/2008 0 20,000 40,000 60,000 B/Florida/4/2006 MFI C IgG - anti-NP - serum 19 dpb FluB-RAM FluB 0 20,000 40,000 B/Florida/4/2006 MFI D IgA - anti-NP - serum 19 dpb RANS FluB att PBS C D IgA - anti-NP - serum 19 dpb Lineage-specific IgG and IgA responses against the NA and the NP in serum at 19 dpb. Lineage-speciﬁc IgG and IgA responses against the NA and the NP in serum at 19 dpb. Figure 4. Lineage-specific IgG and IgA responses against the NA and the NP in serum at 19 dpb. IgG and IgA responses in serum were analyzed using protein microarrays. Sera collected at 19 dpb from mice inoculated with either PBS, FluB-RAM, FluB-RANS, or FluB att (n = 4/group) were tested against purified IBV NA or NP protein antigens purchased from Sino Biological. The results are expressed as the group mean fluorescence intensity (MFI) ± SD. The higher the MFI, the more Abs bound to a particular antigen. (A) Anti-NA IgG responses. (B) Anti-NA IgA responses. (C) Anti-NP IgG responses. (D) Anti-NP IgA responses. MFIs were plotted and analyzed using Prism v9. Statis- tical analysis to compare responses between groups was performed using two-way ANOVA fol- lowed by a Tukey’s test for multiple comparisons. Significant differences between groups are deno- tated by stars (). * = p < 0.01. Figure 4. Lineage-speciﬁc IgG and IgA responses against the NA and the NP in serum at 19 dpb. IgG and IgA responses in serum were analyzed using protein microarrays. Sera collected at 19 dpb from mice inoculated with either PBS, FluB-RAM, FluB-RANS, or FluB att (n = 4/group) were tested against puriﬁed IBV NA or NP protein antigens purchased from Sino Biological. The results are expressed as the group mean ﬂuorescence intensity (MFI) ± SD. 3.6. Qualitative Differences in Humoral and Mucosal Responses among Different Vaccine Groups at 14 dpc HI titers were compared between groups though a two-ay ANOVA followed by a Tukey’s test for multiple comparisons. Signiﬁcant differences between group are denotated by stars (). *** = p < 0.0001. HI responses at 14 dpc were similar among the three vaccine groups, antibody titer increase of about 1 log2 compared with post-boost HI titers, p samples from the rearranged vaccine groups (Figure 5C). No statistically si ferences were observed between vaccine groups with trends like those ob boost. With respect to the rearranged vaccine groups, the data showed better female mice than in male mice. Further analyses of serum and nasal wash lected at 14 dpc revealed recall IgG and IgA anti-HA responses against both and Yamagata-lineage antigens (Figure 6). As expected, the reactivity of se from all vaccine groups against Victoria lineage HA antigens was 1.5–2-fold to those of the Yamagata lineage (Figure 6A,B). Interestingly, the HA1 an Perhaps the most striking differences in IgG and IgA proﬁles were observed in the NW samples (Figure 7). The trend of serum IgG, but not IgA, anti-HA responses at 19 dpb (Figure 3) translated similarly in the NW samples for both IgG and IgA responses (Figure 7). Thus, a trend of higher IgG and IgA anti-HA responses was observed for samples of the FluB-RANS group, whereas those from the FluB-RAM and FluB att had the lowest of such responses and were like each other. The FluB-RANS group displayed signiﬁcantly higher general anti-Victoria IgG readings than both FluB-RAM and FluB att (p < 0.0001) (Figure 7A bottom), and a higher anti-Yamagata IgG response than FluB att (p = 0039). IgA responses detected in the NW material appeared to be more robust than the IgG responses. When comparing groups within the same HA antigen, the FluB-RANS group had signiﬁcantly higher anti-Victoria responses than FluB-RAM and FluB att for B/Massachusetts/03/2010 (p = 0.0019 and p = 0.0156), B/Brisbane/60/2008 (p = 0.0047 and p = 0.0370), B/Malaysia/2506/2004 (p = 0.0294 vs. Flu Batt only), B/Wisconsin/02/2012- HA1 (p = 0.0021 and p = 0.0122), B/Massachusetts/03/2010-HA1 (p = 0.0006 and p = 0.0014), B/Brisbane/60/2008-HA1 (p = 0.0134 vs. FluB att only), and B/Ohio/01/2005-HA1 (p = 0.0153 and p = 0.0322). 3.6. Qualitative Differences in Humoral and Mucosal Responses among Different Vaccine Groups at 14 dpc The higher the MFI, the more Abs bound to a particular antigen. (A) Anti-NA IgG responses. (B) Anti-NA IgA responses. (C) Anti- NP IgG responses. (D) Anti-NP IgA responses. MFIs were plotted and analyzed using Prism v9. Statistical analysis to compare responses between groups was performed using two-way ANOVA followed by a Tukey’s test for multiple comparisons. Signiﬁcant differences between groups are denotated by stars (). * = p < 0.01. Figure 4. Lineage-specific IgG and IgA responses against the NA and the NP in serum at 19 dpb. IgG and IgA responses in serum were analyzed using protein microarrays. Sera collected at 19 dpb from mice inoculated with either PBS, FluB-RAM, FluB-RANS, or FluB att (n = 4/group) were tested against purified IBV NA or NP protein antigens purchased from Sino Biological. The results are expressed as the group mean fluorescence intensity (MFI) ± SD. The higher the MFI, the more Abs bound to a particular antigen. (A) Anti-NA IgG responses. (B) Anti-NA IgA responses. (C) Anti-NP IgG responses. (D) Anti-NP IgA responses. MFIs were plotted and analyzed using Prism v9. Statis- tical analysis to compare responses between groups was performed using two-way ANOVA fol- lowed by a Tukey’s test for multiple comparisons. Significant differences between groups are deno- tated by stars (). * = p < 0.01. Figure 4. Lineage-speciﬁc IgG and IgA responses against the NA and the NP in serum at 19 dpb. IgG and IgA responses in serum were analyzed using protein microarrays. Sera collected at 19 dpb from mice inoculated with either PBS, FluB-RAM, FluB-RANS, or FluB att (n = 4/group) were tested against puriﬁed IBV NA or NP protein antigens purchased from Sino Biological. The results are expressed as the group mean ﬂuorescence intensity (MFI) ± SD. The higher the MFI, the more Abs bound to a particular antigen. (A) Anti-NA IgG responses. (B) Anti-NA IgA responses. (C) Anti- NP IgG responses. (D) Anti-NP IgA responses. MFIs were plotted and analyzed using Prism v9. Statistical analysis to compare responses between groups was performed using two-way ANOVA followed by a Tukey’s test for multiple comparisons. Signiﬁcant differences between groups are denotated by stars (). * = p < 0.01. 13 of 21 hallenge Vaccines 2021, 9, 897 5. (A) Monitoring body weight in male and female mice. After challenge, body weight was monitored for Survival after challenge. 3.6. Qualitative Differences in Humoral and Mucosal Responses among Different Vaccine Groups at 14 dpc Mortality in males and females was recorded until 14 dpc. (C) Post-challenge ermination. Blood samples were collected for serology at 14 dpc. Sera were separated and used to pe comparing males and females. Body weight values were graphed as the group mean ± SD. Survival d d using the log-rank test. HI titers are represented as the group mean ± SD. Data analysis and graphs were rism v9. HI titers were compared between groups though a two-ay ANOVA followed by a Tukey’s test for isons. Significant differences between group are denotated by stars (). * = p < 0.0001. 3.6. Qualitative Differences in Humoral and Mucosal Responses among Different V Groups at 14 dpc 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 0 20 40 60 80 100 Days after challenge Survival (%) Male mice - survival post-challenge 0 1 2 3 4 5 6 7 8 9 10 11 12 60 70 80 90 100 110 Days post challenge Body Weight (% change) Male mice - body weight changes post-challenge Males Females 10 20 40 80 160 320 640 HI titer 14 days post-challenge 0 1 2 3 4 5 6 7 8 9 10 11 12 60 70 80 90 100 110 Days post challenge Body Weight (% change) Female mice - body weight changes post-challenge 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 0 20 40 60 80 100 Days after challenge Survival (%) Female mice - survival post-challenge ** PBS-Challenge FluB-RAM/Ch FluB-RANS/Ch A B C FluB-RAM FluB-RANS FluB att PBS FluB att/Ch Figure 5. (A) Monitoring body weight in male and female mice. After challenge, body weight was monitored for up to 12 dpc. (B) Survival after challenge. Mortality in males and females was recorded until 14 dpc. (C) Post-challenge antibody titer determination. Blood samples were collected for serology at 14 dpc. Sera were separated and used to perform HI assays comparing males and females. Body weight values were graphed as the group mean ± SD. Survival data were analyzed using the log-rank test. HI titers are represented as the group mean ± SD. Data analysis and graphs were prepared using Prism v9. HI titers were compared between groups though a two-ay ANOVA followed by a Tukey’s test for multiple comparisons. Signiﬁcant differences between group are denotated by stars (). 3.6. Qualitative Differences in Humoral and Mucosal Responses among Different Vaccine Groups at 14 dpc * = p < 0.0001. 0 1 2 3 4 5 6 7 8 9 10 11 12 60 70 80 90 100 110 Days post challenge Body Weight (% change) Male mice - body weight changes post-challenge A A 0 1 2 3 4 5 6 7 8 9 10 11 12 60 70 80 90 100 110 Days post challenge Body Weight (% change) Female mice - body weight changes post-challenge PBS-Challenge FluB-RAM/Ch FluB-RANS/Ch FluB att/Ch 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 0 20 40 60 80 100 Days after challenge Survival (%) Male mice - survival post-challenge B B 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 0 20 40 60 80 100 Days after challenge Survival (%) Female mice - survival post-challenge ** Males Females 10 20 40 80 160 320 640 HI titer 14 days post-challenge FluB-RAM FluB-RANS FluB att PBS C (A) Monitoring body weight in male and female mice. After challenge, body weight was monitored fo Survival after challenge. Mortality in males and females was recorded until 14 dpc. (C) Post-challenge rmination. Blood samples were collected for serology at 14 dpc. Sera were separated and used to pe omparing males and females. Body weight values were graphed as the group mean ± SD. Survival d using the log-rank test. HI titers are represented as the group mean ± SD. Data analysis and graphs were sm v9. HI titers were compared between groups though a two-ay ANOVA followed by a Tukey’s test for ons. Significant differences between group are denotated by stars (). *** = p < 0.0001. 3.6. Qualitative Differences in Humoral and Mucosal Responses among Different V Groups at 14 dpc Figure 5. (A) Monitoring body weight in male and female mice. After challenge, body weight was monitored for up to 12 dpc. (B) Survival after challenge. Mortality in males and females was recorded until 14 dpc. (C) Post-challenge antibody titer determination. Blood samples were collected for serology at 14 dpc. Sera were separated and used to perform HI assays comparing males and females. Body weight values were graphed as the group mean ± SD. Survival data were analyzed using the log-rank test. HI titers are represented as the group mean ± SD. Data analysis and graphs were prepared using Prism v9. 3.6. Qualitative Differences in Humoral and Mucosal Responses among Different Vaccine Groups at 14 dpc (C) Anti-Victoria IgA 0 20,000 40,000 60,000 80,000 B/HongKong/05/1972 B/Victoria/02/1987 B/Malaysia/2506/2004 B/Ohio/01/2005 B/Brisbane/60/2008 B/Massachusetts/03/2010 B/Wisconsin/02/2012 B/Malaysia/2506/2004 B/Brisbane/60/2008 B/Massachusetts/03/2010 MFI HA1 Full A IgG - Flu B HA - Victoria lineage - serum 14 dpc 0 20,000 40,000 60,000 80,000 B/HongKong/05/1972 B/Victoria/02/1987 B/Malaysia/2506/2004 B/Ohio/01/2005 B/Brisbane/60/2008 B/Massachusetts/03/2010 B/Wisconsin/02/2012 B/Malaysia/2506/2004 B/Brisbane/60/2008 B/Massachusetts/03/2010 MFI HA1 Full 0 20,000 40,000 60,000 PBS FluB att FluB-RANS FluB-RAM A IgG - Flu B HA - Victoria lineage - serum 14 dpc B 0 20,000 40,000 60,000 80,000 B/Yamagata/16/1988 B/Victoria/504/2000 B/Florida/07/2004 B/Florida/4/2006 B/Brisbane/3/2007 B/Utah/02/2012 B/Phuket/3073/2013 B/Yamagata/16/1988 B/Florida/4/2006 B/Utah/02/2012 B/Phuket/3073/2013 MFI HA1 Full 0 20,000 40,000 60,000 PBS FluB att FluB-RANS FluB-RAM * IgG - Flu B HA - Yamagata lineage - serum 14 dpc B 0 20,000 40,000 60,000 80,000 B/Yamagata/16/1988 B/Victoria/504/2000 B/Florida/07/2004 B/Florida/4/2006 B/Brisbane/3/2007 B/Utah/02/2012 B/Phuket/3073/2013 B/Yamagata/16/1988 B/Florida/4/2006 B/Utah/02/2012 B/Phuket/3073/2013 MFI HA1 Full IgG - Flu B HA - Yamagata lineage - serum 14 dp A B A IgG - Flu B HA - Victoria lineage - serum 14 dpc IgG - Flu B HA - Yamagata lineage - serum 14 dpc MFI 0 20,000 40,000 60,000 PBS FluB att FluB-RANS FluB-RAM * 0 25,000 50,000 B/Yamagata/16/1988 B/Victoria/504/2000 B/Florida/07/2004 B/Florida/4/2006 B/Brisbane/3/2007 B/Utah/02/2012 B/Phuket/3073/2013 B/Yamagata/16/1988 B/Florida/4/2006 B/Utah/02/2012 B/Phuket/3073/2013 MFI HA1 Full 0 10,000 20,000 PBS FluB att FluB-RANS FluB-RAM * D IgA Flu B HA - Yamagata lineage - serum 14 dpc NS FluB att PBS 0 25,000 50,000 B/HongKong/05/1972 B/Victoria/02/1987 B/Malaysia/2506/2004 B/Ohio/01/2005 B/Brisbane/60/2008 B/Massachusetts/03/2010 B/Wisconsin/02/2012 B/Malaysia/2506/2004 B/Brisbane/60/2008 B/Massachusetts/03/2010 MFI HA1 Full 0 12,500 25,000 37,500 50,000 PBS FluB att FluB-RANS FluB-RAM C IgA - Flu B HA - Victoria lineage - serum 14 dpc FluB-RAM FluB-R 0 25,000 50,000 B/Yamagata/16/1988 B/Victoria/504/2000 B/Florida/07/2004 B/Florida/4/2006 B/Brisbane/3/2007 B/Utah/02/2012 B/Phuket/3073/2013 B/Yamagata/16/1988 B/Florida/4/2006 B/Utah/02/2012 B/Phuket/3073/2013 MFI HA1 Full D IgA Flu B HA - Yamagata lineage - serum 14 dpc C D C IgA - Flu B HA - Victoria lineage - serum 14 dpc D IgA Flu B HA - Yamagata lineage - serum 14 dpc MFI 0 10,000 20,000 PBS FluB att FluB-RANS FluB-RAM * FluB att PBS FluB att Figure 6. Lineage-specific IgG and IgA responses against the HA in serum at 14 dpc. IgG and IgA responses in serum challenge were analyzed using protein microarrays. Sera collected at 14 dpc from mice challenged with B/Bris/PB2 (n = 4/group) were tested against a variety of purified IBV full HA or HA1 portion protein antigens purchased from Biological. 3.6. Qualitative Differences in Humoral and Mucosal Responses among Different Vaccine Groups at 14 dpc When comparing general anti-Victoria and -Yamagata re- sponses, FluB-RANS had signiﬁcantly higher IgA responses than the other two groups (Vic- p < 0.0001 and p < 0.0001; Yam- p < 0.0001 and p = 0.0201) (Figure 7C,D bottom). Further, 14 of 21 Vaccines 2021, 9, 897 signals were stronger for the full-length HAs and more prominent for the Victoria-lineage antigens compared with the Yamagata-lineage antigens, as expected (Figure 7A,D). 1 signals were stronger for the full-length HAs and more prominent for the Victoria-lineage antigens compared with the Yamagata-lineage antigens, as expected (Figure 7A,D). 1 Figure 6. Lineage-specific IgG and IgA responses against the HA in serum at 14 dpc. IgG and IgA responses in se challenge were analyzed using protein microarrays. Sera collected at 14 dpc from mice challenged with B/Bris/P (n = 4/group) were tested against a variety of purified IBV full HA or HA1 portion protein antigens purchased f Biological. The results are expressed as the group mean fluorescence intensity (MFI) ± SD. The higher the MFI, Abs bound to a particular antigen. (A) Anti-Victoria IgG responses. (B) Anti-Yamagata IgG response. (C) Ant IgA responses. (D) Anti-Yamagata IgA responses. MFIs were plotted and analyzed using Prism v9. Top graphs f subfigure show the responses from each group against every single HA protein antigen; the bottom graphs su 0 20,000 40,000 60,000 80,000 B/HongKong/05/1972 B/Victoria/02/1987 B/Malaysia/2506/2004 B/Ohio/01/2005 B/Brisbane/60/2008 B/Massachusetts/03/2010 B/Wisconsin/02/2012 B/Malaysia/2506/2004 B/Brisbane/60/2008 B/Massachusetts/03/2010 MFI HA1 Full 0 25,000 50,000 B/HongKong/05/1972 B/Victoria/02/1987 B/Malaysia/2506/2004 B/Ohio/01/2005 B/Brisbane/60/2008 B/Massachusetts/03/2010 B/Wisconsin/02/2012 B/Malaysia/2506/2004 B/Brisbane/60/2008 B/Massachusetts/03/2010 MFI HA1 Full 0 25,000 50,000 B/Yamagata/16/1988 B/Victoria/504/2000 B/Florida/07/2004 B/Florida/4/2006 B/Brisbane/3/2007 B/Utah/02/2012 B/Phuket/3073/2013 B/Yamagata/16/1988 B/Florida/4/2006 B/Utah/02/2012 B/Phuket/3073/2013 MFI HA1 Full 0 20,000 40,000 60,000 PBS FluB att FluB-RANS FluB-RAM 0 12,500 25,000 37,500 50,000 PBS FluB att FluB-RANS FluB-RAM 0 10,000 20,000 PBS FluB att FluB-RANS FluB-RAM * A B C D IgG - Flu B HA - Victoria lineage - serum 14 dpc IgA - Flu B HA - Victoria lineage - serum 14 dpc IgA Flu B HA - Yamagata lineage - serum 14 dpc 0 20,000 40,000 60,000 80,000 B/Yamagata/16/1988 B/Victoria/504/2000 B/Florida/07/2004 B/Florida/4/2006 B/Brisbane/3/2007 B/Utah/02/2012 B/Phuket/3073/2013 B/Yamagata/16/1988 B/Florida/4/2006 B/Utah/02/2012 B/Phuket/3073/2013 MFI HA1 Full 0 20,000 40,000 60,000 PBS FluB att FluB-RANS FluB-RAM * IgG - Flu B HA - Yamagata lineage - serum 14 dpc FluB-RAM FluB-RANS FluB att PBS ure 6. Lineage-specific IgG and IgA responses against the HA in serum at 14 dpc. 3.6. Qualitative Differences in Humoral and Mucosal Responses among Different Vaccine Groups at 14 dpc IgG and IgA responses in serum afte lenge were analyzed using protein microarrays. Sera collected at 14 dpc from mice challenged with B/Bris/PB2 F406Y 4/group) were tested against a variety of purified IBV full HA or HA1 portion protein antigens purchased from Sin ogical. The results are expressed as the group mean fluorescence intensity (MFI) ± SD. The higher the MFI, the mor bound to a particular antigen. (A) Anti-Victoria IgG responses. (B) Anti-Yamagata IgG response. 3.6. Qualitative Differences in Humoral and Mucosal Responses among Different Vaccine Groups at 14 dpc (C) Anti B 0 5,000 10,000 15,000 B/Yamagata/16/1988 B/Victoria/504/2000 B/Florida/07/2004 B/Florida/4/2006 B/Brisbane/3/2007 B/Utah/02/2012 B/Phuket/3073/2013 B/Yamagata/16/1988 B/Florida/4/2006 B/Utah/02/2012 B/Phuket/3073/2013 MFI HA1 Full IgG - Flu B HA - Yamagata lineage - NW 14 dpc B D 0 5,000 10,000 15,000 B/Yamagata/16/1988 B/Victoria/504/2000 B/Florida/07/2004 B/Florida/4/2006 B/Brisbane/3/2007 B/Utah/02/2012 B/Phuket/3073/2013 B/Yamagata/16/1988 B/Florida/4/2006 B/Utah/02/2012 B/Phuket/3073/2013 MFI HA1 Full 0 2,000 4,000 6,000 8,000 PBS FluB att FluB-RANS FluB-RAM IgG - Flu B HA - Yamagata lineage - NW 14 dpc B 0 20,000 40,000 60,000 B/HongKong/05/1972 B/Victoria/02/1987 B/Malaysia/2506/2004 B/Ohio/01/2005 B/Brisbane/60/2008 B/Massachusetts/03/2010 B/Wisconsin/02/2012 B/Malaysia/2506/2004 B/Brisbane/60/2008 B/Massachusetts/03/2010 MFI HA1 Full 0 10,000 20,000 30,000 40,000 PBS FluB att FluB-RANS FluB-RAM ** A IgG - Flu B HA - Victoria lineage - NW 14 dpc A A IgG - Flu B HA - Victoria lineage - NW 14 dpc A IgG - Flu B HA - Victoria lineage - NW 14 dpc IgG - Flu B HA - Yamagata lineage - NW 14 dpc B IgG - Flu B HA - Yamagata lineage - NW 14 dpc 0 20,000 40,000 60,000 B/HongKong/05/1972 B/Victoria/02/1987 B/Malaysia/2506/2004 B/Ohio/01/2005 B/Brisbane/60/2008 B/Massachusetts/03/2010 B/Wisconsin/02/2012 B/Malaysia/2506/2004 B/Brisbane/60/2008 B/Massachusetts/03/2010 MFI HA1 Full A IgG - Flu B HA - Victoria lineage - NW 14 dpc MFI 0 2,000 4,000 6,000 8,000 PBS FluB att FluB-RANS FluB-RAM 0 10,000 20,000 30,000 40,000 PBS FluB att FluB-RANS FluB-RAM 0 10,000 20,000 30,000 40,000 B/Yamagata/16/1988 B/Victoria/504/2000 B/Florida/07/2004 B/Florida/4/2006 B/Brisbane/3/2007 B/Utah/02/2012 B/Phuket/3073/2013 B/Yamagata/16/1988 B/Florida/4/2006 B/Utah/02/2012 B/Phuket/3073/2013 MFI HA1 Full D IgA Flu B HA - Yamagata lineage - NW 14 dpc 0 20,000 40,000 60,000 B/HongKong/05/1972 B/Victoria/02/1987 B/Malaysia/2506/2004 B/Ohio/01/2005 B/Brisbane/60/2008 B/Massachusetts/03/2010 B/Wisconsin/02/2012 B/Malaysia/2506/2004 B/Brisbane/60/2008 B/Massachusetts/03/2010 MFI HA1 Full * * *** * * * C IgA - Flu B HA - Victoria lineage - NW 14 dpc D IgA - Flu B HA - Victoria lineage - NW 14 dpc C D IgA Flu B HA - Yamagata lineage - NW 14 dpc 0 6,000 12,000 18,000 24,000 PBS FluB att FluB-RANS FluB-RAM **** * FluB att PBS 0 10,000 20,000 30,000 40,000 PBS FluB att FluB-RANS FluB-RAM ** ** FluB-RAM FluB-RANS FluB-RANS FluB-RAM FluB att Figure 7. Post-challenge lineage-specific IgG and IgA responses against the HA in nasal wash (NW) material. IgG and IgA responses in NW were analyzed using protein microarrays. 3.6. Qualitative Differences in Humoral and Mucosal Responses among Different Vaccine Groups at 14 dpc The results are expressed as the group mean fluorescence intensity (MFI) ± SD. The higher the MFI, th Abs bound to a particular antigen. (A) Anti-Victoria IgG responses. (B) Anti-Yamagata IgG response. (C) Anti-V IgA responses. (D) Anti-Yamagata IgA responses. MFIs were plotted and analyzed using Prism v9. Top graphs from Figure 6. Lineage-specific IgG and IgA responses against the HA in serum at 14 dpc. IgG and IgA responses in serum after challenge were analyzed using protein microarrays. Sera collected at 14 dpc from mice challenged with B/Bris/PB2 F406Y (n = 4/group) were tested against a variety of purified IBV full HA or HA1 portion protein antigens purchased from Sino Biological. The results are expressed as the group mean fluorescence intensity (MFI) ± SD. The higher the MFI, the more Abs bound to a particular antigen. (A) Anti-Victoria IgG responses. (B) Anti-Yamagata IgG response. (C) Anti-Victoria IgA 15 of 21 Vaccines 2021, 9, 897 responses. (D) Anti-Yamagata IgA responses. MFIs were plotted and analyzed using Prism v9. Top graphs from each subﬁgure show the responses from each group against every single HA protein antigen; the bottom graphs summarize the combined IgG or IgA responses against a particular lineage. Statistical analysis to compare responses between groups was performed using two-way ANOVA followed by a Tukey’s test for multiple comparisons. Signiﬁcant differences between groups are denotated by stars (). = p < 0.05, = p < 0.01, * = p < 0.001. es 2021, 9, 897 16 of 2 responses. (D) Anti-Yamagata IgA responses. MFIs were plotted and analyzed using Prism v9. Top graphs from each subﬁgure show the responses from each group against every single HA protein antigen; the bottom graphs summarize the combined IgG or IgA responses against a particular lineage. Statistical analysis to compare responses between groups was performed using two-way ANOVA followed by a Tukey’s test for multiple comparisons. Signiﬁcant differences between groups are denotated by stars (). = p < 0.05, = p < 0.01, * = p < 0.001. es 2021, 9, 897 16 of 2 e 7. Post-challenge lineage-specific IgG and IgA responses against the HA in nasal wash (NW) material. IgG an nses in NW were analyzed using protein microarrays. 3.6. Qualitative Differences in Humoral and Mucosal Responses among Different Vaccine Groups at 14 dpc NW collected at 14 dpc from mice challenged with B/Bri Y (n = 4/group) was tested against a variety of purified IBV full HA or HA1 portion protein antigens purchased Biological. The results are expressed as the group mean fluorescence intensity (MFI) ± SD. The higher the MF Abs bound to a particular antigen. (A) Anti-Victoria IgG responses. (B) Anti-Yamagata IgG response. (C) Ant gA responses. (D) Anti-Yamagata IgA responses. MFIs were plotted and analyzed using Prism v9. Top graphs ubfigure show the responses from each group against every single HA protein antigen; the bottom graphs su 0 20,000 40,000 60,000 B/HongKong/05/1972 B/Victoria/02/1987 B/Malaysia/2506/2004 B/Ohio/01/2005 B/Brisbane/60/2008 B/Massachusetts/03/2010 B/Wisconsin/02/2012 B/Malaysia/2506/2004 B/Brisbane/60/2008 B/Massachusetts/03/2010 MFI HA1 Full 0 20,000 40,000 60,000 B/HongKong/05/1972 B/Victoria/02/1987 B/Malaysia/2506/2004 B/Ohio/01/2005 B/Brisbane/60/2008 B/Massachusetts/03/2010 B/Wisconsin/02/2012 B/Malaysia/2506/2004 B/Brisbane/60/2008 B/Massachusetts/03/2010 MFI HA1 Full * * *** * * * 0 10,000 20,000 30,000 40,000 B/Yamagata/16/1988 B/Victoria/504/2000 B/Florida/07/2004 B/Florida/4/2006 B/Brisbane/3/2007 B/Utah/02/2012 B/Phuket/3073/2013 B/Yamagata/16/1988 B/Florida/4/2006 B/Utah/02/2012 B/Phuket/3073/2013 MFI HA1 Full 0 10,000 20,000 30,000 40,000 PBS FluB att FluB-RANS FluB-RAM ** 0 10,000 20,000 30,000 40,000 PBS FluB att FluB-RANS FluB-RAM 0 6,000 12,000 18,000 24,000 PBS FluB att FluB-RANS FluB-RAM ** * A B C D IgG - Flu B HA - Victoria lineage - NW 14 dpc IgA - Flu B HA - Victoria lineage - NW 14 dpc IgA Flu B HA - Yamagata lineage - NW 14 dpc 0 5,000 10,000 15,000 B/Yamagata/16/1988 B/Victoria/504/2000 B/Florida/07/2004 B/Florida/4/2006 B/Brisbane/3/2007 B/Utah/02/2012 B/Phuket/3073/2013 B/Yamagata/16/1988 B/Florida/4/2006 B/Utah/02/2012 B/Phuket/3073/2013 MFI HA1 Full 0 2,000 4,000 6,000 8,000 PBS FluB att FluB-RANS FluB-RAM ** IgG - Flu B HA - Yamagata lineage - NW 14 dpc FluB-RAM FluB-RANS FluB att PBS ure 7. Post-challenge lineage-speciﬁc IgG and IgA responses against the HA in nasal wash (NW) material. IgG onses in NW were analyzed using protein microarrays. NW collected at 14 dpc from mice challenged with B/B Y (n = 4/group) was tested against a variety of puriﬁed IBV full HA or HA1 portion protein antigens purchas Biological. The results are expressed as the group mean ﬂuorescence intensity (MFI) ± SD. The higher the e Abs bound to a particular antigen. (A) Anti-Victoria IgG responses. (B) Anti-Yamagata IgG response. 3.6. Qualitative Differences in Humoral and Mucosal Responses among Different Vaccine Groups at 14 dpc Thus, samples from the FluB-RANS group tended to have the lowest IgA responses in both serum and NW samples, whereas those from the FluB-RAM group had the overall highest responses, particularly against NP, although not statistically different (p > 0.05). Overall, the pattern of anti-NA and anti-NP responses post-challenge showed opposite trends with respect to the anti-HA responses at 14 dpc (Figures 6–8). Differences were also observed at 14 dpc in the pattern of IgG and IgA serum and NW reactivity against the NA and NP antigens on the array (Figure 8). Anti-NA and anti- NP IgG serum responses at 14 dpc were similar among vaccine groups (Figure 8A), but were close to background against NA and low against NP in NW at 14 dpc (Figure 8B). The pattern of IgA serum and NW against NA and NP at 14 dpc (Figure 8C,D) followed the patterns observed at 19 dpb, despite their initial low signals (Figure 4B,D). Thus, sam- ples from the FluB-RANS group tended to have the lowest IgA responses in both serum and NW samples, whereas those from the FluB-RAM group had the overall highest re- sponses, particularly against NP, although not statistically different (p > 0.05). Overall, the pattern of anti-NA and anti-NP responses post-challenge showed opposite trends with respect to the anti-HA responses at 14 dpc (Figures 6–8) Figure 8. Post-challenge lineage-specific IgG and IgA responses against the NA and the NP in serum and NW. IgG and IgA responses in serum were analyzed using protein microarrays. Sera and NW collected at 14 dpc from mice challenged with B/Bris/PB2 F406Y (n = 4/group) were tested against purified IBV NA or NP protein antigens purchased from Sino Biological. The results are expressed as the group mean fluorescence intensity (MFI) ± SD. The higher the MFI, the more Abs bound to a particular antigen. (A) Anti-NA and -NP IgG responses in serum. (B) Anti-NA and -NP IgG re- sponses in NW. (C) Anti-NA and -NP IgA responses in serum. (D) Anti-NA and -NP IgA responses in NW. MFIs were plotted and analyzed using Prism v9. Statistical analysis to compare responses between groups was performed using two-way ANOVA followed by a Tukey’s test for multiple comparisons. Significant differences between groups are denotated by stars (). = p < 0.05. 4. 3.6. Qualitative Differences in Humoral and Mucosal Responses among Different Vaccine Groups at 14 dpc NW collected at 14 dpc from mice challenged with B/Bris/PB2 F406Y (n = 4/group) was tested against a variety of purified IBV full HA or HA1 portion protein antigens purchased from Sino Biological. The results are expressed as the group mean fluorescence intensity (MFI) ± SD. The higher the MFI, the more Abs bound to a particular antigen. (A) Anti-Victoria IgG responses. (B) Anti-Yamagata IgG response. (C) Anti-Vic- t i I A (D) A ti Y t I A MFI l tt d d l d i P i 9 T h f Figure 7. Post-challenge lineage-speciﬁc IgG and IgA responses against the HA in nasal wash (NW) material. IgG and IgA responses in NW were analyzed using protein microarrays. NW collected at 14 dpc from mice challenged with B/Bris/PB2 F406Y (n = 4/group) was tested against a variety of puriﬁed IBV full HA or HA1 portion protein antigens purchased from Sino Biological. The results are expressed as the group mean ﬂuorescence intensity (MFI) ± SD. The higher the MFI, the more Abs bound to a particular antigen. (A) Anti-Victoria IgG responses. (B) Anti-Yamagata IgG response. (C) Anti-Victoria 16 of 21 Vaccines 2021, 9, 897 IgA responses. (D) Anti-Yamagata IgA responses. MFIs were plotted and analyzed using Prism v9. Top graphs from each subﬁgure show the responses from each group against every single HA protein antigen; the bottom graphs summarize the combined IgG or IgA responses against a particular lineage. Statistical analysis to compare responses between groups was performed using two-way ANOVA followed by a Tukey’s test for multiple comparisons. Signiﬁcant differences between groups are denotated by stars (). = p < 0.05, = p < 0.01, * = p < 0.001, and **** = p < 0.0001. Vaccines 2021, 9, 897 17 of Differences were also observed at 14 dpc in the pattern of IgG and IgA serum and NW reactivity against the NA and NP antigens on the array (Figure 8). Anti-NA and anti-NP IgG serum responses at 14 dpc were similar among vaccine groups (Figure 8A), but were close to background against NA and low against NP in NW at 14 dpc (Figure 8B). The pattern of IgA serum and NW against NA and NP at 14 dpc (Figure 8C,D) followed the patterns observed at 19 dpb, despite their initial low signals (Figure 4B,D). 3.6. Qualitative Differences in Humoral and Mucosal Responses among Different Vaccine Groups at 14 dpc Discussion 0 20,000 40,000 60,000 B/Florida/4/2006 B/Phuket/3073/2013 B/Brisbane/60/2008 MFI anti-NA anti-NP 0 20,000 40,000 60,000 80,000 B/Florida/4/2006 B/Phuket/3073/2013 B/Brisbane/60/2008 MFI anti-NA anti-NP * * FluB-RAM FluB-RANS FluB att PBS A B C D IgA - anti-NA and anti-NP - serum 14 dpc IgA - anti-NA and anti-NP - NW 14 dpc 0 10,000 20,000 30,000 40,000 B/Florida/4/2006 B/Phuket/3073/2013 B/Brisbane/60/2008 MFI anti-NA anti-NP IgG - anti-NA and anti-NP - serum 14 dpc IgG - anti-NA and anti-NP - NW 14 dpc 0 2,000 4,000 8000 10000 B/Florida/4/2006 B/Phuket/3073/2013 B/Brisbane/60/2008 MFI anti-NA anti-NP Figure 8. Post-challenge lineage-speciﬁc IgG and IgA responses against the NA and the NP in serum and NW. IgG and IgA responses in serum were analyzed using protein microarrays. Sera and NW collected at 14 dpc from mice challenged with B/Bris/PB2 F406Y (n = 4/group) were tested against puriﬁed IBV NA or NP protein antigens purchased from Sino Biological. The results are expressed as the group mean ﬂuorescence intensity (MFI) ± SD. The higher the MFI, the more Abs bound to a particular antigen. (A) Anti-NA and -NP IgG responses in serum. (B) Anti-NA and -NP IgG responses in NW. (C) Anti-NA and -NP IgA responses in serum. (D) Anti-NA and -NP IgA responses in NW. MFIs were plotted and analyzed using Prism v9. Statistical analysis to compare responses between groups was performed using two-way ANOVA followed by a Tukey’s test for multiple comparisons. Signiﬁcant differences between groups are denotated by stars (). = p < 0.05. B 0 10,000 20,000 30,000 40,000 B/Florida/4/2006 B/Phuket/3073/2013 B/Brisbane/60/2008 MFI anti-NA anti-NP IgG - anti-NA and anti-NP - NW 14 dpc 0 20,000 40,000 60,000 80,000 B/Florida/4/2006 B/Phuket/3073/2013 B/Brisbane/60/2008 MFI anti-NA anti-NP * * FluB-R FluB-R FluB a PBS A IgG - anti-NA and anti-NP - serum 14 dpc B A IgG - anti-NA and anti-NP - NW 14 dpc IgG - anti-NA and anti-NP - NW 14 dpc D IgA - anti-NA and anti-NP - NW 14 dpc 0 2,000 4,000 8000 10000 B/Florida/4/2006 B/Phuket/3073/2013 B/Brisbane/60/2008 MFI anti-NA anti-NP 0 20,000 40,000 60,000 B/Florida/4/2006 B/Phuket/3073/2013 B/Brisbane/60/2008 MFI anti-NA anti-NP C IgA - anti-NA and anti-NP - serum 14 dpc D C IgA - anti-NA and anti-NP - NW 14 dpc IgA - anti-NA and anti-NP - NW 14 dpc IgA - anti-NA and anti-NP - serum 14 dpc anti-NA anti-NA Figure 8. 4. Discussion Inﬂuenza virus genome re-arrangement is a viable alternative for the development LAIV vaccines. We previously showed such potential within the background of a H9N2 virus carrying full-length H9 and H5 HA proteins while maintaining a full set of the re- maining viral proteins [31]. Like the approach followed in this study, the NS2 ORF from the H9N2 IAV was inserted downstream the PB1 gene, whereas the NS segment was modiﬁed to carry the NS1 ORF and a prototypic H5 HA ORF with a modiﬁed monobasic cleavage site. The H9N2/H5 virus showed successful protection against lethal highly pathogenic avian inﬂuenza A/Vietnam/1203/04 (H5N1) in mice and ferrets [31]. The same strategy was used to generate H9N2 viruses successfully expressing enhanced green ﬂuorescent pro- tein (eGFP) and secreted Gaussia luciferase (GLuc), as well as a 2009 prototypic H1N1 virus expressing GLuc [31,40]. Based on these previous studies, we developed the FluB-RAM and FluB-RANS LAIV candidates, with the exception that these viruses do not express foreign antigens. The FluB-RAM and FluB-RANS viruses remained stable after six serial passages in ECEs, as shown by RT-PCR and Sanger and NGS sequence analyses. p g y g q y In vitro growth of FluB-RAM and FluB-RANS viruses was impaired under multiple temperature conditions in MDCK cells compared with the WT B/Bris strain. Of the two rearranged viruses, FluB-RANS grew at lower titers than FluB-RAM and its growth at 37 ◦C was barely over the limit of detection at 72 hpi (Figure 1C). However, both rearranged viruses reached titers of at least 108 EID50/mL in ECEs that would make them suitable as vaccine candidates. The growth kinetics results were consistent with the observations during the in vivo safety assessment. Both FluB-RAM and FluB-RANS were attenuated in comparison with the B/Bris WT. The safety proﬁle of FluB-RANS showed more attenuation than another LAIV candidate, FluB att, with four amino acid mutations in PB1 (E48K, K391E, E580G, and S660A), resulting in no noticeable signs of disease and no body weight changes. In contrast, the FluB-RAM virus induced some body weight loss, particularly in male mice, with one of those having to be euthanized. Nevertheless, the clinical signs induced by FluB-RAM inoculation in male mice were signiﬁcantly lower, whereas they were almost nonexistent in female mice compared with those observed with the B/Bris WT strain (Figure 2B). 3.6. Qualitative Differences in Humoral and Mucosal Responses among Different Vaccine Groups at 14 dpc Post-challenge lineage-specific IgG and IgA responses against the NA and the NP in serum and NW. IgG and IgA responses in serum were analyzed using protein microarrays. Sera and NW collected at 14 dpc from mice challenged with B/Bris/PB2 F406Y (n = 4/group) were tested against purified IBV NA or NP protein antigens purchased from Sino Biological. The results are expressed as the group mean fluorescence intensity (MFI) ± SD. The higher the MFI, the more Abs bound to a particular antigen. (A) Anti-NA and -NP IgG responses in serum. (B) Anti-NA and -NP IgG re- sponses in NW. (C) Anti-NA and -NP IgA responses in serum. (D) Anti-NA and -NP IgA responses in NW. MFIs were plotted and analyzed using Prism v9. Statistical analysis to compare responses between groups was performed using two-way ANOVA followed by a Tukey’s test for multiple comparisons. Significant differences between groups are denotated by stars (). = p < 0.05. 4 Discussion Figure 8. Post-challenge lineage-speciﬁc IgG and IgA responses against the NA and the NP in serum and NW. IgG and IgA responses in serum were analyzed using protein microarrays. Sera and NW collected at 14 dpc from mice challenged with B/Bris/PB2 F406Y (n = 4/group) were tested against puriﬁed IBV NA or NP protein antigens purchased from Sino Biological. The results are expressed as the group mean ﬂuorescence intensity (MFI) ± SD. The higher the MFI, the more Abs bound to a particular antigen. (A) Anti-NA and -NP IgG responses in serum. (B) Anti-NA and -NP IgG responses in NW. (C) Anti-NA and -NP IgA responses in serum. (D) Anti-NA and -NP IgA responses in NW. MFIs were plotted and analyzed using Prism v9. Statistical analysis to compare responses between groups was performed using two-way ANOVA followed by a Tukey’s test for multiple comparisons. Signiﬁcant differences between groups are denotated by stars (). = p < 0.05. Figure 8. Post-challenge lineage-specific IgG and IgA responses against the NA and the NP in serum Figure 8. Post-challenge lineage-speciﬁc IgG and IgA responses against the NA and the NP in serum Vaccines 2021, 9, 897 17 of 21 17 of 21 4. Discussion ( g ) It is important to note that, during the process of testing safety and efﬁcacy of the different vaccines, we observed biological sex as a variable for susceptibility to IBV. In our experience, male mice were more prone than female mice to developing more signiﬁcant signs of disease and mortality upon IBV infection (Figures 2B and 5A,B). In addition, female mice, but not male mice showed a biphasic curve of associated clinical signs after IBV challenge, with an initial phase of pronounced body weight loss, recovery close to initial body weight, and then a second phase of mild body weight loss before a second recovery phase (Figure 5A). Sex differences related to susceptibility to IAV have been extensively characterized [41,42]. However, previous studies have determined that female mice are more susceptible than males to IAV infection. In this regard, the differences in the susceptibility of male versus female mice infected with IBV of this report follow the pattern observed in humans where biological males are more prone to hospitalization due to inﬂuenza than biological females. In addition, although non-statistically signiﬁcant, antibody titers after boost vaccination and after challenge showed a trend towards higher responses in female than in male mice (Figures 2 and 5 and data not shown). These observations are consistent with previous studies assessing the response to vaccination in humans and mice that revealed higher antibody responses, higher B cell responses, higher cross-reactive antibodies, and higher CD4+ T cell numbers in females compared with males [41–45]. Thus, understanding sex as a variable to study IBV susceptibility and vaccine responses is warranted, but beyond the scope of this report. Comparing the results from the present study to previous observations with the FluB att virus published elsewhere, but part of the same study, the rearranged FluB-RAM and FluB-RANS viruses induced comparable HI antibody levels within 1 log2 difference of each other, both before and after challenge (Figures 2 and 5). Further, qualitative differences in Vaccines 2021, 9, 897 18 of 21 18 of 21 IgG and IgA responses were observed among different vaccine groups explored using a protein microarray (Figures 3 and 4). It was interesting to observe that the most attenuated virus, FluB-RANS, led to overall higher anti-HA serum IgG responses before challenge. In contrast, anti-HA serum IgG responses from the FluB-RAM were among the weakest before challenge despite the virus being the least attenuated. 4. Discussion The pattern of anti-HA serum IgG in samples from the FluB att was intermediate between the two rearranged vaccine groups. However, serum samples from the FluB att group were consistently higher for IgA against HA and for IgG and IgA against NA and NP antigens in the array. Further, overall IgG and IgA responses against NA and NP from the FluB-RANS group were among the weakest. Despite these qualitative differences, both rearranged viruses protected mice against lethal challenge with the B/Bris/PB2-F406Y strain. NW antibody responses after challenge were of particular interest as they reﬂect recall antibody responses to the site that would most efﬁciently prevent infection. In NW samples from the FluB-RANS group, anti-HA IgG and IgA responses were particularly prominent, but anti-NA/NP IgA responses were the weakest compared with other groups. Interestingly, anti-NA/NP IgA responses were more prominent after challenge in serum and NW samples from the FluB-RAM group. Thus, we observed opposite patterns between anti-HA and anti-NA/NP responses for the FluB-RANS and FluB-RAM groups and intermediate patterns for the FluB att group. These observations are signiﬁcant because they suggest that humoral responses against different IBV antigens are not equally impacted by the different LAIV backbones. Despite the relatively lower attenuation of the FluB-RAM virus, it could be useful in dose sparing situations and/or in the presence of pre-existing immunity as complement boost vaccine. Previous studies have suggested that priming with an LAIV followed by a killed virus vaccine leads to more complete protective responses than prime-boost strategies using a single type of vaccine against the 2009 pandemic H1N1 virus. More relevant to this report, vaccination with a seasonal H1 LAIV (pre-2009 H1N1 antigen) followed by a boost with a pandemic H1 LAIV led to more robust protective responses than either vaccine administered twice [46,47]. Thus, it is tempting to speculate that one or more LAIV platforms could be used in prime-boost approaches that would improve the protective response of currently approved vaccines against IBV. References 1. Francis, T., Jr. A new type of virus from epidemic inﬂuenza. Science 1940, 92, 405–408. [CrossRef] [PubMed] 2 Rit h M B P l P Kilb E D RNA f i ﬂ A B d C i J Vi l 1976 18 738 744 [C R f] [P bM d] ancis, T., Jr. A new type of virus from epidemic inﬂuenza. Science 1940, 92, 405–408. [CrossRef] [PubMed] 1. Francis, T., Jr. A new type of virus from epidemic inﬂuenza. Science 1940, 92, 405 408. [CrossRef] [PubMed] 2. Ritchey, M.B.; Palese, P.; Kilbourne, E.D. RNAs of inﬂuenza A, B, and C viruses. J. Virol. 1976, 18, 738–744. [CrossRef] [PubMed] 3. Betakova, T.; Nermut, M.V.; Hay, A.J. The NB protein is an integral component of the membrane of inﬂuenza B virus. J. Gen. Virol. 1996, 77, 2689–2694. [CrossRef] 2. Ritchey, M.B.; Palese, P.; Kilbourne, E.D. RNAs of inﬂuenza A, B, and C viruses. J. Virol. 1976, 18, 738–7 ; Palese, P.; Kilbourne, E.D. RNAs of inﬂuenza A, B, and C viruses. J. Virol. 1976, 18, 738–744. [CrossRef] [Pu y, ; , ; , , , J , , [ ] [ ] 3. Betakova, T.; Nermut, M.V.; Hay, A.J. The NB protein is an integral component of the membrane of inﬂuenza B virus. J. Gen. Virol. 1996, 77, 2689–2694. [CrossRef] 4. Racaniello, V.R.; Palese, P. Inﬂuenza B virus genome: Assignment of viral polypeptides to RNA segments. J. Virol. 1979, 29, 361–373. [CrossRef] oppin, P.W.; Lamb, R.A. A previously unrecognized inﬂuenza B virus glycoprotein from a bicistronic mRNA viral neuraminidase. Proc. Natl. Acad. Sci. USA 1983, 80, 4879–4883. [CrossRef] 5. Shaw, M.W.; Choppin, P.W.; Lamb, R.A. A previously unrecognized inﬂuenza B virus glycoprotein from also encodes the viral neuraminidase. Proc. Natl. Acad. Sci. USA 1983, 80, 4879–4883. [CrossRef] 6. Briedis, D.J.; Lamb, R.A.; Choppin, P.W. Inﬂuenza B virus RNA segment 8 codes for two nonstructural proteins. Virology 1981, 112, 417–425. [CrossRef] 7. Horvath, C.M.; Williams, M.A.; Lamb, R.A. Eukaryotic coupled translation of tandem cistrons: Identiﬁcation of the inﬂuenza B virus BM2 polypeptide. EMBO J. 1990, 9, 2639–2647. [CrossRef] 8. Bodewes, R.; de Mutsert, G.; van der Klis, F.R.; Ventresca, M.; Wilks, S.; Smith, D.J.; Koopmans, M.; Fouchier, R.A.; Osterhaus, A.D.; Rimmelzwaan, G.F. Prevalence of antibodies against seasonal inﬂuenza A and B viruses in children in Netherlands. Clin. Vaccine Immunol. 2011, 18, 469–476. [CrossRef] 9. 5. Conclusions In this report, we successfully generated and assessed the efﬁcacy of two LAIV IBV vaccines using genome re-arrangement. FluB-RAM and FluB-RANS are attenuated in vitro and in vivo. Regardless of differences in attenuation proﬁles, both LAIV vaccine candidates induced protective antibody responses, and effectively protected mice against lethal challenge. These results warrant more in-depth assessment of the FluB-RAM and FluB-RANS LAIVs in mice and other animal models, to further characterize their protection efﬁcacy and the stimulation of different arms of the immune response. Biological sex-driven differences in responses to vaccination are to be further evaluated. Author Contributions: D.R.P., D.S.R. and S.C.-G. conceptualized the experiment. S.C.-G., D.R.P. and D.S.R. designed the experiments. S.C.-G. performed cloning for the generation of pSCG-PB1BM2, pSCG-PB1BNS2, pSCG-BM1-∆M2, and pSCG-BNS1-∆NEP reverse genetics plasmids, as well as virus rescue and growth kinetics. S.C.-G. and G.G. performed viral sequencing. S.C.-G., C.J.C. and J.-S.M. performed in vivo experiments and sample collection. S.C.-G. performed sample processing. A.J. (Aarti Jain), A.J. (Algimantas Jasinskas), R.N. and D.H.D. performed inﬂuenza antigen microarray. S.C.-G. and D.R.P. performed data analysis. S.C.-G. and D.R.P. wrote the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This study was supported by a subcontract from the Center for Research on Inﬂuenza Pathogenesis (CRIP) to D.R.P. under contract HHSN272201400008C from the National Institute of Allergy and Infectious Diseases (NIAID) Centers for Inﬂuenza Research and Surveillance (CEIRS) and grant 1R21AI146448-01 from NIAID to D.R.P. In addition, D.R.P. receives additional support from the Georgia Research Alliance and from the Caswell S. Eidson endowment funds. Vaccines 2021, 9, 897 19 of 21 Institutional Review Board Statement: Animal studies were approved and conducted in compliance with all the regulations stated by the Institutional Animal Care and Use Committee (IACUC) of the University of Georgia (UGA under AUP A2019 01-004-A2). Vaccination and challenge studies were conducted under BSL-2 conditions at the Davison Life Sciences Complex, University of Georgia. Animal studies and procedures were performed according to the Institutional Animal Care and Use Committee Guidebook of the Ofﬁce of Laboratory Animal Welfare and PHS policy on Humane Care and Use of Laboratory Animals. Animal studies were carried out in compliance with the ARRIVE guidelines (https://arriveguidelines.org) (accessed on 18 January 2021). Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. 5. Conclusions Data Availability Statement: The data discussed in this publication have been deposited in NCBI’s Gene Expression Omnibus [48] and are accessible through GEO Series accession number GSE180642. Acknowledgments: We thank Kristine R. Wilcox and the personnel from the Life Sciences vivarium at the University of Georgia. Conﬂicts of Interest: The authors have ﬁled invention disclosures for RAM and RANS technologies for IAV and IBV viruses as potential vaccines. 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https://openalex.org/W2117795413	https://www.scielo.br/j/jbchs/a/5DL6gMCsX9rYhvXpZWDvxSF/?lang=en&format=pdf	English	null	Stripping voltammetric determination of cadmium in sea water using a carbon paste electrode modified with alginic acid from brown algae	Journal of the Brazilian Chemical Society	2,010	cc-by	2,346	Carolina Muñoz,* Manuel Zúñiga and Verónica Arancibia Carolina Muñoz,* Manuel Zúñiga and Verónica Arancibia Facultad de Química, Pontificia Universidad Católica de Chile, 7820436, Santiago, Chile A concentração do cádmio em um meio aquoso foi determinada usando-se um elétrodo de carbono ácido-modificado de pasta de alginina. O elétrodo de trabalho foi preparado usando-se uma mistura homogênea de pó de grafite, alginina ácida (AA) e óleo mineral. A voltametria de onda quadrada com redissolução anódica (SWASV) usando-se este elétrodo modificado, mostrou uma onda anódica bem definida com pequena oxidação do AA em -0,05 V. O sinal é deslocado para um potencial menos positivo e a corrente máxima aumentou na presença do CdII. Após a otimização das condições experimentais, a corrente máxima anódica do CdII-AA mostrou ser linear com sua concentração até ca. 30.0 µg L-1, com limite de detecção de 0.9 µg L-1 em pH 2.0 (HNO3. tacc = 60 s, Eacc = -0.80 V). O método foi validado com a determinação de CdII em água do mar sintética enriquecida (ASTM D665). O elétrodo modificado mostrou boa estabilidade e repetibilidade. Cadmium concentration in an aqueous medium is quantified using an alginic acid-modified carbon paste electrode. The working electrode was prepared using a homogeneous mixture of graphite powder, alginic acid (AA) and mineral oil. Square wave anodic stripping voltammetry (SWASV) using this modified electrode showed one small well-resolved anodic wave for the oxidation of AA at –0.05 V. This signal shifts toward less positive potential and peak current increases in the presence of CdII. After optimizing the experimental conditions, the anodic peak current of CdII-AA was linearly related to its concentration up to ca. 30.0 µg L-1, with a detection limit of 0.9 µg L-1 at pH 2.0 (HNO3. tacc = 60 s, Eacc = -0.80 V). The method was validated by determining CdII in spiked synthetic sea water (ASTM D665). The modified electrode showed good stability and repeatability. Keywords: alginic acid, carbon paste electrode, cadmium, SWASV J. Braz. Chem. Soc., Vol. 21, No. 9, 1688-1691, 2010. Printed in Brazil - ©2010 Sociedade Brasileira de Química 0103 - 5053 $6.00+0.00 J. Braz. Chem. Soc., Vol. 21, No. 9, 1688-1691, 2010. Printed in Brazil - ©2010 Sociedade Brasileira de Química 0103 - 5053 $6.00+0.00 Article *e-mail: cdmunozc@uc.cl Reagents and solutions The first step of this study was to find out the influence of pH on the peak current in the 2.0 to 10.0 range using Britton Robinson buffers. However, it was found that at acid pH adjusted with nitric acid peak current of Cd‑AA had adequate values. Figure 1A shows the stripping voltammogram of the carbon paste electrode (blank) in a 0.01 mol L-1 HNO3 solution and Figure 1B shows a voltammogram of the alginic acid modified carbon paste electrode (alginate/graphite powder 40/60). As shown in the figure, alginic acid (AA) has an oxidation peak at -0.05 V. When a trace amount of cadmium is added to the electrochemical cell (Figure 1C), the oxidation peak potential of AA shifts to less positive potential values (P1), close to -0.09 V, depending on the cadmium concentration, and the peak current of AA increases greatly, while a new small and broad oxidation peak is seen at more positive potential values (P2, 0.19 V) due to the CdII presence. All solutions were prepared with Milli-Q water (18.2 MW). Standard stock solutions of 1 µg mL-1 of cadmium were prepared from standard cadmium 1000 µg mL-1 solution (Merck, Darmstadt, Germany). Alginic acid sodium salt from brown algae (CAS 9005- 38-3) was obtained from Sigma. Nitric acid was obtained from Merck. Synthetic sea water (ASTM D665 Aldrich, USA) was used for validation of the method. Limit of detection (LOD) All the voltammetric measurements were carried out using a self-made carbon paste electrode modified with alginic acid as a working electrode, Ag/AgCl 3 mol L-1 KCl as the reference electrode, and platinum wire as the auxiliary electrode. SWASV experiments were performed on a CV50W Voltammetric analyzer electrochemical system (Bioanalytical Systems. USA). The measurements were made at room temperature and dissolved oxygen was removed with bubbling Argon. For pH measurements an Orion 430 pH meter was used. The limit of detection was calculated using the approximation of Miller and Miller7 for calibration curves. Minimum signal (ymin) = a + 3Sy/x, where a = intercept and Sy/x = standard deviation of the calibration curve. Introduction When the guluronic acid content is increased, the affinity of alginates for cations such as Pb2+, Cu2+, Cd2+, Zn2+, Ca2+, etc is higher. The major mechanisms include ionic interactions and the formation of complexes between metallic cations and groups contained in the alginic acid. Among the diverse and natural ligand availability, algae have already proved to be the most promising for heavy metal recovery.1-6 Alginic acid is the common name given to a family of linear polysaccharides containing 1,4-linked b‑D‑mannuronic acid (M) and a-L-guluronic acid (G), whose pKa are 3.38 and 3.65, respectively. Alginic acid occurs in all brown algae, where it may be present in both the cell wall matrix and in the mucilage or intercellular material, and it makes up between 10 and 40% of the algae's dry weight (untreated). The ratio of mannuronic and guluronic groups and the affinity for metals ions vary with the type of algae and the part of the plant from which the polysaccharide is extracted. The presence of these G-blocks results in an enhanced selectivity for cadmium or calcium relative to monovalent ions such as sodium as well as to protons and smaller divalent ions such as magnesium. Carbon paste electrodes modified with different ligands have been used to preconcentrate the analytic target (by means of a selective chemical reaction at the surface) and then to quantify the surface-bound species using a voltammetric technique. These electrodes present some advantages, such as easy manufacture, nontoxicity, low price, wider operational potential window, and stability in various solvents. The aim of this study was to optimize stripping voltammetric procedures for the determination of cadmium using a carbon paste electrode coated with alginic acid from brown algae. Muñoz et al. Vol. 21, No. 9, 2010 1689 Electrode preparation Modified carbon paste electrodes (MCPEs) were prepared with different alginate/graphite ratios by thoroughly homogenizing with paraffin in an agate mortar and pestle. The mixture was then packed in a cylindrical plastic tube used for insulin treatment (1.0 mL and internal diameter 4 mm) and connected to a copper wire to provide the electric contact. Before use the electrodes were polished with paper card and used immediately. Figure 1. (A) Stripping voltammogram of carbon paste electrode; (B) carbon paste electrode modified with alginic acid 60/40 (m/m); (C) B in the presence of 4.9 µg L–1 cadmium. pH 2 (HNO3); Eacc = -0.80 V; tacc = 60s. P1: -0.09 V; P2: 0.19 V. Experimental a frequency of 15 Hz. The method was validated by determining CdII in spiked synthetic sea water (ASTM D665). Procedure 10.0 mL of water, 100 µL of 1 mol L-1 HNO3, and different volumes of 1.0 µg mL-1 cadmium solution were pipetted into the voltammetric cell. The air present in the solution was removed with bubbling argon (saturated with water vapor) for 5 min and then an accumulation potential (Eacc) was applied to the freshly prepare modified carbon paste electrode while the solution was stirred (700 rpm). After the accumulation period (tacc), the stirring was stopped, and after 10 s the Osteryoung square wave stripping voltammogram was recorded by applying a positive-going scan between -0.80 and 1.00 V with 5 mV step amplitude, 50 mV pulse amplitude, and Figure 1. (A) Stripping voltammogram of carbon paste electrode; (B) carbon paste electrode modified with alginic acid 60/40 (m/m); (C) B in the presence of 4.9 µg L–1 cadmium. pH 2 (HNO3); Eacc = -0.80 V; tacc = 60s. P1: -0.09 V; P2: 0.19 V. Stripping Voltammetric Determination of Cadmium in Sea Water using a Carbon Paste Electrode 1690 J. Braz. Chem. Soc. Figure 3. Effect of varying accumulation time (0 to 120 s) on peak stripping current of 4.9 µg L–1 cadmium. Eacc = -0.80 V; pH 2.0. P1: peak current to -0.09 V; P2: peak current to 0.19 V. Alginic acid can be oxidized to the corresponding polymeric dialdehyde acid, which undergoes hydrolysis in dilute acid.8 These measurements were made at different alginate/graphite ratios, and the best results were obtained at 40/60 (m/m). When the amount of alginate is higher the modified electrode becomes swollen and the results obtained are not reproducible. Linear range, limit of detection, reproducibility The calibration graph for the determination of CdII was obtained under the optimized conditions: 40/60 alginate/ graphite ratio, pH 2.0 (adjusted with HNO3 solution), Eacc = - 0.80 V and tacc = 60 s. The peak currents P1 and P2 were proportional to the concentration of cadmium over the 0.0-40.0 µg L-1 and 0.0-30.0 µg L-1 range, respectively (R = 0.991 and R = 0.997), and the detection limit obtained was 0.9 µg L-1 (R = 0.997, n = 6, Epeak = - 0.09 V). Figure 4 shows the calibration curve in Milli-Q water. Figure 2. Effect of varying accumulation potential (1.00 to - 0.80 V) on peak stripping current of 4.9 µg L–1 cadmium. tacc = 60 s; pH 2.0. P1: peak current to - 0.09 V; P2: peak current to 0.19 V. Interference studies and validation of the method Figure 4. Effect of varying cadmium concentration (2.0; 3.9; 5.9; 7.9; 9.8 and 11.8 µg L–1) in Milli-Q water. Modified carbon paste electrode with alginic acid (60/40 m/m). tacc = 60 s; Eacc = - 0.80 V; pH = 2.0. Figure 2. Effect of varying accumulation potential (1.00 to - 0.80 V) on peak stripping current of 4.9 µg L–1 cadmium. tacc = 60 s; pH 2.0. P1: peak current to - 0.09 V; P2: peak current to 0.19 V. Figure 4. Effect of varying cadmium concentration (2.0; 3.9; 5.9; 7.9; 9.8 and 11.8 µg L–1) in Milli-Q water. Modified carbon paste electrode with alginic acid (60/40 m/m). tacc = 60 s; Eacc = - 0.80 V; pH = 2.0. Effect of accumulation potential The influence of the variation of the accumulation potential on the peak current (P1 and P2), examined over the 1.00 to -1.00 V range, is shown in Figure 2. While the peak current P2 is almost constant over the whole potential range, with a slight increase when an accumulation potential of 0.10 V was applied, the largest peak current P1 was obtained at more positive accumulation potentials (1.00 V) and it decreased substantially if the accumulation potential was more negative approaching -0.20 V, and then the peak current started increasing again. If an accumulation potential of 1.0 V is applied, the peak current obtained is more intense, however the signal is very broad and reproducibility is poor. A value of -0.80 V was applied to later measurements and the potential was scanned in a positive direction (-0.80 to 1.00 V). Figure 3. Effect of varying accumulation time (0 to 120 s) on peak stripping current of 4.9 µg L–1 cadmium. Eacc = -0.80 V; pH 2.0. P1: peak current to -0.09 V; P2: peak current to 0.19 V. References 1 Davis, T. A.; Volesky, B.; Mucci, A.; Water Res. 2003, 37, 4311. 1 Davis, T. A.; Volesky, B.; Mucci, A.; Water Res. 2003, 37, 4311. 2 Haug A.; Acta Chem. Scand. 1961, 15, 1794. 2 Haug A.; Acta Chem. Scand. 1961, 15, 1794. 3 Haug A.; Larsen B.; Smidsrod O.; Carbohyd. Res. 1974, 32, 217. 3 Haug A.; Larsen B.; Smidsrod O.; Carbohyd. Res. 1974, 32, 217. Effect of accumulation time As shown in Figure 3, the peak current of signal P2 is independent of accumulation time, while the peak current of signal P1 increased linearly with increasing accumulation time from 0 to 120 s. Subsequent experiments were performed with an accumulation time of 60 s in order to avoid extending the time of analysis. Interference studies and validation of the method 10.0 mL of synthetic sea water spiked with cadmium (4.0 µg L-1) was added in the electrochemical cell (0.01 mol L-1 HNO3). Anodic stripping voltammograms Muñoz et al. Vol. 21, No. 9, 2010 1691 Acknowledgements were obtained and then aliquots of CdII standard solution were added using the standard addition method. The results obtained for cadmium were 3.8 ± 0.3 µg L-1. For the interference study an aliquot of synthetic Milli-Q water was contaminated with CdII, PbII, CuII, ZnII, AlIII and AsIII standard solution at equal concentration (10.0 μg L−1), and the analysis of CdII was carried out using the standard addition method. Under these conditions CuII, ZnII, AlIII and AsIII did not show signals between -0.80 to 1.0 V, but PbII showed a signal at 0.26 V. If PbII concentration is 50 times higher than that of CdII, only one signal is seen for the two metal ions. Financial support from the Fondo Nacional de Desarrollo Científico y Tecnológico (FONDECYT. Project No 1080524) and from the Comisión Nacional de Investigación Científica y Tecnológica (CONICYT) for a PhD scholarship is gratefully acknowledged. Conclusions 4 Haug A.; Smidsrod O.; Acta Chem. Scand. 1965, 19, 341. 4 Haug A.; Smidsrod O.; Acta Chem. Scand. 1965, 19, 341. This paper shows the feasibility of determining cadmium in contaminated sea water samples using an alginate-modified carbon paste electrode. Other advantages of the proposed method are the low cost and easy preparation of the electrode. This method appears to be a promising analytical tool for the determination of cadmium in contaminated natural waters. 5. Davis, T.; Llanes, F.; Volesky, B.;Mucci, A.; Environ. Sci. Technol. 2003, 37, 261. 5. Davis, T.; Llanes, F.; Volesky, B.;Mucci, A.; Environ. Sci. Technol. 2003, 37, 261. 6. Fourest, E. and Volesky, B.; Environ. Sci. Technol. 1996, 30, 277. 6. Fourest, E. and Volesky, B.; Environ. Sci. Technol. 1996, 30, 277. 7 Miller, J. C.; Miller, J. N.; Statistics for Analytical Chemistry, 2nd edition, Ellis Horwood: London, 1988. 8 Lucas, H. J.; Stewart, W. T.; J. Am. Chem. Soc. 1940, 62, 1792. Submitted: September 9, 2009 Published online: May 20, 2010 Published online: May 20, 2010
https://openalex.org/W2579834268	https://www.nature.com/articles/cddis2016467.pdf	English	null	HIV-1 Nef is released in extracellular vesicles derived from astrocytes: evidence for Nef-mediated neurotoxicity	Cell death and disease	2,017	cc-by	13,045	HIV-1 Nef is released in extracellular vesicles derived from astrocytes: evidence for Nef-mediated Stephanie Cicalese1,2, Taha Mohseni Ahooyi1, Kamel Khalili1, Shohreh Amini1 and Ilker Kudret Sariyer,1 Sami Saribas1,2, Stephanie Cicalese1,2, Taha Mohseni Ahooyi1, Kamel Khalili1, Shohreh Amini1 and Ilker Human immunodeficiency virus-associated neurological disorders (HANDs) affect the majority of AIDS patients and are a significant problem among HIV-1-infected individuals who live longer because of combined anti-retroviral therapies. HIV-1 utilizes a number of viral proteins and subsequent cytokine inductions to unleash its toxicity on neurons. Among HIV-1 viral proteins, Nef is a small protein expressed abundantly in astrocytes of HIV-1-infected brains and has been suggested to have a role in the pathogenesis of HAND. In order to explore its effect in the central nervous system, HIV-1 Nef was expressed in primary human fetal astrocytes (PHFAs) using an adenovirus. Our results revealed that HIV-1 Nef is released in extracellular vesicles (EVs) derived from PHFA cells expressing the protein. Interestingly, HIV-1 Nef release in EVs was enriched significantly when the cells were treated with autophagy activators perifosine, tomaxifen, MG-132, and autophagy inhibitors LY294002 and wortmannin suggesting a novel role of autophagy signaling in HIV-1 Nef release from astrocytes. Next, Nef-carrying EVs were purified from astrocyte cultures and neurotoxic effects on neurons were analyzed. We observed that HIV-1 Nef-containing EVs were readily taken up by neurons as demonstrated by immunocytochemistry and immunoblotting. Furthermore, treatment of neurons with Nef-carrying EVs induced oxidative stress as evidenced by a decrease in glutathione levels. To further investigate its neurotoxic effects, we expressed HIV-1 Nef in primary neurons by adenoviral transduction. Intracellular expression of HIV-1 Nef caused axonal and neurite degeneration of neurons. Furthermore, expression of HIV-1 Nef decreased the levels of phospho-tau while enhancing total tau in primary neurons. In addition, treatment of primary neurons with Nef-carrying EVs suppressed functional neuronal action potential assessed by multielectrode array studies. Collectively, these data suggested that HIV-1 Nef can be a formidable contributor to neurotoxicity along with other factors, which leads to HAND in HIV-1-infected AIDS patients. 1Department of Neuroscience, Center for Neurovirology, Lewis Katz School of Medicine, Temple University, Philadelphia, 19140 PA, USA Corresponding author: IK Sariyer, Department of Neuroscience, Center for Neurovirology, Lewis Katz School of Medicine at Temple University, 3500 North Broad Street, Medical Education and Research Building, 7th Floor, Philadelphia 19140, PA 19140, USA. Tel: +1 215 707 6337; Fax: +1 215 707 4888; E-mail: isariyer@temple.edu 2These authors contributed equally to this work. Received 31.8.16; revised 15.11.16; accepted 18.11.16; Edited by A Verkhratsky Citation: Cell Death and Disease (2017) 8, e2542; doi:10.1038/cddis.2016.467 Ofﬁcial journal of the Cell Death Differentiation Association Citation: Cell Death and Disease (2017) 8, e2542; doi:10.1038/cddis.2016.467 Ofﬁcial journal of the Cell Death Differentiation Association OPEN www.nature.com/cddis HIV-1 Nef is released in extracellular vesicles derived from astrocytes: evidence for Nef-mediated Nef exhibits its toxic effects when it reaches its destination26 and can alter endosomal morphology27 leading to the accumulation of multivesicular bodies (MVBs) and lysosomes. MVBs are in turn released as EVs.26 HIV-1 Nef appears to be responsible for many events leading to neurological impairments in the HIV-1-infected brain such as neuronal degeneration by inducing IP-10 release,28 cytokine production and negatively affecting cellular pathways.29,30 Neurotoxic effects of HIV-1 Nef were shown using recombinant Nef on human glial cells and neurons.31 Furthermore, animal studies revealed that HIV-1 Nef-induced neurocognitive impairments in rats.32,33 The effect of HIV-1 infection on the brain depends on the subtype of virus. Interestingly, some AIDS patients exhibited the presence of HIV-1 DNA in their infected brains, while the others had no detectable viral DNA after autopsies.34 HIV-1 Nef modulates progression of AIDS in patients with HAD compared with those without and subtype D is most likely associated with HAD.35 Although the neurotoxicity of HIV-1 gp120 and Tat has been widely studied and better understood,36 the neurotoxicity of HIV-1 Nef is still not clear despite some recent reports. In this study, we elucidate the neurotoxic effects of HIV-1 Nef utilizing both EVs and by directly expressing this viral protein in primary human neurons. from the supernatants of PHFA cells using differential centrifugation. Cell extracts from the transduced cells and the purified EVs were analyzed by western blotting (Figure 1c). Interestingly, crude EV lysates from cells transduced with adenovirus expressing Nef-GFP contained only a trace amount of Nef-EGFP (Figure 1c, higher exposure) suggesting that EGFP fusion with Nef may interfere with Nef release in EVs. Role of autophagy in Nef release in EVs. Several lines of evidence suggest a close interaction between autophagy pathways and the biogenesis and secretion of EVs.54 We have recently reported that expression of HIV-1 Nef compromises the autophagic pathways in primary human fetal astrocytes (PHFAs) by inducing autophagosome forma- tion and blocking the assembly of autophagolysosomes.39 As shown in Figure 1b, Nef was readily detected in EV lysates obtained from PHFA cells expressing HIV-1 Nef. Therefore, we sought to investigate the possible role of autophagic signaling in Nef release in EVs. PHFA cells were plated in six- well tissue culture dishes and transduced with either Ad-null or Ad-Nef constructs. Cells were treated with several well- defined autophagy activators and inhibitors as described in Materials and Methods section. HIV-1 Nef is released in extracellular vesicles derived from astrocytes: evidence for Nef-mediated At 24 h post-treatments, culture media were collected and processed for EV purifica- tion by ExoQuick from System Biosciences (System Biosciences, CA, USA). As shown in Figures 2a and b, treatment of astrocytes with autophagy inhibitors perifosine (inhibitor of AKT phosphorylation), tomaxifen (estrogen receptor antagonist), MG-132 (proteasome inhibitor) and autophagy activators LY294002 (PI3 kinase inhibitor) and worthmannin (PI3 kinase inhibitor) caused a significant increase in Nef release in EVs. In parallel to the biochemical analysis of EVs, the possible impact of the treatments at indicated concentrations on cellular viabilities were also analyzed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte- trazolium bromide) assay. The MTT assay results indicated that none of the drugs at the concentrations used in panel A caused any significant toxicity (Supplementary Figure 1). Moreover, we have titrated the key autophagy activators (perifosine and MG-132) and autophagy inhibitors (LY294002 and wortmannin) as low, moderate and high doses. As shown in Supplementary Figure 2, all the drugs showed dose- responsive induction of Nef release in EVs suggesting their specific impact on endosomal biogenesis. In parallel to the culture media for EV purification presented in Figure 2a, cells were also harvested for whole-cell protein extraction and analyzed by western blotting. As shown in Figure 2c, HIV-1 Nef expression was mostly consistent with all treatments except MG-132, which caused a slight increase in total Nef protein levels in astrocytes (lane 8) suggesting that Nef turnover may be regulated by proteasomal degradation. Interestingly, Nef expression in astrocytes caused a signifi- cant increase in pAKT (S476) and mTOR (S2448) phosphor- ylation that was suppressed by perifosine, MG-132 and wortmannin treatments. LC3-I and its lipidated form LC3-II levels were also analyzed to monitor autophagic flux in accordance with treatments. Interestingly, the subsequent increase in Nef release in EVs did not correlate with LC3-I lipidation and activation of LC3-II HIV-1 Nef is released in extracellular vesicles derived from astrocytes: evidence for Nef-mediated Cell Death and Disease (2017) 8, e2542; doi:10.1038/cddis.2016.467; published online 12 January 2017 Human immunodeficiency virus-1 (HIV-1), the etiological agent of AIDS, not only wreaks havoc on the immune system,1–3 but also inflicts the central nervous system (CNS)4 leading to HIV-associated neurological disorders (HANDs).5–10 Before combined anti-retroviral therapies (cARTs), HIV-1 infections were responsible for neuroinflam- mation leading to encephalitis (HIVE) as evidenced by astrocytosis, neuronal loss, activated microglia and infiltration of macrophages in to infected brains.7,11 The introduction of cART reduced the neuronal damage inflicted by HIV infection.12 The paradigm of AIDS neuropathogenesis was changed to less severe forms of HAND such as mild neurocognitive disorder and impairments in neurocognition, but it did not completely prevent the more severe form of HAND, HIV-1-associated dementia (HAD).7,13 that neurons bear surface receptors, HIV-1 does not infect these cells. However, there are reports indicating the presence of HIV-1 nucleic acids in neurons obtained from some AIDS patients.16,17 In addition, HIV-1 was found to infect neuronal cell lines such as SK-N-MC in a CD4-independent manner.18 In fact, human neurons are more prone to the toxic effects of HIV-1 viral proteins and, subsequently released cytokines and chemokines.19 HIV-1 regulatory proteins such as Tat and Vpr, and viral envelope protein gp120 are known to damage the BBB.20 HIV-1 infection is known to induce inflammatory cytokines such as TNF-alpha, IL-1 and IL6.21,22 In this respect, HIV-1 viral proteins dysregulate several CNS processes, such as chemokine production, glutamate transport and cellular pathways, to cause neurotoxicity.19 There are indications that HIV-1 Nef also has a role in neuronal toxicity but the extent of its effects in the CNS remains unknown. Nef is one of HIV-1’s auxiliary proteins with a molecular weight of 27–34 kD. It is myristylated on its amino terminus by post-translational modification.23 Expression of Nef occurs during early HIV-1 infection of cells including astrocytes,24,25 macrophages and CD4+ T cells, and is released from these infected cells as a cargo protein in HIV-1 infects CD4+ T cells first and then proceeds to invade the brain by infiltrating the blood–brain barrier (BBB), and exhibiting its devastating effects at later stages to develop HAND in nearly half of all AIDS patients.14 HIV-1 infection in the brain generally occurs in macrophages and microglia in a receptor-mediated manner, whereas a limited number of astrocytes are infected nonproductively.15 Despite the fact Nef-mediated neurotoxicity AS Saribas et al 2 extracellular vesicles (EVs). Results HIV-1 Nef is released in EVs derived from primary human astrocytes. In order to investigate whether HIV-1 Nef is released in EVs from astrocytes, PHFA cells were transduced with an adenovirus construct expressing Nef as described in Materials and Methods section. Cells were fixed and stained for glial fibrillary acidic protein (GFAP) and Nef by immuno- cytochemistry (Figure 1a). Ad-Nef-transduced cells were positive for the astrocyte marker GFAP in green. After 24 h post-transduction, EVs were prepared from the supernatants of PHFA cells using differential centrifugation as well as ExoQuick method as described in Materials and Methods section. Cell extracts from the transduced cells and the purified EVs were analyzed by western blot and compared with each other. Ad-Nef-transduced PHFA lysates had high levels of HIV-1 Nef protein as shown in the western blot (Figure 1b). EVs derived from these cells also contained HIV-1 Nef protein indicating that Nef protein is indeed associated with these EVs. We also analyzed the exosome- depleted astrocyte supernatants for HIV-1 Nef and found that there was no free Nef protein detectable (data not shown). Astrocytic EVs are also known to carry HSP-70.37 We have observed high levels of HSP-70 in both Null and Nef EVs. GAPDH was used as a loading control for cell lysates. Both Null and Nef-EVs were positive for Alix, which is an exosome marker. Alix was not at detection range in cell lysates most likely due to the low levels of this protein in cell lysates but more in EVs. It has been recently shown that crude EVs from Jurkat cells expressing Nef-EGFP did not incorporate Nef into the EVs.38 In order to test whether primary astrocytes could release Nef-EGFP in EVs, PHFA cells were transfected with an expression vector encoding either EGFP alone or Nef-EGFP. After 48 h post-transfection, EVs were prepared Cell Death and Disease Nef-mediated neurotoxicity AS Saribas et al 3 e 1 (a) Detection of HIV-1 Nef in EVs derived from PHFA. (a) Immunocytochemistry (ICC) of the PHFA cells transduced with Ad-Nef. The PHFA cells were grown in two- amber slides and transduced with Ad-Nef as described in Materials and Methods section. The cells were fixed, mounted and visualized by immunocytochemistry. First (upper left) shows GFAP – an astrocyte marker – distribution in astrocytes in green color. HIV-1 Nef expression in PHFA is shown in red color. Results Bottom left illustrates DAPI tom right is the illustration of merging GFAP, HIV-1 Nef and DAPI. (b) Western blot analyses of PHFA lysates and astrocytic EVs. PHFA cells were either transduced with or Ad-Nef for 24 h. Whole-cell protein lysates and EV lysates purified from growth media were separated on SDS-PAGE, transferred on a nitrocellulose membrane and d by western blotting for the detection of Nef, HSP-70 and Alix using specific antibodies. GAPDH was also probed in the same membranes as a loading control. (c) n blot analysis of PHFA lysates and astrocytic EVs. PHFA cells were transfected with an expression vector encoding either GFP or Nef-GFP for 48 h. Whole-cell protein and EV lysates purified from growth media were separated on SDS-PAGE, transferred on a nitrocellulose membrane, and analyzed by western blotting for the detection of ef-GFP and HSP-70. Both lower (left panels) and higher (right panels) exposures of the blots were shown and compared Figure 1 (a) Detection of HIV-1 Nef in EVs derived from PHFA. (a) Immunocytochemistry (ICC) of the PHFA cells transduced with Ad-Nef. The PHFA cells were grown in two- well chamber slides and transduced with Ad-Nef as described in Materials and Methods section. The cells were fixed, mounted and visualized by immunocytochemistry. First picture (upper left) shows GFAP – an astrocyte marker – distribution in astrocytes in green color. HIV-1 Nef expression in PHFA is shown in red color. Bottom left illustrates DAPI and bottom right is the illustration of merging GFAP, HIV-1 Nef and DAPI. (b) Western blot analyses of PHFA lysates and astrocytic EVs. PHFA cells were either transduced with Ad-Null or Ad-Nef for 24 h. Whole-cell protein lysates and EV lysates purified from growth media were separated on SDS-PAGE, transferred on a nitrocellulose membrane and analyzed by western blotting for the detection of Nef, HSP-70 and Alix using specific antibodies. GAPDH was also probed in the same membranes as a loading control. (c) Western blot analysis of PHFA lysates and astrocytic EVs. PHFA cells were transfected with an expression vector encoding either GFP or Nef-GFP for 48 h. Whole-cell protein lysates and EV lysates purified from growth media were separated on SDS-PAGE, transferred on a nitrocellulose membrane, and analyzed by western blotting for the detection of GFP, Nef-GFP and HSP-70. Results Both lower (left panels) and higher (right panels) exposures of the blots were shown and compared EVs carrying HIV-1 Nef are taken up by primary human neurons . Detection of HIV-1 Nef in EVs suggested that Nef could be delivered from astrocytes into the neurons by these EVs. To analyze possible uptake of Nef-containing EVs by neurons, PHFN cells were treated with EVs derived from PHFA cells either transduced with Ad-Null or Ad-Nef for approximately 48 h to ensure the uptake by neuronal cells. In order to analyze whether Nef is delivered into the neurons by EVs, cell lysates from PHFN cells treated with EVs were analyzed by western blotting. PHFN cells were harvested after extensive PBS washing followed by trypsinization to eliminate any surface-bound vesicles. As shown in Figure 3a, HIV-1 Nef protein was clearly detected in Nef EV-treated neuronal lysates suggesting that Nef was delivered into the neurons by EVs derived from PHFA cells expressing HIV-1 Nef. To gain more insight into the delivery of HIV-1 Nef into the neurons by EVs released from PHFA cells expressing Nef, immunocytochemistry was done on neurons treated with astrocyte-derived exosomes. PHFNs were cultured in two- well chamber slides and treated with Ad-Null or Ad-Nef EVs purified from PHFA, as described in Materials and Methods section. After 48 h of EV treatment, neurons were fixed, incubated with rabbit polyclonal anti-MAP-2, anti-neurofila- ment, anti-class III β-tubulin, double stained for monoclonal anti-Nef and processed by immunocytochemistry. As shown in Figure 3c, as expected, primary neurons showed high levels of MAP-2, neurofilament and class III β-tubulin expression (in red fluorescein color). On the other hand, several neurons showed immunoreactivity and typical cyto- plasmic and perinuclear staining for HIV-1 Nef (in green fluorescein color) suggesting that Nef was delivered to neurons. Interestingly, neurons with Nef uptake showed the neurons by EVs released from PHFA cells expressing Nef, immunocytochemistry was done on neurons treated with astrocyte-derived exosomes. PHFNs were cultured in two- well chamber slides and treated with Ad-Null or Ad-Nef EVs purified from PHFA, as described in Materials and Methods section. After 48 h of EV treatment, neurons were fixed, incubated with rabbit polyclonal anti-MAP-2, anti-neurofila- ment, anti-class III β-tubulin, double stained for monoclonal anti-Nef and processed by immunocytochemistry. As shown in Figure 3c, as expected, primary neurons showed high levels of MAP-2, neurofilament and class III β-tubulin expression (in red fluorescein color). Results (c) In parallel to culture supernatants, whole-cell protein lysates were also prepared from the same studies presented in panel a and processed by western blotting for the detection of HIV-1 Nef, LC3, pAKT (S476), total AKT, p44/42 MAPK (Thr202/Tyr204), total MAPK, pmTOR (S2448) and total mTOR expression levels. β- Actin, GAPDH and tubulin were also probed in same membranes as loading controls. Po0.05 Figure 2 Effect of autophagy signaling on Nef release in EVs from PHA cells. (a) PHFA cells were transduced with Ad-Null and Ad-Nef constructs and treated with various autophagy activators and inhibitors as described in Materials and Methods section. EVs were purified from culture supernatants, lysed with TNN buffer containing 1% NP40 and analyzed by western blotting for the detection of HIV-1 Nef and HSP-70 proteins. In lanes 1 and 2, whole-cell protein lysates from PHFA cells transduced either with Ad-Nef or Ad- Null were loaded as positive and negative controls of Nef detection. (b) Protein bands from panel a on the western blot membranes were used to quantitate HIV-1 Nef levels and HSP-70 by ImageJ program and the relative values were plotted after normalizing Nef with HSP-70 using Excel program. The results are shown in bar graphs averages from the three independent experiments. (c) In parallel to culture supernatants, whole-cell protein lysates were also prepared from the same studies presented in panel a and processed by western blotting for the detection of HIV-1 Nef, LC3, pAKT (S476), total AKT, p44/42 MAPK (Thr202/Tyr204), total MAPK, pmTOR (S2448) and total mTOR expression levels. β- Actin, GAPDH and tubulin were also probed in same membranes as loading controls. Po0.05 decreased levels of MAP-2 and class III β-tubulin (open arrows) staining, suggesting a possible impact of Nef in neuronal cytoskeleton structure. Unlike MAP-2 and class III β-tubulin, neurons with Nef uptake showed normal neurofila- ment expression with perfect HIV-1 Nef colocalization (middle panel, closed arrows). These results suggest that HIV-1 Nef can be delivered by astrocytes to the neurons by EVs and may be associated with neurotoxicity. whereas Ad-Null-treated neurons looked normal (Figure 3a). At 48 h post-transduction, most of the Ad-Nef-transduced PHFN cells exhibited the neurotoxic effect of the HIV-1 Nef losing most of their axons and dendrites and forming beaded structures, whereas the majority of the Ad-Null-transduced neurons remained unaffected. Results On the other hand, several neurons showed immunoreactivity and typical cyto- plasmic and perinuclear staining for HIV-1 Nef (in green fluorescein color) suggesting that Nef was delivered to neurons. Interestingly, neurons with Nef uptake showed Cell Death and Disease Nef-mediated neurotoxicity AS Saribas et al 4 Figure 2 Effect of autophagy signaling on Nef release in EVs from PHA cells. (a) PHFA cells were transduced with Ad-Null and Ad-Nef constructs and treated with various autophagy activators and inhibitors as described in Materials and Methods section. EVs were purified from culture supernatants, lysed with TNN buffer containing 1% NP40 and analyzed by western blotting for the detection of HIV-1 Nef and HSP-70 proteins. In lanes 1 and 2, whole-cell protein lysates from PHFA cells transduced either with Ad-Nef or Ad- Null were loaded as positive and negative controls of Nef detection. (b) Protein bands from panel a on the western blot membranes were used to quantitate HIV-1 Nef levels and HSP-70 by ImageJ program and the relative values were plotted after normalizing Nef with HSP-70 using Excel program. The results are shown in bar graphs averages from the three independent experiments. (c) In parallel to culture supernatants, whole-cell protein lysates were also prepared from the same studies presented in panel a and processed by western blotting for the detection of HIV-1 Nef, LC3, pAKT (S476), total AKT, p44/42 MAPK (Thr202/Tyr204), total MAPK, pmTOR (S2448) and total mTOR expression levels. β- Actin, GAPDH and tubulin were also probed in same membranes as loading controls. *Po0.05 Figure 2 Effect of autophagy signaling on Nef release in EVs from PHA cells. (a) PHFA cells were transduced with Ad-Null and Ad-Nef constructs and treated with various autophagy activators and inhibitors as described in Materials and Methods section. EVs were purified from culture supernatants, lysed with TNN buffer containing 1% NP40 and analyzed by western blotting for the detection of HIV-1 Nef and HSP-70 proteins. In lanes 1 and 2, whole-cell protein lysates from PHFA cells transduced either with Ad-Nef or Ad- Null were loaded as positive and negative controls of Nef detection. (b) Protein bands from panel a on the western blot membranes were used to quantitate HIV-1 Nef levels and HSP-70 by ImageJ program and the relative values were plotted after normalizing Nef with HSP-70 using Excel program. The results are shown in bar graphs averages from the three independent experiments. Results The test is based on conversion n derivative into luciferin by GSH in a couple of eactions where the luciferin produced is measured cence, and GSH levels are proportional to luciferin. n GSH levels in cells are indicative of oxidative can lead to apoptosis and cell death. We found a decline of GSH levels in PHFN cells treated with dicating that HIV-1 Nef may induce oxidative stress human neurons (Figure 5a). To gain more insight ediated neurotoxicity, the effect of HIV-1 Nef on total tau (t-tau) and phosphor-tau (p-tau) was PHFN and SH-SY5Y neuroblastoma cells. PHFN 5Y cells were transduced with either Ad-Null or Ad- Nef as described above. The whole-cell extracts obtained from the transduced cells were analyzed by immunoblotting. We observed that HIV-1 Nef is expressed both in PHFN and SH-SY5Y cells after Ad-Nef transduction; however, in these cells the expression levels of Nef protein was slightly less than that of primary human astrocytes (Figures 5b and c). HIV-1 Nef expression caused a significant decline in p-tau levels, whereas enhancing the t-tau levels in primary neurons (Figure 4a, compare lanes 1 and 2). When the p-tau/t-tau levels were compared with the control (Ad-Null), Nef-induced effect on p-tau was more significant (Figure 5c). HIV-1 Nef decreased the p-tau levels nearly 30%. In contrast, SH-SY5Y cells, which contain already lower p-tau levels were not affected by HIV-1 Nef expression. Both p-tau and t-tau levels were nearly identical in both Ad-Null and Ad-Nef-transduced cells (Figure 5b, compare lanes 3 and 4). Cell Death and Dis Nef as described above. The whole-cell extracts obtained from the transduced cells were analyzed by immunoblotting. We observed that HIV-1 Nef is expressed both in PHFN and SH-SY5Y cells after Ad-Nef transduction; however, in these cells the expression levels of Nef protein was slightly less than that of primary human astrocytes (Figures 5b and c). HIV-1 Nef expression caused a significant decline in p-tau levels, whereas enhancing the t-tau levels in primary neurons (Figure 4a, compare lanes 1 and 2). When the p-tau/t-tau levels were compared with the control (Ad-Null), Nef-induced effect on p-tau was more significant (Figure 5c). HIV-1 Nef decreased the p-tau levels nearly 30%. In contrast, SH-SY5Y cells, which contain already lower p-tau levels were not affected by HIV-1 Nef expression. Both p-tau and t-tau levels were nearly identical in both Ad-Null and Ad-Nef-transduced cells (Figure 5b, compare lanes 3 and 4). Results Finally, at 72 h post-transduc- tion, cells transduced with Ad-Nef showed a total loss of axons and dendrites leaving only the cell body structures compared with Ad-Null control. In order to gain more insight into Nef-mediated neurotoxicity, neurofilament middle chain expression as a neuronal marker was also monitored in cells expressing HIV-1 Nef by immunocytochemistry. These experiments were conducted in slides by transducing primary neurons with adeno constructs; Ad-Nef or Ad-Null as described above and the slides were fixed at 48 h post- transduction. As shown in Figure 4b, Nef expression in primary neurons caused a marked change in neuronal morphology and resulted in re-distribution of neurofilament expression. These results have suggested that Nef expres- sion in neurons is detrimental to neuronal processes, axons and dendrites. HIV-1 Nef causes axonal and neurite degeneration in primary human neurons. We have tested the neurotoxic effect of HIV-1 Nef by intracellularly co-expressing the Nef protein with EGFP in neurons. For this purpose, we co- transduced PHFN with Ad-Nef and Ad-EGFP or Ad-Null and Ad-EGFP, and followed the expression of EGFP fluorescence over time. As EGFP is expressed throughout the transduced neurons, it serves as a tool to monitor structural integrity of neurons. Twenty-four hours post-transduction, EGFP expres- sion was clearly seen distributed in neurons from the cell body to axons and dendrites (Figure 4a). In neurons transduced with Ad-Nef, cells started to display degeneration, Cell Death and Disease Nef-mediated neurotoxicity AS Saribas et al 5 igure 3 PHFA-derived EVs can deliver Nef to primary human neurons. (a) Western blot analysis of PHFN lysates treated with control and Nef-EVs. Whole-cell protein sates from EV-treated PHFNs were prepared and resolved on SDS-PAGE (lanes 3 and 4). After transferring the gel on a nitrocellulose membrane, expression of HIV-1 Nef was nalyzed by western blotting. Beta-actin was also probed in the same membranes as a loading control. In lanes 1 and 3, whole-cell lysates from PHFA cells transduced with Ad- ull and Ad-Nef were also loaded as negative and positive controls, respectively. (b) PHFNs were cultured in two-well chamber slides. Exosomes were purified from PHFA ansduced with either Ad-Null or Ad-Nef and then used to treat neurons for 4 h. PHFNs were fixed at 48 h and processed by immunocytochemistry for the detection and labeling f MAP-2, neurofilament, class III β-tubulin and Nef. Open arrows point Nef positive, but MAP-2 or class III β-tubulin-negative cells. Results Closed arrows point cells with Nef double aining with MAP-2 and class III β-tubulin Figure 3 PHFA-derived EVs can deliver Nef to primary human neurons. (a) Western blot analysis of PHFN lysates treated with control and Nef-EVs. Whole-cell protein lysates from EV-treated PHFNs were prepared and resolved on SDS-PAGE (lanes 3 and 4). After transferring the gel on a nitrocellulose membrane, expression of HIV-1 Nef was analyzed by western blotting. Beta-actin was also probed in the same membranes as a loading control. In lanes 1 and 3, whole-cell lysates from PHFA cells transduced with Ad- Null and Ad-Nef were also loaded as negative and positive controls, respectively. (b) PHFNs were cultured in two-well chamber slides. Exosomes were purified from PHFA transduced with either Ad-Null or Ad-Nef and then used to treat neurons for 4 h. PHFNs were fixed at 48 h and processed by immunocytochemistry for the detection and labeling of MAP-2, neurofilament, class III β-tubulin and Nef. Open arrows point Nef positive, but MAP-2 or class III β-tubulin-negative cells. Closed arrows point cells with Nef double staining with MAP-2 and class III β-tubulin Figure 3 PHFA-derived EVs can deliver Nef to primary human neurons. (a) Western blot analysis of PHFN lysates treated with control and Nef-EVs. Whole-cell protein lysates from EV-treated PHFNs were prepared and resolved on SDS-PAGE (lanes 3 and 4). After transferring the gel on a nitrocellulose membrane, expression of HIV-1 Nef was analyzed by western blotting. Beta-actin was also probed in the same membranes as a loading control. In lanes 1 and 3, whole-cell lysates from PHFA cells transduced with Ad- Null and Ad-Nef were also loaded as negative and positive controls, respectively. (b) PHFNs were cultured in two-well chamber slides. Exosomes were purified from PHFA transduced with either Ad-Null or Ad-Nef and then used to treat neurons for 4 h. PHFNs were fixed at 48 h and processed by immunocytochemistry for the detection and labeling of MAP-2, neurofilament, class III β-tubulin and Nef. Open arrows point Nef positive, but MAP-2 or class III β-tubulin-negative cells. Closed arrows point cells with Nef double staining with MAP-2 and class III β-tubulin HIV-1 Nef expression on tau protein levels in SH-SY5Y cells. To analyze further whether Nef- ent of neurons caused neurotoxicity, we utilized luthatione assay. Results Effect of HIV-1 Nef expression on tau protein levels in PHFN and SH-SY5Y cells. To analyze further whether Nef- EV treatment of neurons caused neurotoxicity, we utilized GSH-Glo Gluthatione assay. The test is based on conversion of a luciferin derivative into luciferin by GSH in a couple of enzymatic reactions where the luciferin produced is measured by luminescence, and GSH levels are proportional to luciferin. Changes in GSH levels in cells are indicative of oxidative stress that can lead to apoptosis and cell death. We found a significant decline of GSH levels in PHFN cells treated with Nef-EVs indicating that HIV-1 Nef may induce oxidative stress in primary human neurons (Figure 5a). To gain more insight into Nef-mediated neurotoxicity, the effect of HIV-1 Nef expression on total tau (t-tau) and phosphor-tau (p-tau) was analyzed in PHFN and SH-SY5Y neuroblastoma cells. PHFN and SH-SY5Y cells were transduced with either Ad-Null or Ad- Nef as described above. The whole-cell extracts obtained from the transduced cells were analyzed by immunoblotting. We observed that HIV-1 Nef is expressed both in PHFN and SH-SY5Y cells after Ad-Nef transduction; however, in these cells the expression levels of Nef protein was slightly less than that of primary human astrocytes (Figures 5b and c). HIV-1 Nef expression caused a significant decline in p-tau levels, whereas enhancing the t-tau levels in primary neurons (Figure 4a, compare lanes 1 and 2). When the p-tau/t-tau levels were compared with the control (Ad-Null), Nef-induced effect on p-tau was more significant (Figure 5c). HIV-1 Nef decreased the p-tau levels nearly 30%. In contrast, SH-SY5Y cells, which contain already lower p-tau levels were not affected by HIV-1 Nef expression. Both p-tau and t-tau levels were nearly identical in both Ad-Null and Ad-Nef-transduced cells (Figure 5b, compare lanes 3 and 4). Cell Death and Disease Nef-mediated neurotoxicity AS Saribas et al 6 igure 4 Nef expression in PHFN cells is detrimental. (a) PHFN cells were transduced with Ad-Null+Ad-EGFP or Ad-Nef+Ad-EGFP as described in Materials and Methods ection. The expression of EGFP was followed for 3 days in live PHFN cultures by fluorescence microscopy. Left panel represents the effect of the HIV-1 Nef expression on euronal architecture as monitored by EGFPexpression as well as bright field microscopy. (b) ICC of the PHFNs transduced with Ad-Nef or Ad-Null, and effect of HIV-1-Nef on the eurofilament levels. Results The PHFN cells were seeded on two-well chamber slides, transduced with either Ad-Nef or Ad-Null, and fixed at 48 h post-transductions. The slides were obed with monoclonal HIV-1 Nef (SF2) and polyclonal neurofilament middle chain antibodies and visualized using a Leica Fluorescence Microscope. Top panel show the ICC of e Ad-Null, bottom panel shows the ICC of the Ad-Nef-transduced PHFNs. From left to right: neurofilament expression in green color, HIV-1-Nef expression in purple, DAPI aining in blue and merging of the neurofilaments, HIV-1-Nef and DAPI Figure 4 Nef expression in PHFN cells is detrimental. (a) PHFN cells were transduced with Ad-Null+Ad-EGFP or Ad-Nef+Ad-EGFP as described in Materials and Methods section. The expression of EGFP was followed for 3 days in live PHFN cultures by fluorescence microscopy. Left panel represents the effect of the HIV-1 Nef expression on neuronal architecture as monitored by EGFPexpression as well as bright field microscopy. (b) ICC of the PHFNs transduced with Ad-Nef or Ad-Null, and effect of HIV-1-Nef on the neurofilament levels. The PHFN cells were seeded on two-well chamber slides, transduced with either Ad-Nef or Ad-Null, and fixed at 48 h post-transductions. The slides were probed with monoclonal HIV-1 Nef (SF2) and polyclonal neurofilament middle chain antibodies and visualized using a Leica Fluorescence Microscope. Top panel show the ICC of the Ad-Null, bottom panel shows the ICC of the Ad-Nef-transduced PHFNs. From left to right: neurofilament expression in green color, HIV-1-Nef expression in purple, DAPI staining in blue and merging of the neurofilaments, HIV-1-Nef and DAPI Nef-EVs interfere with neuronal action potential assessed by multielectrode array (MEA) studies. In order to gain insight into the impact of Nef-carrying EVs on neurophysiological functions, we used MEA approach. Primary embryonic rat neurons (PERNs) were plated and cultured in MEA dishes as described in Materials and Methods section. Meanwhile, EVs were purified and char- acterized from culture supernatants of PHFA either trans- duced with Ad-Nef or Ad-Null. PERNs were treated with EVs released and obtained from same number of astrocytes over a period of 96 h. MEA extracellular action potential recordings were done at 0, 24, 48 and 96 h post-treatments. The recorded electrophysiological activities are shown for a 60-s time span (60–120 s); as the first 60 s is the time required for the MEA amplifier recovery after substation of the MEA. Results The results are shown in bar graphs averages from the three independent experiments was found in neurons isolated from some HIV-1-infected brains,16,17 there was no evidence of Nef expression in these cells. Nevertheless, exposing neurons directly to HIV-1 Nef is crucial for understanding its neurotoxicity. HIV-1 Nef is primarily expressed in astrocytes,45 macrophages and microglia and exits these cells by means of EVs carrying a number of host biomolecules such as proteins, enzymes and RNA. We found that adenoviral Ad-Nef transduction of astrocytes not only generated high levels of HIV-1 Nef but also led to production of EVs carrying HIV-1 Nef. EVs have sometimes been ignored because it has been assumed that they are a mechanism of cellular garbage disposal. However, EVs have recently gained more attention after the discovery of their role in cell-to-cell communication46,47 and cancer proliferation.48 In the CNS, a majority of cells, such as astrocytes, neurons, oligodendrocytes and microglia, release EVs in order to setablish neuronal communication.49 EVs also contribute to neuropathology by delivering the toxic form of proteins in, for example, Alzheimers’ and Parkinson’s diseases.50,51 However, the EV machinery is also vulnerable to viral infections. A recent study demonstrated that HIV-1 exploits EVs to corrupt cell-to-cell communication in order to favor its spread in infected cells.52 HIV-1 Nef-containing was found in neurons isolated from some HIV-1-infected brains,16,17 there was no evidence of Nef expression in these cells. Nevertheless, exposing neurons directly to HIV-1 Nef is crucial for understanding its neurotoxicity. HIV-1 Nef is primarily expressed in astrocytes,45 macrophages and microglia and exits these cells by means of EVs carrying a number of host biomolecules such as proteins, enzymes and RNA. We found that adenoviral Ad-Nef transduction of astrocytes not only generated high levels of HIV-1 Nef but also led to production of EVs carrying HIV-1 Nef. EVs have sometimes been ignored because it has been assumed that they are a mechanism of cellular garbage disposal. However, EVs have recently gained more attention after the discovery of their role in cell-to-cell communication46,47 and cancer proliferation.48 In the CNS, a majority of cells, such as astrocytes, neurons, oligodendrocytes and microglia, release EVs in order to setablish neuronal communication.49 EVs also contribute to neuropathology by delivering the toxic form of proteins in, for example, Alzheimers’ and Parkinson’s diseases.50,51 However, the EV machinery is also vulnerable to viral infections. Results A recent study demonstrated that HIV-1 exploits EVs to corrupt cell-to-cell communication in order to favor its spread in infected cells.52 HIV-1 Nef-containing activity continues, and spiking activity in this culture totally disappears, whereas the number of recorded bursts of the negative and positive control returns to its basal activity within the succeeding 48 h. Results PHFN cells were grown in poly-D-lysine coated six-well plates in neuronal basal growth media at 37 oC under 5% CO2 maintained by changing 1/2 media every 4 days. Nef and Null EVs were added to neurons as described before and incubated for 4 days before the harvest. PHFNs were harvested after trypsin treatment and GSH levels were measured according to the manufacturer’s instructions. (b) Western blot analysis of the cell lysates obtained from the PHFN and SH-SY5Y neuroblastoma cells transduced with Ad-Nef or Ad-Null. Whole-cell lysates of the transduced PHFNs or SH-SY5Y cells were analyzed by western blotting for the detection of Nef, tau and phopsho-tau levels by using specific antibodies. GAPDH was also probed in the same membranes as a loading control. (c) Quantification of the proteins in Ad-Nef or Ad-Null-transduced PHFNs. Protein bands on the western blot membranes were used to quantitate tau and phospho-tau levels and loading controls, GAPDH by ImageJ program and the relative values were plotted after normalizing the tau and phospho-tau with GAPDH using Excel program. The results are shown in bar graphs averages from the three independent experiments Figure 5 Effect of Nef on Tau and phospho-tau in primary neurons. (a) GSH-Glo Gluthatione assay for the EV-treated PHFNs. PHFN cells were grown in poly-D-lysine coated six-well plates in neuronal basal growth media at 37 oC under 5% CO2 maintained by changing 1/2 media every 4 days. Nef and Null EVs were added to neurons as described before and incubated for 4 days before the harvest. PHFNs were harvested after trypsin treatment and GSH levels were measured according to the manufacturer’s instructions. (b) Western blot analysis of the cell lysates obtained from the PHFN and SH-SY5Y neuroblastoma cells transduced with Ad-Nef or Ad-Null. Whole-cell lysates of the transduced PHFNs or SH-SY5Y cells were analyzed by western blotting for the detection of Nef, tau and phopsho-tau levels by using specific antibodies. GAPDH was also probed in the same membranes as a loading control. (c) Quantification of the proteins in Ad-Nef or Ad-Null-transduced PHFNs. Protein bands on the western blot membranes were used to quantitate tau and phospho-tau levels and loading controls, GAPDH by ImageJ program and the relative values were plotted after normalizing the tau and phospho-tau with GAPDH using Excel program. Results All recordings were performed at 37 °C and 7% CO2; the same condition employed to maintain the cells during the culturing period. As a reference, the activity of neurons was recorded right before starting the treatments (pretreatment condition of t = 0 h). As depicted by Figure 6, a day after the treatment (t = 24 h), the MEAs under the mentioned conditions experi- enced an increase of 55%, 33% and 115% compared with their basal pretreatment activity. This trend more or less holds for the controls in the following day, however, it is reversed for the Nef-treated cells. According to the recordings at t = 48 h post treatment, the activities of controls increased by 25% compared with the preceding day. Nef-treated neurons showed a considerable decrease of 83%. The dropping of Cell Death and Disease Nef-mediated neurotoxicity AS Saribas et al ect of Nef on Tau and phospho-tau in primary neurons. (a) GSH-Glo Gluthatione assay for the EV-treated PHFNs. PHFN cells were grown in poly-D-lysine coated neuronal basal growth media at 37 oC under 5% CO2 maintained by changing 1/2 media every 4 days. Nef and Null EVs were added to neurons as described bated for 4 days before the harvest. PHFNs were harvested after trypsin treatment and GSH levels were measured according to the manufacturer’s instructions. analysis of the cell lysates obtained from the PHFN and SH-SY5Y neuroblastoma cells transduced with Ad-Nef or Ad-Null. Whole-cell lysates of the transduced Y5Y cells were analyzed by western blotting for the detection of Nef, tau and phopsho-tau levels by using specific antibodies. GAPDH was also probed in the es as a loading control. (c) Quantification of the proteins in Ad-Nef or Ad-Null-transduced PHFNs. Protein bands on the western blot membranes were used to nd phospho-tau levels and loading controls, GAPDH by ImageJ program and the relative values were plotted after normalizing the tau and phospho-tau with xcel program. The results are shown in bar graphs averages from the three independent experiments 7 5 Eff t f N f T d h h t i i ( ) GSH Gl Gl th ti f th EV t t d PHFN PHFN ll i l l i t d Figure 5 Effect of Nef on Tau and phospho-tau in primary neurons. (a) GSH-Glo Gluthatione assay for the EV-treated PHFNs. Discussion Middle row: Ad-Null EVs treated neurons at t = 0, 24, 48 and 96 h. Bottom row: the Ad-Nef-EVs treated neurons at t = 0, 24, 48 and 96 h 8 Figure 6 Extracellular action potential recordings of primary rat neurons treated with Nef-EVs by MEA. Rat hippocampal neurons were dissociated from the hippocampi of E18 prenatal rat embryos and placed onto the MEA dishes. For the best recording, quality neurons are required to stay and develop processes on the MEA for at least 12–14 days. Experimental recordings were started when the cultures were 14 days old after the cells were treated by control and Nef-EVs obtained from PHFAs. After placing the MEAs on the amplifier, recordings were performed using the MC_Rack software at a sampling frequency of 2000 kHz. The recorded spiking data (in μV versus time) were transferred to the MATLAB environment for further offline analysis. Top row: untreated control at t = 0, 24, 48 and 96 h. Middle row: Ad-Null EVs treated neurons at t = 0, 24, 48 and 96 h. Bottom row: the Ad-Nef-EVs treated neurons at t = 0, 24, 48 and 96 h HIV-1 Nef was readily detected in the EV-treated neurons, which was surprising and indicates that Nef delivery by EVs is sufficient for it to accumulate and be detected by immunoblot- ting. Our immunocytochemistry data also indicated that Nef EVs were taken up by human neurons. Using EVs to deliver Nef to the neurons also resulted in increased oxidative stress as measured by a decrease in GSH levels, an indication of neurotoxicity. We noticed significant neurotoxicity by expres- sing HIV-1 Nef in primary human neurons. Axonal and neurite degeneration is significantly higher in the presence of HIV-1 Nef in these cells indicating the toxic effect of the viral protein. Prolonged exposure to Nef completely destroyed nearly all axons and dendrites in the transduced cells. Furthermore, significant changes were also observed in tau proteins. Our finding shows that intracellular expression of HIV-1 Nef in primary neurons enhanced the t-tau while downregulating the p-tau levels. The relationship between HIV-1 infection, and t-tau and p-tau proteins in the brain are complex and not completely understood. Anthony et al.55 have reported that HIV-1-infected subjects have elevated levels of p-tau in particular in HAART-treated patients suggesting Alzheimers’ like neuropathology. Discussion HIV-1 does not infect human neurons, but instead it targets neurons by its proteins, Tat, Vpr, gp120 and Nef. Effects of HIV-1 Tat and gp120 on neurons have been studied extensively; however, toxicity caused by these viral proteins in the CNS is not completely understood.40 It has been shown that expres- sion of HIV-1 Tat in astrocytes can lead to astrocytosis and subsequent death of neurons,41 and similar effects have also been reported with gp120-mediated neurotoxicity. Both Tat and gp120 appear to be responsible for serious neurotoxicity in HIV-1-infected brains.40,42–44 Despite the gp120 and Tat, little is known about the possible impact of HIV-1 Nef on neurons. In our work, we have elucidated some of the neurotoxic effects of HIV-1 Nef on human neurons using a two-pronged approach: first to deliver the Nef protein to neurons by means of astrocytic exosomes and second to transduce the neuronal cells with Ad- Nef to express Nef protein intracellularly. Although the Nef gene HIV-1 does not infect human neurons, but instead it targets neurons by its proteins, Tat, Vpr, gp120 and Nef. Effects of HIV-1 Tat and gp120 on neurons have been studied extensively; however, toxicity caused by these viral proteins in the CNS is not completely understood.40 It has been shown that expres- sion of HIV-1 Tat in astrocytes can lead to astrocytosis and subsequent death of neurons,41 and similar effects have also been reported with gp120-mediated neurotoxicity. Both Tat and gp120 appear to be responsible for serious neurotoxicity in HIV-1-infected brains.40,42–44 Despite the gp120 and Tat, little is Cell Death and Disease Nef-mediated neurotoxicity AS Saribas et al Figure 6 Extracellular action potential recordings of primary rat neurons treated with Nef-EVs by MEA. Rat hippocampal neurons were dissociated from the hippocampi of E18 prenatal rat embryos and placed onto the MEA dishes. For the best recording, quality neurons are required to stay and develop processes on the MEA for at least 12–14 days. Experimental recordings were started when the cultures were 14 days old after the cells were treated by control and Nef-EVs obtained from PHFAs. After placing the MEAs on the amplifier, recordings were performed using the MC_Rack software at a sampling frequency of 2000 kHz. The recorded spiking data (in μV versus time) were transferred to the MATLAB environment for further offline analysis. Top row: untreated control at t = 0, 24, 48 and 96 h. Discussion In the differential centrifugation method,59 approximately 30 ml supernatants from the transduced PHFA supernatants were first centrifuged at 3000× g for 30 min at 4 oC (Eppendorf Centrifuge, 5804R) to clear cell debris followed by a centrifugation at 10 000 × g for 30 min at 4 oC (HB-6 rotor, Sorval Centrifuge, RC6+, Thermo Scientific) followed by filtration (Corning Incorp., NY, USA). At this step, clear supernatants were either stored at 4 oC or being proceeded for ultracentrifuga- tion. Ultracentrifugation was performed at 100 000 × g either using SW55Ti rotor at 35 000 r.p.m. for 3 h or SW24 at 24 000 r.p.m. for 4 h in a Beckman Ultracentrifuge. The EV pellets at this stage were small and had a transparent appearance with brownish color. After centrifugation, the tubes were inverted to remove the remaining liquid then the pellets were resuspended in 1 x PBS buffer. The EVs prepared by differential centrifugations are very similar to those obtained using ExoQuick method as determined by immunoblotting. The resuspended EV solutions were either used immediately or stored at −30 oC until use. Protein contents of the EVs were determined by Bradford Protein Assay. HANDs manifest itself at many different levels in neuronal activities, such as deficiencies in cognition, motor functions and behavioral abnormalities, which are somewhat similar to other neurological diseases. Nearly half of the HIV-1 infection results with HAND. Overall, our results have suggested that EVs derived from primary astrocytes can deliver Nef into the neurons and contribute to the neurotoxicity associated with HAND development in patients infected with HIV-1. Materials and Methods Ethics statement. All samples were obtained and utilized in accordance with Temple University Human Subjects Protections and the approval of the institutional review board. Plasmids and adenovirus constructs. Ad-Nef was constructed by cloning the HIV-1 Nef (SF2) gene from pcDNA3.1-SF2-Nef (NIH-AIDS reagent program, Germantown, MD, USA) into pDC515 (Ad-5) and propagated in HEK293A cells before purification using Opti-Prep gradient. Purified Ad-Nef had a titer of 1 × 109 p.f.u./ml. To construct recombinant adenoviral vector harboring EGFP, EGFP cDNA was cloned in the adenovirus-shuttle plasmid pDC515 under the control of the murine cytomegalovirus promoter (purchased from Microbix Inc., Ontario, Canada). Adeno-EGFP recombinant shuttle containing EGFP sequence was transfected into HEK-293 cells with pBHGfrt (del) E1, 3FLP, and a plasmid that provides adenovirus type 5 genome deleted in E1 and E3 genes. Discussion The growth medium was changed every 3– 4 days. PHFA were plated at densities of 2 × 106 cells/100 mm dish for adenovirus transduction experiments. SH-SY5Y neuroblastoma cells were obtained from American Tissue Culture Collection (ATCC, Manassas, VA, USA, CRL-2266). They were plated in the poly-D-lysine-coated six-well dishes and maintained in astrocyte growth medium (described above) at 37 oC under 5% CO2 atmosphere. (Gibco/Thermo Fisher Inc. Waltham, MA, USA), 0.05% GlutaMAX (Gibco) and gentamicin (10 μg/ml) at 37 oC under 5% CO2. Half of the neuronal growth medium was replaced with fresh medium every 3–4 days. PHFAs were also obtained from CNAC tissue culture core. Cells were maintained in astrocyte growth media (Dulbecco’s modified Eagle's medium (DMEM): F12 medium supplemented with 10% fetal bovine serum, 10% GlutaMAX, insulin and gentamicin (10 μg/ml)) at 37 oC under 5% CO2 atmosphere. The growth medium was changed every 3– 4 days. PHFA were plated at densities of 2 × 106 cells/100 mm dish for adenovirus transduction experiments. SH-SY5Y neuroblastoma cells were obtained from American Tissue Culture Collection (ATCC, Manassas, VA, USA, CRL-2266). They were plated in the poly-D-lysine-coated six-well dishes and maintained in astrocyte growth medium (described above) at 37 oC under 5% CO2 atmosphere. Purification of the EVs (exosomes) from PFHA. Supernatants were obtained either from the un-transduced PHFA or PHFA transduced with Ad-Nef or Ad- Null. EVs were purified from these supernatants utilizing either ExoQuick (System Biosciences (SBI)) or differential centrifugation. In the ExoQuick method, 5 ml supernatant obtained from the Ad-Null or Ad-Nef-transduced PFHA cells was centrifuged at 3000 r.p.m. (Eppendorf Centrifuge, Eppendorf North America, Hauppauge, NY, USA, 5804R) for 30 min at 4 oC, then treated with 1 ml ExoQuick reagent by gently mixing in a 15 ml sterile culture tube. The mixture was allowed to precipitate EVs overnight at 4 oC. On the next day, the precipitated EVs were recovered by centrifugation at 3000 r.p.m. for 30 min at 4 oC (Eppendorf Centrifuge, 5804R). The liquid phase was removed and tubes centrifuged briefly to collect the remaining liquid that was carefully removed by pipetting without disturbing the pellet. Finally, the pellets were gently rinsed with 1 ml cold 1 x PBS to remove the remnants of the reagent. The white colored exosome pellet obtained using ExoQuick was resuspended in 1 x PBS buffer. The exosome solution was either used directly or stored at −30 oC. Discussion Plaques of recombinant adenovirus arising as a result of frt/FLP recombination were isolated, grown and purified by cesium chloride density equilibrium banding. Empty shuttle plasmid, pDC515, was used to construct control Ad-Null, a virus without a transgene (Barrero et al., 2013).57 The titers for Ad-.EGFP and Ad-Null were 3 × 1010 p.f.u./ml. Nef-EGFP construct was a kind gift from Dr. Michael D Powell (Campbell et al., 2008).58 Treatment of PHFA cells with autophagy inducers and inhibitors. PHFA cells were cultured in 60 mm tissue culture dishes (800 000 cells per dish). Cells were transduced with either Ad-Null or Ad-Nef and treated with autophagy activators rapamycin (50 nM), perifosine (50 μM), resveratrol (75 μM), tomaxifen (5 μM), MG-132(10 μM), valproic acid (0.6 mM) and autophagy inhibitors Baf-A1 (10 nM), 3-MA (10 mM), LY294002 (50 μM), wortmannin (2 nM) and chloroquine (50 μM). All chemicals and drugs were purchased from InvivoGen, San Diego, CA, USA. Supernatants were obtained either from the un-transduced PHFA or PHFA transduced with Ad-Nef or Ad-Null at 48 h post-transduction and 36 h post- treatments. EVs were purified from these supernatants utilizing ExoQuick (System Biosciences (SBI)), lysed in TNN buffer containing 1% NP40, 30 μg total EV lysates from each treatment were separated on SDS-PAGE, and analyzed by western blotting for the detection of HIV-1 Nef and HSP-70. In parallel to the purification of EVs from culture supernatants, whole-cell lysates were also collected from the same studies, separated on SDS-PAGE, and analyzed by western blotting for the detection of HIV-1 Nef, LC3, pAKT(Ser476), total AKT, p44/42 MAPK, total MAPK, pmTOR (Ser2448) and total mTOR. Reagents. Antibodies were obtained from the following sources: anti-Nef (SF2), EH1 from NIH-AIDS Reagent Program, anti-GAPDH and anti-HSP-70 from Santa Cruz (Dallas, TX, USA), anti-tau from Santa Cruz and anti-PHF-tau from Pierce/ Thermo Fisher, Inc., (Waltham, MA, USA), anti-beta tubulin class III from Sigma- Aldrich (St. Louis, MO, USA), anti-Alix Cell Signaling, Inc. (Beverly, MA, USA), LC3B from Sigma-Aldrich, β-tubulin from LI-COR, Odyssey (Lincoln, NE, USA), anti-neurofilament medium chain from Cell Signaling, Inc., anti-pAKT (Ser476), anti-AKT, p44/42 MAPK (Thr202/204), anti-MAPK, pmTOR (Ser2448), and anti- mTOR were purchased from Cell Signaling Technology (Danvers, MA, USA). Mammalian protease inhibitors were obtained from Sigma-Aldrich. Bradford reagent was from BioWorld (Dublin, OH, USA). GSH-Glo Glutathione Assay kit was purchased from Promega Corp. (Madison, WI, USA). Treatment of PHFN with astrocyte-derived EVs. Discussion Gisslén et al.56 have analyzed t-tau and p-tau levels in 86 HIV-1+subjects including 21 of them with AIDS dementia complex (ADC) in a cross-sectional study EVs are released from the infected cells such as astrocytes and macrophages.53 These Nef-containing EVs can reach neurons where they deliver Nef’s toxic effect. We chose human astrocyte-derived EVs to study Nef neurotoxicity, as the astrocytes are known to release EVs to support neurons. Our second approach was intracellular expression of Nef in neurons that exhibits this viral protein’s ultimate neurotoxicity. p y Several lines of evidence suggest that autophagy and secretion of EVs are coordinated mechanisms.54 We have recently reported that expression of HIV-1 Nef compromises the autophagic pathways in PHFAs by inducing autophagosome formation and blocking the assembly of autophagolysosomes.39 Here, we studied possible role of autophagy signaling in Nef release associated with EVs. Interestingly, autophagy inhibitors Perifosine (inhibitor of AKT phosphorylation), tomaxifen (estro- gen receptor antagonist), MG-132 (proteasome inhibitor) and autophagy activators LY294002 (PI3 kinase inhibitor) and worthmannin (PI3 kinase inhibitor) caused a significant increase in Nef release in EVs. These results clearly provided evidence that autophagy pathways influenced by HIV-1 Nef may contribute to EV biogenesis and release of Nef in EVs from astrocytes. The exact role of autophagy in EV biogenesis and role of HIV-1 Nef in molecular crosstalk between autophagy and EV biogenesis remained to be determined. Here, we studied possible role of autophagy signaling in Nef release associated with EVs. Interestingly, autophagy inhibitors Perifosine (inhibitor of AKT phosphorylation), tomaxifen (estro- gen receptor antagonist), MG-132 (proteasome inhibitor) and autophagy activators LY294002 (PI3 kinase inhibitor) and worthmannin (PI3 kinase inhibitor) caused a significant increase in Nef release in EVs. These results clearly provided evidence that autophagy pathways influenced by HIV-1 Nef may contribute to EV biogenesis and release of Nef in EVs from astrocytes. The exact role of autophagy in EV biogenesis and role of HIV-1 Nef in molecular crosstalk between autophagy and EV biogenesis remained to be determined. Cell Death and Disease Nef-mediated neurotoxicity AS Saribas et al Nef-mediated neurotoxicity AS Saribas et al 9 demonstrating that while some ADC and HIV-1+infected patients had high t-tau in their CSF, there was no increase in p-tau levels unlike those seen in Alzheimers’ patients. Discussion Interest- ingly, a recent study showed that amyotrophic lateral sclerosis patients have low levels of p-tau in their CSF and these low levels are correlated with neurocognitive dysfunction seen in this disease and p-tau/t-tau can be used as a biomarker for the disease progression. These results are intriguing as we also observed low p-tau in HIV-1 Nef-expressing primary neurons. In our case, the neurons are of fetal origin and not myelinated, therefore they do not represent adult human neurons. In SH- SY5Y neuroblastoma cells, however, we did not see any change in p-tau or t-tau levels. As these cells are transformed, the pathways governing the tau pathology could be different and therefore not affected by HIV-1 Nef. Possible impact of Nef- EVs on neuronal electrophysiology was also analyzed by MEA studies. Consistent with neurotoxicity assays, Nef-EVs inter- fered with neuronal action potential assessed by MEA studies. demonstrating that while some ADC and HIV-1+infected patients had high t-tau in their CSF, there was no increase in p-tau levels unlike those seen in Alzheimers’ patients. Interest- ingly, a recent study showed that amyotrophic lateral sclerosis patients have low levels of p-tau in their CSF and these low levels are correlated with neurocognitive dysfunction seen in this disease and p-tau/t-tau can be used as a biomarker for the disease progression. These results are intriguing as we also observed low p-tau in HIV-1 Nef-expressing primary neurons. In our case, the neurons are of fetal origin and not myelinated, therefore they do not represent adult human neurons. In SH- SY5Y neuroblastoma cells, however, we did not see any change in p-tau or t-tau levels. As these cells are transformed, the pathways governing the tau pathology could be different and therefore not affected by HIV-1 Nef. Possible impact of Nef- EVs on neuronal electrophysiology was also analyzed by MEA studies. Consistent with neurotoxicity assays, Nef-EVs inter- fered with neuronal action potential assessed by MEA studies. (Gibco/Thermo Fisher Inc. Waltham, MA, USA), 0.05% GlutaMAX (Gibco) and gentamicin (10 μg/ml) at 37 oC under 5% CO2. Half of the neuronal growth medium was replaced with fresh medium every 3–4 days. PHFAs were also obtained from CNAC tissue culture core. Cells were maintained in astrocyte growth media (Dulbecco’s modified Eagle's medium (DMEM): F12 medium supplemented with 10% fetal bovine serum, 10% GlutaMAX, insulin and gentamicin (10 μg/ml)) at 37 oC under 5% CO2 atmosphere. Discussion After primary antibody incubation, cells were washed three times with PBS and then incubated in a secondary solution containing 1500 FITC mouse secondary and 1:500 rhodamine rabbit secondary, in 5% BSA for 2 h. Wells were then washed five times with PBS, and mounted with Vectashield mounting solution containing DAPI. Then glass coverslips were added before imaging on a Leica Fluorescence Microscope. (Leica Biosystems Inc., Buffalo Grove, IL, USA). MEA recordings. All the recordings were performed using the MEA-1060 system (Multichannel Systems, Reutlingen, BW, Germany). The core component of this system is an MEA consisting of 60 titanium nitrite (TiN) electrodes covering a rectangular grid. Each electrode contains a circular TiN pad of 30 μm diameter where the array spacing between each two neighboring electrodes varies in the range of 100–500 μm. Before plating the cells, the MEAs underwent sterilization via applying 70% ethanol and then exposing the arrays to UV light for 60 min. As the MEA surface is originally hydrophobic, poly-D-lysine was used to hydrophilize the MEAs, as well as to provide a layer to enhance cell adhesion to the MEAs. To this end, poly-D-lysine (Sigma-Aldrich) was diluted in PBS with final concentration of 1 mg/ml and applied to the MEA surface for 2 h at 37 °C. In addition to poly-D-lysine, laminin (Invitrogen/Thermo Fisher, Inc., Waltham, MA, USA) was also introduced to the MEA surface (overnight at 37 °C) to promote long-lasting cellular adhesion (to culture cells for 410 days) and to improve the neural processes development. Once the MEAs were sterilized and coated, rat neural cells were plated on them with the average density of 5000 cells/mm2. Rat hippocampal cells were dissociated from the hippocampi of E18 prenatal rat embryos. After preparations, the suspended cells were directly placed onto the MEA surface where they settled adhered to the array within the next 3–4 h. For the best recording, quality neurons are required to stay and develop processes on the MEA for at least 12– 14 days. During this period, neurons were maintained and fed using an appropriate medium in a regular manner. Experimental recordings were started when the cultures were 14 days old after the cells were treated by control and Nef-EVs obtained from PHFAs. After placing the MEAs on the amplifier, recordings were performed using the MC_Rack software (Reutlingen , BW, Germany) at a sampling frequency of 2000 kHz. Discussion PHFN were grown in 2 ml neurobasal growth media (Gibco) in six-well plates or in two-well chamber slides at 37 oC under 5% CO2. For treatments, Null or Nef-EVs were first diluted and gently suspended in 1 × PBS to prevent self-aggregation. Then half of the neuronal growth medium was removed from the cell culture and aliquots of diluted EVs solutions were added to reach a final total protein concentration of approximately 20 μg total proteins per well. The control cells were treated with same volume of 1 × PBS. The cells were Cell culture. Primary human fetal neurons (PHFN) were obtained in six-well plates or two-chamber slides from the Comprehensive Neuro-AIDS Center (CNAC) tissue culture core at Temple University Lewis Katz School of Medicine. Cells were maintained in Neurobasal medium (Gibco) containing B-27 supplement Cell Death and Disease Nef-mediated neurotoxicity AS Saribas et al 10 then incubated at 37 oC for 1 h followed by addition of 1 ml fresh neurobasal growth medium to each well. The EV-treated neurons were incubated 48 h at 37 oC under 5% CO2 before harvesting for whole-cell protein extraction. chloroquine (50μM). Twenty-four hours post-treatments, cells were incubated with 1 ml of MTTworking solution (DMEM with 0.5 mg/ml MTT) for 2 h at 37 °C. The converted dye was solubilized with 1 ml acidic isopropanol (0.004 M HCL in isopropanol). Absorbance of the converted dye was measured at a wavelength of 570 nm with background subtraction at 650 nm. Immunocytochemistry for Nef uptake by neurons. PHFNs were cultured in two-well chamber slides (500 000 cells per well). PHFNs were treated with Null or Nef-EVs by adding 20 μg EVs in PBS (purified from PHFA media) to 400 μl Opti-MEM and incubating at 37 oC for 4 h. Following EV incubation, Opti- MEM was removed, and neurons were supplemented with neuronal media for 48 h. After incubation, neurons were fixed with cold acetone:methanol (50 : 50) for 1 min, washed three times with PBS, then, blocked with 10% BSA in PBS for 1 h, and incubated with rabbit polyclonal anti-MAP-2, anti-neurofilament, anti-class III β-tubulin, and monoclonal anti-Nef antibodies. Primary antibody dilutions were 1:300 in 5% BSA overnight at 4 °C with gentle rocking. Discussion To reduce the amount of noise while making sure to acquire the important information from neural spiking, an online built-in bandwidth filter was used throughout the recordings. The recorded spiking data (in μV versus time) were transferred to the MATLAB environment for further offline analysis. Preparation of the PHFN and PHFA cell lysates, and SDS-PAGE/ western blotting. The PHFA lysates were prepared 24–48 h after adenovirus transduction using TNN buffer with 1% NP40 supplemented with mammalian protease inhibitors. For 10% SDS-PAGE/western blot, 40 μg proteins per well were used. The PHFN lysates were prepared as follows. At the end of incubations, cells were washed in PBS and harvested by trypsinization. PHFN were then lysed in TNN buffer with 1% NP40 supplemented with mammalian protease and phosphatase inhibitors, 0.4 mM NaF and 2 mM Na3VO4. The protein concentrations were determined by Bradford Protein Quantification assay. Protein samples were first denatured in SDS loading dye, heated at 95 oC for 5 min, then separated on 10% SDS-PAGE. After electrophoresis, gels were transferred onto 0.45 μm nitrocellulose membranes for 2 h at 250 mA, alternatively overnight at 40 mA in 1 × cold transfer buffer (100 mM TrisHCl, pH 7.4, 150 mM NaCl, 20% methanol). Membranes were blocked in either 1 x PBST buffer containing 10% milk or 1 x LI- COR blocking buffer at least 1 h at RT. The primary monoclonal antibodies, anti- HIV-1 Nef (sf2), anti-tau, anti-phospho-tau, anti-GAPDH, anti-alix, anti-beta tubulin class III, anti-HSP-70, anti-pAKT (Ser476), anti-AKT, p44/42 MAPK (Thr202/204), anti-MAPK, pmTOR (Ser2448) and anti-mTOR were diluted 1/1000 in buffer containing 5% milk and added to membranes where needed and were incubated overnight at 4 oC with gentle shaking. After washing the membranes with 1 X PBST buffer three times, the secondary antibodies (LI-COR goat against rabbit 680 for polyclonal, LI-COR goat against mouse 800 for monoclonal) were added to the membranes and incubated for 45 min at RT followed by three times (5 min) washing with 1 X PBST buffer and a final wash with 1 X PBS buffer. Washed membranes were stored in 1 X PBS before scanning images on an ODISSEY Clx instrument. The intensity of each protein band was calculated using ImageJ (NIH) software (https://imagej.nih.gov/ij) and bar graphs were produced in Microsoft Excel program. Statistical analysis. All of the values presented on the graphs are given as means ± S.E.M. Discussion ANOVA and unpaired Student’s t-tests were used to analyze the statistical significance, and P-values o0.05 were considered statistically significant. Conflict of Interest The authors declare no conflict of interest. Acknowledgements. We thank the past and present members of the Department of Neuroscience and Center for Neurovirology for sharing their ideas and reagents. This study utilized services offered by core facilities of the Comprehensive Neuro-AIDS Center (CNAC NIMH Grant Number P30MH092177) at Temple University Lewis Katz School of Medicine. This work was made possible by grants awarded by NIH to KK, SA and IKS. GSH-Glo glutathione assay. The assay kit containing all necessary reagents was purchased from Promega Corp. PHFN were treated with Null and Nef-containing EVs separately as described above. After 4 days, PHFN were washed with PBS, trpysinized and resuspended in 200 μl PBS buffer. The assay was carried out according to the manufacturer’s guideline. Twenty microliters of Luciferin-NT and 20 μl GSTwas first mixed with 2 ml GSH-Glo reaction buffer. From this mixture, 10 μl resuspended neuronal samples were mixed with 100 μl GSH reaction buffer containing Luciferin-NT and GST. 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https://openalex.org/W2083751277	https://zenodo.org/record/1931927/files/article.pdf	German	null	Bemerkungen zu dem Aufsatz F. Frankes „Ueber Epicondylitis humeri” in No. 1 dieser Wochenschrift	Deutsche medizinische Wochenschrift/Deutsche Medizinische Wochenschrift	1,910	public-domain	880	Vün Prof. M. Bern bar dt in Berlin. An die Spitze seines Aufsatzes: ,,TJeber Epicondylitis humeri" in dieser Wochenschrift 1910, No.1, S. 13, hat F. Franke folgenden Satz gestellt: Unter obigem Titel hatte ich für den 37. Kongreß der Deutschen Gesellschaft für Chirurgie 1908 einen Vortrag angemeldet über ein eigen- tümliches schmerzhaftes Leiden an der Außenseite hauptsächlich des rechten Elibogengelenkes, das ich schon seit über zehn Jahren bei einer ganzen Reihe von Personen, besonders des mittleren Alters beobachtet hatte, über das mir aber die Literatur keinen Aufschluß gab. b h i h d f f k h d ß i h g Demgegenüber möchte ich darauf aufmerksam machen, daß ich im Jahre 1896 im Neurologischen Zentralbiatt 1896, No. leine Mitteilung veröffentlicht habe, betitelt: ,,Ueber eine wenig bekannte Form der Beschäftigungsneuralgie." In der Einleitung zu diesem Auf- satze hob ich hervor:,, Bei der Umschau über die etwa vorliegende Literatur fand ich außer einer denselben Gegenstand, wenn auch kurz behandelnden Notiz von E. Remak nichts, weshalb ich mich für berechtigt halte, im folgenden die Aufmerksamkeit der Fachgenossen auf diesen Gegenstand zu lenken." g Es liegt mir fern, das, was ich damals gesagt, hier noch einmal ausführlich zu wiederholen. Wer sich für diesen Gegenstand interessiert, mag an dem betreffenden Ort nachlesen. Er wird finden, daß sich meine nunmehr vor 14 Jahren gemachten Bemerkungen im wesentlichen mit denen von Franke decken, wenngleich sie in mancher Beziehung auch wieder davon abweichen. So habe ich den besonderen Einfluß der Influenza damals nicht nachweisen können und vielmehr Ueberarbeitung und besondere Anstrengung als ätiologische Momente in den Vorder- grund gestellt. Ich erwähnte damals schon, daß sich das Leiden vor- wiegend bei Männern, weiter vorwiegend rechtseitig fand; ich sagte weiter, daß es möglicherweise auch einmal am Epicondylus humeri medialis vorkommen könnte, daß ich es auch als an der -linken Seite vor- kommend und zweimal doppelseitig vorhanden feststellen konnte. A f d i h i b f P d Th i ill Auf das, was ich in bezug auf Prognose und Therapie gesagt, will ich hier nicht weiter eingehen, da ich sonst den ganzen kleinen Aufsatz von damals wiederholen müßte. Nur in bezug auf die Aetiologie -und die Diagnose möchte ich meine damaligen Bemerkungen hier noch einmal wörtlich wiedergeben. g Ich sagte: Unter den ätiologischen Momenten spielt - offenbar die Ueberanstrengung gewisser Muskelgruppen, der Strecker der Hand und Finger, die erste Rolle. DEUTSCHE MEDIZINISCHE WOCHENSCHRIFT. DEUTSCHE MEDIZINISCHE WOCHENSCHRIFT. DEUTSCHE MEDIZINISCHE WOCHENSCHRIFT. 3. Februar 1910. 221 Heruntergeladen von: NYU. Urheberrechtlich geschützt. in No. 1 dieser Woehenschrit. in No. 1 dieser Woehenschrit. Vün Prof. M. Bern bar dt in Berlin. Vün Prof. M. Bern bar dt in Berlin. Diese Muskeln werden nicht nur bei allen den Bewegungen innerviert, welche eine Extension bezwecken, sondern sie treten auch bei Tätigke-itsäußerungen, welche scheinbar von den anta- gonistisehen Muskeln allein ausgeführt werden, dem Beugen der Hand und Finger, in die energischste Aktion. Ich erinnere nur an die auf- Tho5 DEUTSCHE MEDIZINISCHE WOCHESCHRIFT. Tho5 222 fallende Schwäche des Händedrucks derjenigen, welche an einer Korn- pressions- oder Blei-Radialislähmung leiden. Eine ungemein große Anzahl von Muskeln nimmt von dem Epi- condylus lateralis oder dessen nächster, am Oberarm oder am Radius- köpfchen gelegener sehniger Umgebung ihren Ursprung. Der M. supi- nator longus (brachioradialis), der Supinator brevis, der M. externus carpi radialis longus und brevis, der Extensor digitorum commuiiis, der Extensor carpi ulnaris, der Anconaeus, sie alle entspringen vom Epi- condylus lateralis allein oder von ihm und den Gelenkbändern, welche ihn mit dem Radiusköpfchen verbinden, und setzen bei ihrer wiederholten und übermäßigen Kontraktion den periostalen Ueberzug dieses Knochen- vorsprungs einer oft nicht unbedeutenden Zerrung aus. Ob in einigen Fällen, wo ein Trauma auf diese Gegend eingewirkt hat oder wo, wie es mir einigemal (namentlich bei der doppelseitigen Affektion) schien, refrigeratorische Einflüsse (Erkältung, sogenannte rheumatische Affektion) vorhanden waren, eine geringe Entzündung des Periostes vorliegt, wage ich nicht zu entscheiden. Jedenfalls können diese Entzündungen kaum bedeutender Natur sein, da, wie gesagt, selbst in ausgesprochenen Fällen Röte, Schwellung, Temperaturerhöhung und oft auch spontane Schmerzhaftigkeit vermißt wird. p g Was ich bisher mitgeteilt, wird wohl genügen, um die wesent- liche Uebereinstimmung (abgesehen von der durch Franke in den Vordergruifd gerückten ätiologischen Wirksamkeit der Influenza) der Frankeschen Beobachtungen mit den meinigen darzutun. Zu bedauern ist, daß Franke in der von ihm durchforschten Literatur, wie er sagt, keinen Aufschluß gefunden. Außer meiner eingangs erwähnten Arbeit im Neurologischen Zentralblatt, in der ich der von R e ma k publizierten Beobachtungen Erwähnung tat, habe ich über die in Rede stehende Affektion auch in meinem Buche: ,,Die Erkrankungen der peripherischen Nerven" Bd. 2, zweite Auflage aus dem Jahre 1904, S. 350 u. 351 die bis dahin bekannten Tatsachen über das Leiden zusammengestellt und die von F é r é über denselben Cegenstand gemachten Beobachtungen sowie die Rivières hinzugefügt, auch die von Clado gemachten Mit- teilungen über den Tennis-Arm beigebracht. Féré hat übrigens die durch R e ma k und mich schon seit langer Zeit bekannten Zustände mit dem Namen der Epikondylalgie belegt. Heruntergeladen von: NYU. Urheberrechtlich geschützt. Vün Prof. M. Bern bar dt in Berlin. Ich unterlasse weitere Auseinandersetzungen, hebe aber zum Schluß hervor, daß ich der von Franke für einige seiner Fälle vorgeschlagenen Therapie (Abmeißelung des Epicondylus) nach allem, was ich von der wenn auch oft erst in längerer Zeit erfolgenden Spontanheilung des Leidens gesehen habe, nicht zustimmen kann.
https://openalex.org/W4391692670	https://journal.multitechpublisher.com/index.php/ijasr/article/download/1258/1186	English	null	The Effect of Classroom Debate on Students' Academic Achievement in Higher Education: an Overview	International Journal of Applied and Scientific Research	2,024	cc-by	6,053	International Journal of Applied and Scientific Research (IJASR) Vol. 2, No. 1, 2024 : 123-136 International Journal of Applied and Scientific Research (IJASR) Vol. 2, No. 1, 2024 : 123-136 123 ( DOI: https://doi.org/10.59890/ijasr.v2i1.1258 https://journal.multitechpublisher.com/index.php/ijasr/ INTRODUCTION Debate is a time-honoured tradition that dates back 2,400 years (Garrett et al., 1996). Protagoras, known as "the father of debate," first established it as a teaching approach in Ancient Greece (Darby, 2007). Later, in the twelfth century, Muslim academics at colleges used this pedagogy to instruct Islamic jurisprudence (Makdisi, 1981). Debate is an intellectual exercise that entails engaging in discussions with others who have divergent and/or conflicting viewpoints. In order to effectively vote on their thoughts, participants must possess open-mindedness, enabling them to carefully consider and evaluate various viewpoints presented during discussions. Having an open mind is a fundamental characteristic of being a critical thinker. Another characteristic of debate is the audacity to express one's views. Some people believe that debate is connected to democracy and freedom of expression, which may explain why (Ericson et al., 2003). “Innovation refers to the inclination to produce or identify concepts, options, or opportunities that may be beneficial in problem-solving, communication, and entertainment” (Franken, 1994, p. 396). The capacity to surpass traditional concepts, systems, patterns, relationships, or similar entities and to generate significant novel ideas and information using human sensory perception and evaluative capabilities. Creativity is the ability to think and create in a way that is unconventional, using both insight and intellect, and resulting in the highest quality results. Creativity is an essential characteristic for educators in the field of education. Nowadays, teaching heavily relies on statistics, such as test results, and guides instructors with established methods, causing them to doubt their own ability to be creative (Bunting, 2006). Facilitating communication and collaboration between both teachers and students is a crucial first step in integrating creativity into education. Teaching at the tertiary level necessitates lecturers having a closer connection with the learners rather than just delivering lectures. Educators should demonstrate adaptability in embracing ideas and enriching the topics to facilitate discussions and express perspectives (Collard & Looney, 2014). Instructors get advantages from reflecting on their initial motivations for pursuing a career in teaching. What objectives do they anticipate achieving? What satisfaction do they expect? When creating lesson plans, educators should possess the ability to discern the long-term impact these plans will have on the learners for the remainder of the semester. Therefore, instructors must possess the ability to premeditate and cultivate originality in advance. INTRODUCTION Alternative resources, such as audio-video substances, innovative distribution via technological advances, and interactive presentations, should be organised in a suitable manner. To successfully execute this, faculty members must be open-minded and willing to explore unconventional and innovative ideas The Effect of Classroom Debate on Students' Academic Achievement in Higher Education: an Overview Zanyar Nathir Ghafar Pharmacy department,Bright Technical and Vocational Institute, Sulaimaneah, Kurdistan Region- Iraq Corresponding Author: Zanyar Nathir Ghafar zanyar.ghafar@btvi.edu.iq A R T I C L E I N F O Keywords: Academic Debate, Education, Discourse, Tertiary Education, Scholastic Achievement Received : 15, November Revised : 17, December Accepted: 20, January ©2024 Ghafar: This is an open-access article distributed under the terms of the Creative Commons Atribusi 4.0 Internasional. A R T I C L E I N F O Keywords: Academic Debate, Education, Discourse, Tertiary Education, Scholastic Achievement Received : 15, November Revised : 17, December Accepted: 20, January ©2024 Ghafar: This is an open-access article distributed under the terms of the Creative Commons Atribusi 4.0 Internasional. A R T I C L E I N F O Keywords: Academic Debate, Education, Discourse, Tertiary Education, Scholastic Achievement A R T I C L E I N F O Keywords: Academic Debate, Education, Discourse, Tertiary Education, Scholastic Achievement Received : 15, November Revised : 17, December Accepted: 20, January A B S T R A C T The use of classroom debate as an instructional technique is prevalent, particularly in secondary and higher education. There have been several studies examining the effects of classroom discussion in areas such as second language acquisition, philosophical thinking, psychology, and pure science, there is a lack of particular research in the subject of administrative science. Multidisciplinary fields that focus on critical and analytical examination of administrative ideas, management principles, and methods. The review papers covered organisational and leadership theories. Therefore, the study aims is to provide a thorough examination of how classroom debate affects students' academic performance in higher education, in various academic settings, and across all departments and disciplines of study. The research findings indicate classroom debates are beneficial and relevant to many academic subjects because they allow students to think more critically and express creative ideas, which improve their academic performance. A theoretical framework shows a clear connection between student academic success and the advantages of conversation in the classroom. ©2024 Ghafar: This is an open-access article distributed under the terms of the Creative Commons Atribusi 4.0 Internasional. 123 ( DOI: https://doi.org/10.59890/ijasr.v2i1.1258 https://journal.multitechpublisher.com/index.php/ijasr/ ( DOI: https://doi.org/10.59890/ijasr.v2i1.1258 https://journal.multitechpublisher.com/index.php/ijasr/ 123 Ghafar METHODOLOGY The utilization of classroom debate as a pedagogical approach is widespread, especially in secondary and tertiary education. While numerous studies have explored the impact of classroom discussions in domains such as second language acquisition, philosophical reasoning, psychology, and pure science, there is a noticeable gap in research concerning administrative science. 124 International Journal of Applied and Scientific Research (IJASR) Vol. 2, No. 1, 2024 : 123-136 This interdisciplinary field involves the comprehensive and analytical scrutiny of administrative concepts, management principles, and methodologies. Existing literature reviews have predominantly focused on organizational and leadership theories. This study endeavors to conduct a comprehensive investigation into the influence of classroom debate on the academic performance of higher education students, encompassing diverse academic environments and spanning all departments and fields of study. The Influence of Debating Skills on Students' Academic Performance The Influence of Debating Skills on Students' Academic Performance The Influence of Debating Skills on Students Academic Performance According to Doody and Condon (2012), there are six essential abilities required for engaging in an argument. The abilities included are analysis, interpretation, assessment, deduction, clarification, and autonomy. Interpretation skill refers to the aptitude of students to recognise and elucidate fundamental matters in a conversation. Analysis entails the capability to collect and arrange information. Evaluation, on the other hand, involves the ability of students to assess the worth of information based on its precision, relevance, and the existence of diverse perspectives. Meanwhile, inference pertains to the capacity of pupils to draw conclusions, understand values, and express perspectives. Explanation pertains to the logical clarifications provided by students on a particular matter, whereas self-regulation involves the capacity to evaluate and critique the performance of others while actively being part of the team. This article will focus on discussing three out of the six talents. The talents include interpretation, analysis, and inference. The subsequent sections will elucidate these talents. The study, on the other hand, found a statistically significant link between learning through discussions and the development of important graduate skills like analytical thinking and interpersonal skills. Furthermore, the study revealed that intellectual difficulties, motivation, and learning in depth facilitated the learning process. Not all pupils have a favourable disposition towards debate. (D'souza, 2013) RESEARCH RESULT AND DISCUSSION Utilise Debate As A Method Of Fostering Imaginative Education Classroom discussions cultivate creativity in the learning process. Debating has a long and significant history since it promotes students' ability to advocate for their ideas and express themselves via competitive debates between schools. Debate is a competitive activity involving two teams: the positive team, which supports the resolutions, and the negative team, which opposes them (Noonan, 2011). Debate is a structured method of presenting and discussing arguments that facilitates direct contact and representation. Debate inherently involves the use of manipulation, with each argument including an element of persuasion. Persuasion often relies on evoking emotional responses from the audience, which ultimately influence their reactions and interactions with the subject at hand. Debate is considered a pedagogical approach that involves taking a definite stance, in favour of or in opposition to, on a subject, topic, or problem with the aim of teaching and learning. While there have been some studies conducted on the advantages of debates, there is currently a lack of research that directly connects debates with enhanced academic achievement (Omelicheva & Avdeyeva, 2008; Onen, 2016). Hence, given the multitude of advantages that arguing offers in terms of students' academic performance, this research seeks to evaluate the influence of debate on students' scholastic achievements. Debate creates an environment of active learning and encourages cooperation among teams by presenting convincing facts (Doody & Condon, 2012). According to Warner & Bruschke (2001), classroom arguments have the potential to enhance students' academic achievement by promoting active participation and involvement among learners. Catterall (2002) found that learners who participate in debate events have a 25% broader spectrum of academic abilities compared to their counterparts who do not engage in such activities. Debate activities foster competitiveness, motivating students to deepen their knowledge in order to confidently defend their points. Participation in debating events enables students to enhance their writing abilities. Oral conversations will significantly enhance their reading comprehension compared to their counterparts who do not engage in such activities. Colbert and Biggers (1985) were among the first to claim the positive effects of engaging in debating activities on one's well-being. Carr (2002) claimed that students engaged in arguing were more adept at understanding new ideas and foreign terminology. RESEARCH RESULT AND DISCUSSION Additionally, they had the capability to acquire a broader range of knowledge, including subjects such as history, philosophy, laws and regulations, and current events at the collegiate 125 Ghafar Ghafar level. They gained confidence, improved their listening skills, and enhanced their strategic ability. Students who participate in discussion events are more likely to seek out leadership roles in their communities and schools. Better yet, they are known to take the lead on their debate teams (Bradley, 1959). Abilities in Analyzing Analytical skills are another set of abilities that might have an effect on the performance of pupils. Students' analytical abilities are characterised by their capacity to make connections between concepts and to critically examine the significance of those ideas. One of the most important criteria that should be considered when evaluating a debate programme is the extent to which it helps its participants improve their analytical skills. The demands of a changing society necessitate students to assess and evaluate ideas, and these abilities are accessible in debating activities (see Doody & Condon, 2012; Omelicheva & Avdeydeva, 2008). Many writers believe that students should be able to demonstrate these skills through debates. In a society where there is an abundance of information, the capacity to think critically serves as an exceptionally important skill. Bellon (2000) suggests that students can develop and form intellectual curiosity by engaging in "thinking" with their classmates. Through a comprehensive thinking process that indirectly fosters critical thinking, students will gain experience in reasoning processes and develop practice in using those reasoning processes. Their research conducted in 1999 supports this conclusion. Participating in debating activities enhanced students' capacity to reason and defend topics relevant to their subject areas, as discovered by Mike, Berkowitz, Hunt, and Louden (1999). According to Bellon (2000), Tous, and Haghighi (2016), critical thinking is one of the most essential skills because it compels students to engage in rigorous thought processes in an effort to establish connections between phrases and concepts that give them a deeper level of significance. Overall, the students saw the classroom discussion as a beneficial educational exercise. The learners held the belief that the discussions contributed to the enhancement of their critical thinking abilities and proficiency in oral communication. Furthermore, according to the students' assertions, the discussions also yielded advantages such as acquiring proficiency in the subject matter, enhancing self-assurance, conquering performance anxiety, and refining collaborative abilities. Zare & Othman (2015). Novice and medium ESL learners often avoid debate due to the perception that it is an activity only suited for advanced learners. Nevertheless, this research primarily concentrated on using argument as a means of enhancing the speaking abilities of students who have a lesser level of competency in the English language. Competence in the area of communication The ability to understand or clarify the significance of a message, subject, or piece of information is referred to as an interpretation skill. Tilus (2012) described interpretation skills as the ability to understand and successfully communicate the meaning of supplied information to others. Students must actively participate in the interpretation of the information via discussion and reasoning activities in order for classroom debate to take place (Collard & Looney, 2014). Furthermore, students must acquire the skills to research current topics, formulate well-reasoned arguments, consider multiple perspectives, differentiate between subjective and fact-based information, and incorporate relevant data (Darby, 2007). During a debate practice, students are expected to analyse a topic critically and comment on it. Debate improves a student's ability to critically evaluate a subject, provide reasoned arguments, and articulate their points of view (Tumposky, 2004). In addition to developing their persuasive skills and organising their ideas for efficient delivery and acceptance, students are urged to convey and present their ideas in the most captivating manner possible. Students must study a certain curriculum in order to do this. 126 International Journal of Applied and Scientific Research (IJASR) Vol. 2, No. 1, 2024 : 123-136 Abilities in Analyzing As a result, the study selected two Malaysian ESL learners from a boarding school: a male student with a low level of English proficiency and a female student with an intermediate level of ability. The study admits the restriction of having just two samples, but it provided valuable insights into the experiences of these students as participants in the debate competition phenomenon. Skills in Making Inferences Inferential abilities refer to the capacity to develop a systematic approach that leads to the accurate recognition of a consistent pattern via repeated exposure to both examples and non-examples, with subsequent feedback provided after each answer (Shafrir et al., 1990, p. 506). Students with advanced 127 Ghafar inferential skills possess the capacity to recognise problems or disagreements and may provide resolutions based on important occurrences (Doody & Condon, 2012). Debate activities require students to engage in active listening to the logic and arguments of others. This process trains students to assess and contrast arguments from many viewpoints before formulating their own opinions (Omelicheva & Avdeydeva, 2008). This approach encourages students to pay close attention to specific elements, engaging in the simultaneous practice of several abilities, including active listening, effective communication (both verbal and non-verbal), and analytical thinking (Onen, 2016). Developing inferential abilities will improve students' capacity to draw logical conclusions about a given topic. During group conversations, the ability to make inferences is crucial, as it enables students to form judgements and draw conclusions (Doody & Condon, 2012). The authors argue that the advantages of participating in debate exercises may be implemented into a variety of programmes as a teaching tool to develop proficiency with these soft skills. This is in response to the fact that higher education is continuing its attempts to fulfil the expectations of employers. The debate in a particular course on organisational issues includes the perspectives of the educational institution's participants and the earliest indications of how this experience has positively influenced their talents. (Chikeleze et al., 2018). Likewise, it is appropriate to expose students to debates since they encompass many contextual situations, including both official and informal settings, as well as both one-on-one and group interactions. Debate may facilitate learners expressing their opinions by integrating understandable input along with results (Othman et al., 2013). Debate is an organised form of communication that enables learners to assume different roles and develop fundamental interaction abilities. During this phase, students will acquire the capacity to begin and sustain a discussion and articulate their viewpoint. Brice (1992) conducted research indicating that ESL learners typically struggle with their speaking abilities. The Impact of Critical Thinking Ondebate Recently, academics, employers, educators, and the media have all paid close attention to the idea of critical thinking skills and higher-order thinking. Indeed, having the capacity for critical thought is becoming increasingly important for workers in the twenty-first century. Advanced critical thinking skills, negotiation and problem-solving abilities, and outstanding communication talents are becoming more and more valued in staff members and workers (Gervey et al., 2009; Halpern, 2004). In academic and professional contexts, people with strong critical thinking abilities and effective communication skills display behavioural tendencies that are highly appreciated and valued (Mason, 2007; Rudd, 2007; Kosciulek & Wheaton, 2003). Training future workers in communication and critical thinking techniques is still a contentious and continuing matter of discussion. Researchers and academics have started looking into various methods and techniques that could improve and cultivate oral communication and critical thinking skills in the classroom setting, given the increasing emphasis on these abilities and their 128 International Journal of Applied and Scientific Research (IJASR) Vol. 2, No. 1, 2024 : 123-136 increased demand in the changing job market (Halpern, 2003). According to Browne & Freeman (2000), subjects that concentrate on enhancing critical thinking skills have to include a significant number of evaluative learning exercises. Bringing up controversial subjects in the classroom, according to Browne & Freeman (2000), creates an atmosphere of cognitive tension that encourages the investigation of other viewpoints, logical reasoning, and critical thinking. According to research, having discussions is a good way to develop and preserve your ability to think critically and communicate orally (Camp & Schnader, 2010; Paul & Elder, 2007; Ryan & College, 2006; Roy & Macchiette, 2005; Ng et al., 2004). Getting ready to contribute to a conversation is necessary in order to improve a thorough comprehension of the topic and promote active learning. Preparation for a debate improves the capacity to arrange and communicate information clearly, acquire and use facts and evidence to support a concept, critically assess and refute opposing arguments, and articulate an argument using exact language. Critical thinking skills are in line with these competencies (Rudd, 2007; Kosciulek & Wheaton, 2003). increased demand in the changing job market (Halpern, 2003). According to Browne & Freeman (2000), subjects that concentrate on enhancing critical thinking skills have to include a significant number of evaluative learning exercises. The Impact of Critical Thinking Ondebate Bringing up controversial subjects in the classroom, according to Browne & Freeman (2000), creates an atmosphere of cognitive tension that encourages the investigation of other viewpoints, logical reasoning, and critical thinking. According to research, having discussions is a good way to develop and preserve your ability to think critically and communicate orally (Camp & Schnader, 2010; Paul & Elder, 2007; Ryan & College, 2006; Roy & Macchiette, 2005; Ng et al., 2004). Getting ready to contribute to a conversation is necessary in order to improve a thorough comprehension of the topic and promote active learning. Preparation for a debate improves the capacity to arrange and communicate information clearly, acquire and use facts and evidence to support a concept, critically assess and refute opposing arguments, and articulate an argument using exact language. Critical thinking skills are in line with these competencies (Rudd, 2007; Kosciulek & Wheaton, 2003). p ( ) Since critical thinking is such a broad topic, experts have provided several definitions and terminologies. Finding a widely accepted precise definition can be challenging, and the variety of interpretations can sometimes pose problems for teachers when explaining it to their pupils (Rear, 2010). Ennis (1987) offers an often-used definition of critical thinking as "sensible contemplation that is centred on deciding what to believe or do" (p. 10). Comparably, critical thinking is the logical process of choosing what to accept or not believe, according to Norris (1985). Critical thinking is the process of being cautious and introspective while forming opinions about ideas or behaviours, according to Ennis and Norris. According to Gieve (1998), students must investigate and assess the justifications for their behaviours, beliefs, and expertise claims in order to participate in critical thinking. In this process, they must defend and critically examine their own health as well as the health of others, including experts, teachers, and authoritative texts (p. 126). Certain experts consider critical thinking to be the same as scepticism. McPeck (1981) described critical thinking as the propensity and capacity to engage in an activity with deliberate scepticism and analysis (p. 8). According to Sofo (2004), critical thinking entails challenging and reassessing our widely held assumptions and views. Critical thinkers, according to Sofo (2004), are also individuals who evaluate their behaviours to improve how they approach things. They are people who attentively consider other points of view and have an open mind. The Impact of Critical Thinking Ondebate Verbal Communication Proficiency as a Catalyst for Initiating Debates Verbal Communication Proficiency as a Catalyst for Initiating Debates According to Burns and Joyce (1997), speaking is a communication activity that focuses on meaning-building and entails the creation, acquisition, and processing of information. Speaking ability, on the other hand, refers to the capacity for accurate, precise, and useful verbal communication in the target language. Speaking is a crucial ability and the primary means of communication for instructors and ESL/EFL students alike. Colleges and 129 Ghafar Ghafar universities have recently given oral skills, also known as communicative competency, a specific position in the English teaching curriculum. However, throughout the semester, instructors allocate minimal time and effort to assignments and activities that necessitate students to engage in communication with each other in a second or foreign language. Liao (2009) states that oral abilities are evaluated more highly in real-world scenarios, particularly speaking. Speaking is essential for daily communication, and most of the time, one's capacity to communicate clearly and concisely forms the basis of one's first assessment of that person (Liao, 2009). p ( ) Rebecca (2006) claims that speaking is the first method by which youngsters begin to learn a language. According to Rebecca (2006), speaking has a significant role in driving changes in a language and encompasses a substantial part of daily engagement in language-related activities for almost all individuals. Liao (2009) asserts that the primary objective of ESL learners is to achieve proficiency in speaking, since there is a prevailing notion that English language acquisition is closely associated with oral communication. When someone claims that some language learners excel in English, it is often assumed that they possess a high level of proficiency in the language. Furthermore, the acquisition of oral proficiency may greatly facilitate the development and enhancement of other essential abilities. Nevertheless, the speaking skill holds great importance and significance for students, particularly those learning English as a second or foreign language. However, various studies conducted by Kim (2006), Cheng et al. (2004), Morita (2000), and Ferris (1998) have raised concerns about the overall speaking ability of students, especially ESL learners. Ferris (1998) conducted a study examining the perspectives of ESL students from three American tertiary institutions regarding the difficulties they encounter with listening and speaking skills. The study found that the learners expressed the highest level of anxiety towards oral presentations and whole classroom discussions. However, the students reported minimal difficulties when engaging in small-group discussions. Verbal Communication Proficiency as a Catalyst for Initiating Debates In her research, Kim (2006) aimed to investigate the perspectives of Asian foreign graduate students on their academic speaking and listening abilities in university courses, as well as the difficulties they encounter in attaining these standards. Debate in the Classroom Thus, learners have limited opportunities to actively engage in the learning process, express their perspectives, and enhance their critical thinking skills. According to Paul (1990), learners in these educational institutions only acquire lower-order learning, namely associative learning. This style of learning focuses on memorising course knowledge, which may lead to biases, misunderstandings, and confusion. As a result, students may get demotivated and lose their enthusiasm for studying, leading them to rely on short-term memorising techniques and prioritise immediate performance. According to a study conducted by DEROUICHE (2019), classroom debate improves students' ability to comprehend topics from many viewpoints, engage in critical thinking, and thus arrive at clear conclusions and produce reliable evidence. These talents are the primary characteristics associated with critical thinking. Many first-year master students and their lecturers hold the belief that classroom discussion is a good method for promoting critical thinking. Based on these findings, educators should enhance students' understanding of the significance of critical thinking ability and facilitate its development by including classroom debates. Debate in the Classroom Debate serves to develop proficiencies in critical thinking, analysing, synthesising, and spontaneous speaking. According to Krieger (2005), several students demonstrated significant improvement in their capacity to articulate and justify their views by engaging in debate exercises. Furthermore, the students often and promptly identified the shortcomings in one another's arguments. This talent is regarded as a fundamental aspect of critical thinking abilities, whereby students are anticipated to review and scrutinise the information they acquire in a critical manner. According to Nisbett (2003), debate is a significant educational technique that helps develop analytical thinking abilities and encourages individuals to critically evaluate the validity 130 International Journal of Applied and Scientific Research (IJASR) Vol. 2, No. 1, 2024 : 123-136 of their views (p. 210). Facione & Facione (2008) state that critical thinking abilities consist of analytic thinking skills and the ability to consciously reflect on and monitor one's own ideas. In the conventional method of instruction, such as using a chalkboard and lecturing, students adopt a passive role and receive lectures from their educators. Thus, learners have limited opportunities to actively engage in the learning process, express their perspectives, and enhance their critical thinking skills. According to Paul (1990), learners in these educational institutions only acquire lower-order learning, namely associative learning. This style of learning focuses on memorising course knowledge, which may lead to biases, misunderstandings, and confusion. As a result, students may get demotivated and lose their enthusiasm for studying, leading them to rely on short-term memorising techniques and prioritise immediate performance. According to a study conducted by DEROUICHE (2019), classroom debate improves students' ability to comprehend topics from many viewpoints, engage in critical thinking, and thus arrive at clear conclusions and produce reliable evidence. These talents are the primary characteristics associated with critical thinking. Many first-year master students and their lecturers hold the belief that classroom discussion is a good method for promoting critical thinking. Based on these findings, educators should enhance students' understanding of the significance of critical thinking ability and facilitate its development by including classroom debates. of their views (p. 210). Facione & Facione (2008) state that critical thinking abilities consist of analytic thinking skills and the ability to consciously reflect on and monitor one's own ideas. In the conventional method of instruction, such as using a chalkboard and lecturing, students adopt a passive role and receive lectures from their educators. CONCLUSIONS AND RECOMMENDATIONS Classroom discussion facilitates the development of critical thinking and oral communication abilities, among several other talents. Students acquire the skills to combine, examine, and assess claims and arguments. Debate also fosters active learning, enabling students to actively engage in the learning process. Additionally, it aids kids in developing their oral communication abilities. Furthermore, the findings validate that students had a positive inclination towards the debate experience and recognised it as a novel and captivating method. Participating in discussion events boosts students' self- confidence and cultivates their creative thinking. Through the implementation of efficient oversight, the inclusion of discussion activities has the potential to enhance the educational setting for students. This is because engaging in debate requires students to pay close attention to the arguments and defences presented by their classmates. Undoubtedly, participating in discussions is an exceptional platform for nurturing creativity. It improves students' skills and self-assurance, namely by using their ability to understand, analyse, and draw conclusions. These skills provide students with an edge when they transition into the professional domain after graduation. Classroom debate is a dynamic instructional approach that enhances learning across several disciplines. For example, it assists learners in acquiring proficiency in the course material, enhancing critical thinking abilities, and developing oral communication skills. Debate facilitates the development of critical thinking skills in learners via activities such as examining arguments, engaging in research, gathering information, doing analysis, questioning assumptions, assessing arguments, 131 Ghafar and demonstrating interpersonal abilities. It fosters an environment where learners relinquish their passive positions and become actively involved in the educational process. Learners can utilise these talents and skills in various settings. Similarly, engaging in classroom debates facilitates the development of oral communication skills, which are important for success in almost any occupation. ADVANCED RESEARCH Classroom debate is a valuable instructional technique that positively impacts students‟ academic performance in higher education. It promotes critical thinking, enhances communication skills, and fosters a collaborative learning environment. As such, it should be incorporated into the curriculum across all departments and disciplines of study in order to maximize students‟ learning outcomes and prepare them for future success. 132 International Journal of Applied and Scientific Research (IJASR) Vol. 2, No. 1, 2024 : 123-136 REFERENCES Anderson, S., & Mezuk, B. (2012). Participating in a policy debate program and academic achievement among at-risk adolescents in an urban public school district: 1997–2007. Journal of Adolescence, 35(5), 1225-1235. Bellon, J. (2000). 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(1990). Learning disability, inferential skills, and postfailure reflectivity. Journal of learning disabilities, 23(8), 506-514. What is Social Science? Sofo, F. (2004). Open Your Mind: The 7 keys to thinking Critically. Crows Nest, NSW: Allen & Unwin. Tilus, G. (2012). 6 Critical Thinking Skills You Need to Master Now. Tous, M. D., & Haghighi, S. (2016). Developing Critical Thinking with Debate: Evidence from Iranian Male and Female Students. Informal Logic, 36(1), 64-82. Tumposky, N. R. (2004). The debate. The Clearing House: A Journal of Educational Strategies, Issues and Ideas, 78(2), 52-56. Warner, E., & Bruschke, J. (2001). Gone on debating. Competitive Academic Debate As A Tool Of Empowerment. Contemporary Argumentation and Debate, 22, 1-21. Zare, P., & Othman, M. (2015). REFERENCES Students' perceptions toward using classroom debate to develop critical thinking and oral communication ability. Asian Social Science, 11(9), 158. 136
https://openalex.org/W2964802528	https://europepmc.org/articles/pmc6695243?pdf=render	English	null	A Rare Case of Pituitary Apoplexy Secondary to Dengue Fever-induced Thrombocytopenia	Curēus	2,019	cc-by	2,560	DOI: 10.7759/cureus.5323 A Rare Case of Pituitary Apoplexy Secondary to Dengue Fever-induced Thrombocytopenia Mathew Thomas , Alex Robert , Pavan Rajole , Priya Robert 1 2 2 3 Mathew Thomas , Alex Robert , Pavan Rajole , Priya Robert 1 2 2 3 1. Department of Breast Medical Oncology, Cleveland Clinic, Cleveland, USA 2. Internal Medicine, Church of South India Holdsworth Memorial Hospital, Mysore, IND 3. Internal Medicine, Government Medical College, Kottayam, IND  Corresponding author: Mathew Thomas, thomasm4@ccf.org Disclosures can be found in Additional Information at the end of the article © Copyright 2019 Thomas et al. This is an open access article distributed under the terms of the Creative Commons Attribution License CC-BY 3.0., which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Thomas et al. This is an open access article distributed under the terms of the Creative Commons Attribution License CC-BY 3.0., 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 Open Access Case Report Abstract Pituitary apoplexy (PA) is an endocrine emergency resulting from hemorrhage or infarction within a pituitary tumor or non-tumorous pituitary gland. The most important predisposing factors for PA are cerebral angiographic procedures, systemic hypertension, surgeries, head injury, coagulopathies, and drugs. Thrombocytopenia is a risk factor for PA. Dengue fever causes thrombocytopenia and there are reported cases of dengue hemorrhagic fever predisposing to PA. But there are no reported cases of dengue fever per se predisposing to PA, and we report such a case in an 85-year-old elderly male who presented with features suggestive of a hypertensive emergency and, on evaluation, was found to have a pituitary incidentaloma and dengue fever. During the hospital course, he developed acute IIIrd nerve palsy and, when evaluated, was found to have PA. He responded well to medical management with steroids and thyroxine. Prompt initiation of treatment is of utmost importance in pituitary apoplexy, as it can result in adverse events, including loss of vision and even death from hemodynamic compromise. Pituitary apoplexy (PA) is an endocrine emergency resulting from hemorrhage or infarction within a pituitary tumor or non-tumorous pituitary gland. The most important predisposing factors for PA are cerebral angiographic procedures, systemic hypertension, surgeries, head injury, coagulopathies, and drugs. Thrombocytopenia is a risk factor for PA. Dengue fever causes thrombocytopenia and there are reported cases of dengue hemorrhagic fever predisposing to PA. But there are no reported cases of dengue fever per se predisposing to PA, and we report such a case in an 85-year-old elderly male who presented with features suggestive of a hypertensive emergency and, on evaluation, was found to have a pituitary incidentaloma and dengue fever. During the hospital course, he developed acute IIIrd nerve palsy and, when evaluated, was found to have PA. He responded well to medical management with steroids and thyroxine. Prompt initiation of treatment is of utmost importance in pituitary apoplexy, as it can result in adverse events, including loss of vision and even death from hemodynamic compromise. Categories: Endocrinology/Diabetes/Metabolism, Internal Medicine, Neurology Keywords: pituitary apoplexy, dengue, thrombocytopenia Introduction Pituitary apoplexy (PA) is an acute clinical syndrome characterized by sudden-onset headache, vomiting, visual disturbances, altered sensorium, and ophthalmoplegia, secondary to hemorrhage or infarction within a pituitary tumor or non-tumorous pituitary gland [1]. PA may occur spontaneously or as a result of multiple risk factors [2]. Dengue fever causes thrombocytopenia, which, in turn, can precipitate PA. A review of the literature showed five reported cases of dengue hemorrhagic fever predisposing to PA [1-5]. However there are no reported cases of dengue fever per se predisposing to PA, and we report such a case of PA in the setting of dengue fever-induced thrombocytopenia. Received 07/26/2019 Review began 08/01/2019 Review ended 08/02/2019 Published 08/05/2019 How to cite this article Thomas M, Robert A, Rajole P, et al. (August 05, 2019) A Rare Case of Pituitary Apoplexy Secondary to Dengue Fever-induced Thrombocytopenia. Cureus 11(8): e5323. DOI 10.7759/cureus.5323 Received 07/26/2019 Review began 08/01/2019 Review ended 08/02/2019 Published 08/05/2019 Case Presentation An 85-year-old elderly male presented with a low-grade fever for three days, generalized headache for two days, and one day of giddiness. His headache was gradual in onset and of a dull, aching type. There was no history of vomiting, altered sensorium, seizures, head trauma, or weakness. Past medical history was significant for presbycusis and hypertension (on 5 mg PO amlodipine). He denied any history of smoking, excessive alcohol use, or substance abuse. On 2019 Thomas et al. Cureus 11(8): e5323. DOI 10.7759/cureus.5323 examination, he was alert and oriented, with a pulse rate of 68/min, blood pressure (BP) of 210/120 mmHg, and respiratory rate of 18/min. The physical examination was unremarkable except for bilateral sensorineural hearing loss. Investigations and treatment Investigations and treatment Labs at presentation (Table 1) were significant for thrombocytopenia. Variable Reference values Measurement Hemoglobin (g/dL) 13.5-17.5 14 Total leucocyte count (TLC) (/mm3) 4,500-11,000 4400 Platelet count (/mm3) 150,000 - 400,000 9000 MCV (μm3) 80-100 85 Sodium (mEq/L) 136-145 141 Potassium (mEq/L) 3.5-5.0 4.3 Blood urea nitrogen (mmol/dL) 8–24 26 Creatinine (mg/dL) 0.6-1.2 1.2 TSH (µU/mL) 0.5-5 0.63 TABLE 1: Labs at presentation MCV: mean corpuscular volume; TSH: thyroid stimulating hormone His electrocardiogram (ECG) showed sinus rhythm with first-degree heart block. His CT brain showed a sellar lesion favoring a pituitary macroadenoma (25 x 24 mm) (Figure 1). In view of his fever and thrombocytopenia, he was evaluated for dengue fever and was found to have dengue immunoglobulin M (IgM) positive, and hence his platelet count was monitored daily (Table 2). Thus, his differential diagnoses were a hypertensive emergency, severe thrombocytopenia, most probably post viral, and pituitary incidentaloma. Labs at presentation (Table 1) were significant for thrombocytopenia. Labs at presentation (Table 1) were significant for thrombocytopenia. Variable Reference values Measurement Hemoglobin (g/dL) 13.5-17.5 14 Total leucocyte count (TLC) (/mm3) 4,500-11,000 4400 Platelet count (/mm3) 150,000 - 400,000 9000 MCV (μm3) 80-100 85 Sodium (mEq/L) 136-145 141 Potassium (mEq/L) 3.5-5.0 4.3 Blood urea nitrogen (mmol/dL) 8–24 26 Creatinine (mg/dL) 0.6-1.2 1.2 TSH (µU/mL) 0.5-5 0.63 TABLE 1: Labs at presentation MCV: mean corpuscular volume; TSH: thyroid stimulating hormone MCV: mean corpuscular volume; TSH: thyroid stimulating hormone His electrocardiogram (ECG) showed sinus rhythm with first-degree heart block. His CT brain showed a sellar lesion favoring a pituitary macroadenoma (25 x 24 mm) (Figure 1). In view of his fever and thrombocytopenia, he was evaluated for dengue fever and was found to have dengue immunoglobulin M (IgM) positive, and hence his platelet count was monitored daily (Table 2). Thus, his differential diagnoses were a hypertensive emergency, severe thrombocytopenia, most probably post viral, and pituitary incidentaloma. His electrocardiogram (ECG) showed sinus rhythm with first-degree heart block. His CT brain showed a sellar lesion favoring a pituitary macroadenoma (25 x 24 mm) (Figure 1). In view of his fever and thrombocytopenia, he was evaluated for dengue fever and was found to have dengue immunoglobulin M (IgM) positive, and hence his platelet count was monitored daily (Table 2). Thus, his differential diagnoses were a hypertensive emergency, severe thrombocytopenia, most probably post viral, and pituitary incidentaloma. 2 of 9 2019 Thomas et al. Cureus 11(8): e5323. DOI 10.7759/cureus.5323 2019 Thomas et al. Cureus 11(8): e5323. DOI 10.7759/cureus.5323 FIGURE 1: Computed tomography brain showing an iso to hypodense lesion in the sella (arrowhead), causing sellar widening, suggestive of a pituitary lesion, likely a pituitary macroadenoma FIGURE 1: Computed tomography brain showing an iso to hypodense lesion in the sella (arrowhead), causing sellar widening, suggestive of a pituitary lesion, likely a pituitary macroadenoma 3 of 9 2019 Thomas et al. Cureus 11(8): e5323. DOI 10.7759/cureus.5323 TABLE 2: Daily platelet recordings He showed clinical improvement with symptomatic management. But unfortunately on Day 3 of his hospital stay, he became restless, his headache reappeared, and it was associated with neck stiffness. A neurological examination revealed the presence of ptosis and a divergent squint in the right eye; pupils were mid-dilated and sluggish in reaction, the left eye was normal, and there were no signs of meningeal irritation. In view of his incidentaloma, serum prolactin assay (Table 3) and a magnetic resonance imaging (MRI) plus MR angiogram of the brain was done, which revealed a pituitary macroadenoma with normal MR angiogram findings (Figures 2-3). He was treated with mannitol and responded well. Variable Reference value Measurement Day 1 Day 4 Cortisol (µg/dL) 6.7-22.6 6.88 TSH (µU/mL) 0.5-5 0.63 0.22 f T4 (µg/dL) 5-12 0.75 0.67 Prolactin (ng/mL) < 20 1.67 TABLE 3: Pituitary hormonal assay f T4: free T4; TSH: thyroid-stimulating hormone TABLE 3: Pituitary hormonal assay 4 of 9 FIGURE 2: MRI brain showing pituitary macroadenoma MRI: magnetic resonance imaging FIGURE 2: MRI brain showing pituitary macroadenoma MRI: magnetic resonance imaging 5 of 9 2019 Thomas et al. Cureus 11(8): e5323. DOI 10.7759/cureus.5323 FIGURE 4: MRI brain showing pituitary macroadenoma with hemorrhagic areas. MRI: magnetic resonance imaging FIGURE 4: MRI brain showing pituitary macroadenoma with hemorrhagic areas. MRI: magnetic resonance imaging He responded well to steroids, and hence there was no indication for surgical intervention. He was also supplemented with thyroxine 50 µg PO daily. He was discharged with PO medications of hydrocortisone (20 mg in the morning and 10 mg in the evening) and thyroxine 50 µg daily. A follow-up visit at two weeks showed a significant improvement of ptosis and full recovery in two months. He responded well to steroids, and hence there was no indication for surgical intervention. He was also supplemented with thyroxine 50 µg PO daily. He was discharged with PO medications of hydrocortisone (20 mg in the morning and 10 mg in the evening) and thyroxine 50 µg daily. A follow-up visit at two weeks showed a significant improvement of ptosis and full recovery in two months. FIGURE 3: Normal MR angiogram MR: magnetic resonance FIGURE 3: Normal MR angiogram MR: magnetic resonance But on Day 4, his sensorium worsened, BP dropped to 90/60 mmHg, serum sodium was 128 mEq/L, and platelet count dropped to 10,000. Due to the acute onset development of hypotension from the initial presentation of hypertension, associated with hyponatremia in the background of a pituitary incidentaloma, secondary adrenal insufficiency was suspected. Hence, mannitol was replaced with hydrocortisone (after serum cortisol analysis), and four units of platelet transfusions were given. With steroids, he showed significant clinical and hemodynamic improvement, but his ptosis persisted. Repeat brain MRI was done and showed a pituitary macroadenoma (24 x 22mm) with hemorrhagic areas, consistent with the diagnosis of pituitary apoplexy (Figure 4). His pituitary hormonal assay showed decreased levels of thyroxine and prolactin and a lower limit of normal of cortisol (Table 3). 6 of 9 2019 Thomas et al. Cureus 11(8): e5323. DOI 10.7759/cureus.5323 2019 Thomas et al. Cureus 11(8): e5323. DOI 10.7759/cureus.5323 Disclosures Human subjects: Consent was obtained 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. ensues, which makes PA an endocrine emergency. The most common hormonal deficiency is corticotrophin deficiency, occurring in up to 80% of cases, resulting in severe hemodynamic instability and hyponatremia [7]. PA can also result in other hormonal deficits like growth hormone (GH), thyrotropic, and gonadotropic deficiency [7]. CT brain is used for the initial evaluation; MRI is the investigation of choice. PA can be managed both medically and surgically, but the most appropriate approach in the acute phase is controversial [7]. Medical management is aimed at hemodynamic stabilization, correction of electrolyte imbalance, and empiric parenteral steroids (preferably after a blood draw for cortisol assessment), as secondary adrenal insufficiency can occur from acute corticotrophin deficiency [7-8]. Surgery is considered if the patient has a progressive loss of vision and deterioration of consciousness [6]. Transsphenoidal surgery is the recommended surgical approach [7]. Our patient was managed medically with steroids and thyroxine and showed drastic improvement, and hence surgery was not indicated. Pituitary apoplexy is thus an endocrine emergency and if corticosteroids are not started immediately, death may follow as a result of adrenal failure or other neurological complications. Discussion We report a case of pituitary apoplexy in an elderly male from dengue fever-induced thrombocytopenia. The incidence of pituitary apoplexy in pituitary tumors is about 2% - 12% [6]. The important risk factors for PA are cerebral angiographic procedures, systemic hypertension, surgeries (cardiac and orthopedic), head injury, coagulopathies, and drugs (GnRH analogs, dopamine receptor analogs, etc.) [7]. The pathophysiological process involved in PA are: (a) reduced blood flow resulting in infarction, (b) acute increase in blood flow, (c) stimulation of the pituitary gland from stress tests, and (d) coagulopathies from thrombocytopenia or anticoagulation [8]. Our patient had thrombocytopenia from dengue fever, which precipitated apoplexy. The clinical features of PA include headache, signs of meningeal irritation, visual disturbances (due to sudden hemorrhage-related changes, or inflammation of the optic nerve from the bleed), and features of oculomotor (IIIrd) nerve palsy [7]. Acute endocrine deficiency also 7 of 9 2019 Thomas et al. Cureus 11(8): e5323. DOI 10.7759/cureus.5323 Conclusions Dengue fever is a known risk factor for thrombocytopenia, which, in turn, can precipitate pituitary apoplexy. When a patient with thrombocytopenia in the background of a pituitary adenoma (known case of adenoma or incidentaloma) develops vomiting, headache, or meningeal irritation, with features of acute IIIrd nerve palsy, pituitary apoplexy should be kept in mind and intervened immediately, as it is an endocrine emergency that can result in adverse clinical outcomes. 2019 Thomas et al. Cureus 11(8): e5323. DOI 10.7759/cureus.5323 References 1. Tan SK, Seow CJ, Tan E, Chau YP, Dalan R: Pituitary apoplexy secondary to thrombocytopenia due to dengue hemorrhagic fever: a case report and review of the literature. Endocr Pract. 2014, 20:58-64. 10.4158/ep13319.Cr 2. Wildemberg LE, Neto LV, Niemeyer P, Gasparetto EL, Chimelli L, Gadelha MR: Association of dengue hemorrhagic fever with multiple risk factors for pituitary apoplexy. Endocr Pract. 2012, 18:97-101. 10.4158/ep11341.Cr 3. Balaparameswara Rao SJ, Savardekar AR, Nandeesh BN, Arivazhagan A: Management dilemmas in a rare case of pituitary apoplexy in the setting of dengue hemorrhagic fever. Surg Neurol Int. 2017, 8:4. 10.4103/2152-7806.198731 4. Mishra SS, Panigrahi S, Das S: Dengue hemorrhagic fever: a rare cause of pituitary apoplexy . Neurol India. 2014, 62:92-93. 10.4103/0028-3886.128350 5. Kumar V, Kataria R, Mehta VS: Dengue hemorrhagic fever: a rare cause of pituitary tumor 2019 Thomas et al. Cureus 11(8): e5323. DOI 10.7759/cureus.5323 8 of 9 2019 Thomas et al. Cureus 11(8): e5323. DOI 10.7759/cureus.5323 hemorrhage and reversible vision loss. Indian J Ophthalmol. 2011, 59:311-312. 10.4103/0301- 4738.82002 6. Albani A, Ferrau F, Angileri FF, Esposito F, Granata F, Ferreri F, Cannavo S: Multidisciplinary management of pituitary apoplexy. Int J Endocrinol. 2016, 2016:7951536. 10.1155/2016/7951536 7. Briet C, Salenave S, Bonneville JF, Laws ER, Chanson P: Pituitary apoplexy. Endocr Rev. 2015, 36:622-645. 10.1210/er.2015-1042 8. Glezer A, Bronstein MD: Pituitary apoplexy: pathophysiology, diagnosis and management . Arch Endocrinol Metab. 2015, 59:259-264. 10.1590/2359-3997000000047 9 of 9
https://openalex.org/W2904488985	https://www.intechopen.com/citation-pdf-url/64400	English	null	Microbial Ecology in the Atmosphere: The Last Extreme Environment	IntechOpen eBooks	2,021	cc-by	12,222	Ángeles Aguilera, Graciela de Diego-Castilla, Susana Osuna, Rafael Bardera, Suthyvann Sor Mendi, Yolanda Blanco and Elena González-Toril Ángeles Aguilera, Graciela de Diego-Castilla, Susana Osuna, Rafael Bardera, Suthyvann Sor Mendi, Yolanda Blanco and Elena González-Toril Ángeles Aguilera, Graciela de Diego-Castilla, Susana Osuna, Rafael Bardera, Suthyvann Sor Mendi, Yolanda Blanco and Elena González-Toril Abstract The atmosphere is an extreme environment where organisms are subject to low temperatures and high radiation. Many of the microorganisms detected there appear in resistant forms or show mechanisms of adaptation designed to withstand these extreme conditions. Airborne microorganisms may play an important role in the global climate system, biogeochemical cycling, and health. Dust storms are the atmo- spheric phenomenon that move more topsoil through the Earth’s atmosphere, and numerous microorganisms attached to dust particles are thus transported. The Iberian Peninsula is periodically affected by this phenomenon as African dust frequently reaches southern Europe and the Mediterranean basin. There are numerous methods for sampling airborne microbes, but factors such as low biomass and high variability of the atmosphere render them not yet sufficiently efficient. Very few studies have been conducted directly in the atmosphere via sampling using airborne platforms. The National Institute for Aerospace Technology has two CASA C-212-200 aircraft that have been suitably modified to operate as airborne research platforms. These aircraft are a unique tool for the study of atmospheric microbial diversity and the different environments where they can be found. A study of the airborne microbial diversity in a Saharan dust event from four aerobiology sampling flights is provided in advance. Keywords: extremophiles, aerobiology, airborne, aerial platforms, aircraft, dust storm, Saharan dust 1. Introduction Extremophile organisms capable of growing in extreme conditions draw consider- able attention since they show that life is robust and adaptable and help us under- stand its limits. In addition, they show a high biotechnological potential [1, 2]. Most of the best-characterized extreme environments on Earth are geophysical constraints (temperature, pressure, ionic strength, radiation, etc.) in which opportunistic micro- organisms have developed various adaptation strategies. Deep-sea environments, hot springs and geysers, extreme acid waters, hypersaline environments, deserts, and permafrost or ice are some or the most recurrent examples of extreme environ- ments [3]. However, the atmosphere is rarely thought of as an extreme habitat. In the 1 Extremophilic Microbes and Metabolites - Diversity, Bioprospecting and Biotechnological... atmosphere, the dynamics of chemical and biological interactions are very complex, and the organisms that survive in this environment must tolerate high levels of UV radiation, desiccation (wind drying), temperature (extremely low and high tem- peratures), and atmospheric chemistry (humidity, oxygen radicals, etc.) [4]. These factors turn the atmosphere (especially its higher layers) into one of the most extreme environments described to date and the airborne microorganisms into extremophiles or, at least, multiresistant ones [5]. It is known that airborne cells can maintain viability during their atmospheric residence and can exist in the air as spores or as vegetative cells thanks to diverse molecular mechanisms of resistance and adaptation [2, 6]. The big question is whether some of them can be metabolically active and divide. Bacterial residence times can be several days, which facilitate transport over long distances. This fact, together with the extreme conditions of the atmosphere, has led researchers to think for years that they do not remain active during their dispersion. However, recent studies strongly sug- gest that atmospheric microbes are metabolically active and were aerosolized organic matter and water in clouds would provide the right environment for metabolic activity to take place. Thus, the role played by microorganisms in the air would not only be passive but could also influence the chemistry of the atmosphere. In any case, only a certain fraction of bacteria in the atmosphere would be metabolically active [2, 7]. p y Despite recognizing its ecological importance, the diversity of airborne micro- organisms remains largely unknown as well as the factors influencing diversity levels. 1. Introduction 2 Microbial Ecology in the Atmosphere: The Last Extreme Environment DOI: http://dx.doi.org/10.5772/intechopen.81650 Microbial Ecology in the Atmosphere: The Last Extreme Environment DOI: http://dx.doi.org/10.5772/intechopen.81650 Microbial Ecology in the Atmosphere: The Last Extreme Environment DOI: http://dx.doi.org/10.5772/intechopen.81650 In this chapter, we approach the atmosphere as an extreme environment and make use of some advanced data from an example of an in situ study of the atmo- sphere: the analysis of bacterial diversity of the low troposphere of the Iberian Peninsula during an intrusion of Saharan dust using a C-212 aircraft adequately improved for aerobiological sampling. 1. Introduction Recent studies on airborne microbial biodiversity have reported a diverse assemblage of bacteria and fungi [4, 8–12], including taxa also commonly found on leaf surfaces [13, 14] and in soil habitats [15]. The abundance and composition of airborne microbial communities are variable across time and space [11, 16–19]. However, the atmospheric conditions responsible for driving the observed changes in microbial abundances have not been thoroughly established. One reason for these limitations in the knowledge of aerobiology is that until recently, microbiological methods based on culture have been the standard, and it is known that such meth- ods capture only a small portion of the total microbial diversity [20]. In addition, because pure cultures of microorganisms contain a unique type of microbes, culture-based approaches miss the opportunity to study the interactions between different microbes and their environment. Another limitation for the study of aerial microbial ecology at higher altitudes or in open ocean areas is the difficulty of repeated and dedicated use of airborne platforms (i.e., aircraft or balloons) to sample the air. Most studies to date on the atmospheric microbiome are restricted to samples collected near the Earth’s surface (e.g., top of mountains or buildings). Aircraft, unmanned aerial systems (UASs), balloons or even rockets, and satellites could represent the future in aerobiology knowledge [5, 21, 22]. These platforms could open the door to conducting microbial studies in the stratosphere and troposphere at high altitudes and in open-air masses, where long-range atmospheric transport is more efficient, something that is still poorly characterized today. The main challenge in conducting these kinds of studies stems from the fact that microbial collection systems are not sufficiently developed. There is a need for improvement and implementation of suitable sampling systems for platforms capable of sampling large volumes of air for subsequent analyses using multiple techniques, as this would provide a wide range of applications in the atmospheric, environmental, and health sciences. g pp p In aerobiology, dust storms deserve special mention. Most of them originate in the world’s deserts and semideserts and play an integral role in the Earth system [23, 24]. They are the result of turbulent winds, including convective haboobs [25]. This dust reaches concentrations in excess of 6000 μg m−3 in severe events [26]. Dust and dust-associated bacteria, fungal spores, and pollen can be transported thousands of kilometers in the presence of dust [9]. 2. Atmosphere, an extremophile environment It is well known that there is a biota in the atmospheric air. The first study dates back to the nineteenth century, which speak about the presence and dispersion of microorganisms and spores in the atmosphere [27, 28]. Although the atmosphere represents a large part of the biosphere, the density of airborne microorganisms is very low. Estimates suggest that from the ground surface up to about 18 km above sea level (troposphere), there is less than a billionth of the number of cells Figure 1. Diagram displaying atmosphere layers, temperature and airborne emission sources. Yellow line marks atmospheric temperature. Bottom of the figures shows the common sources of aerosolized bacteria, with special attention to dust storms. Figure 1. d Figure 1. Diagram displaying atmosphere layers, temperature and airborne emission sources. Yellow line marks atmospheric temperature. Bottom of the figures shows the common sources of aerosolized bacteria, with special attention to dust storms. 3 Extremophilic Microbes and Metabolites - Diversity, Bioprospecting and Biotechnological... found in the oceans, soils, and subsurface. Between approximately 18 and 50 km above sea level (stratosphere), temperature, oxygen, and humidity decrease and with them the number of cells. Above the ozone layer (between 18 and 35 km into stratosphere), ultraviolet (UV) and cosmic radiation become lethal factors. Once in the mesosphere (above 50 km), life is difficult to imagine; however microorganisms of terrestrial origin could arrive to the stratosphere from lower layers via different phenomena (human activity, thunderstorms, dust storms, or volcanic activity), and bacteria have been found isolated up to 41 km or in dust samples from the International Space Station (Figure 1) [6, 29]. Therefore, airborne microbes are always present in the atmosphere [11, 30, 31], and their permanence is dynamic, resulting in an environment with enormous variability. Estimates calculate that over 1021 cells are lifted into the atmosphere every year, leading to considerable trans- port and dispersal around the atmosphere, with a large portion of these cells return- ing to the surface due to different atmospheric events as part of a feedback cycle. Undoubtedly, airborne microbes play an important role in meteorological processes. They have been linked to the nucleation phenomena that lead to the formation of clouds, rain, and snow and to the alteration of precipitation events [32–34]. Their presence is essential to understand long-range dispersal of plant and potential pathogens [7, 35, 36] and maintain diversity in ground systems and could interfere with the productivity of natural ecosystems [17, 18]. On the other hand, airborne bacteria can have important effects on human health, being responsible for differ- ent phenomena such as seasonal allergies and respiratory diseases. Based on data from terrestrial environments, the global abundance of airborne bacteria has been estimated to range between 104 and 106 m−3 [37]. However, more recent studies incorporating direct counting by microscopy or quantitative PCR have provided more accurate estimates of the number of airborne microbes, which apparently point to a higher number of cells present in the atmosphere [38–41]. 3. Microbial sampling There is a great variety of airborne microorganism sampling systems, allowing us to select the most suitable one depending on our objectives [42]. On the other hand, no standardized protocols exist, which is a major pitfall when developing our objectives. This fact has led some authors to propose the creation of consortiums of interested parties for establishing standardized protocol reproducibility [20], as well as the need to establish global networks of aerobiological studies [11]. Two approaches are proposed: particles or cells can be collected passively or directly from the atmosphere. Passive media usually involves decanting [43] and collecting particles over snow [44] or through the collection of atmospheric water [45]. On the other hand, active method- ologies entail three major approaches: filtration, impaction, and liquid impingement. All three approaches are very efficient when developing culture-dependent techniques. In contrast, culture-independent approaches produce some serious problems that make the work difficult: the high variability of the system and the low biomass mean that sampling campaigns are, in many cases, extremely inefficient [20]. Lastly, the use of airborne platforms is not very extended, but they represent a good opportunity to conduct a more direct study of the atmosphere [5, 19, 31]. 3.3 Impaction In this system, the particles generally impact into a petri dish with an enrich- ment medium. It is, possibly, the most efficient and most used method to conduct studies based on culture. Airflow impacting onto the plates is controlled by slots that allow the homogeneous distribution of the air. The system can be single stage or several stages in cascade, causing the particles to be distributed by size in the different petri dishes [20]. Some variants replace petri dishes with agarose filters or Vaseline strips, in order to carry out independent culture methodologies, but effi- ciency is very low. The original and more popular impactor is the Andersen cascade impactor (Figure 2C) [48]. 3.2 Impingement In impingement, particles are collected in a liquid matrix [20]. Normally a buf- fer is used such as phosphate buffer saline (PBS) that helps maintain the viability of the cells. One of the more widely used liquid impingers is BioSampler SKC (Figure 2B). In this case, the tangential movement of the particles inside the flow impinger retains the particles in the collecting liquid. The suspension obtained could be used for culturing or for molecular ecology assays [20]. One of the advan- tages of impingement collection is that it facilitates quantitative techniques such as flow cytometry or in situ hybridization [47]. 3.1 Filtration Filtration is a simple and cheap method that is often efficient. It involves pumping air through a filter where the mineral and biological particles are trapped. Filters of different materials and porosity are available made of cellulose, nylon, polycarbonate 4 Microbial Ecology in the Atmosphere: The Last Extreme Environment DOI: http://dx.doi.org/10.5772/intechopen.81650 Microbial Ecology in the Atmosphere: The Last Extreme Environment DOI: http://dx.doi.org/10.5772/intechopen.81650 or fiberglass, or quartz. Sizes used range from 0.2 to 8 μm, depending on the size of the particles to be captured and the capacity of the pump. In many cases, a PM10 filter can give better results when collecting smaller bacteria, as it allows greater airflow. Airflow filtration rates generally range between 300 and 1000 L/minute [4, 46]. Microorganisms trapped in the filter can be cultured, or the filters can be directly used for DNA extraction. In addition, filters are a very suitable support for microscopy, and countless holders for filters are available (an example is shown in Figure 2A). 3.4. Airborne platforms This operation, which can be very simple in the laboratory or at ground level, becomes tremendously complicated on an airplane, since air intakes that are part of the fuselage of the aircraft are often difficult to steril- ize. It is therefore necessary to develop robust sterilization protocols. The spectacular work of DeLeon-Rodríguez of 2013 has been criticized in this aspect [40, 59]; (3) sampling time. A possible solution to the low biomass of the atmosphere is to increase sampling time, but in the case of flights, we are limited to the flight autonomy of the aircraft. Although scarce, some studies from airplanes have been conducted. The first studies that were conducted in airplanes were carried out by impaction on a petri plate with enrichment means, which allowed isolating microorganisms from the upper troposphere and even from the stratosphere [21, 57, 60]. However, advances in molecular ecology have caused the most recent studies to favor filtration [40, 58]. must be in a location on the airplane that avoids chemical contamination from the operation of the device. Previous studies have used wing-mounted air samplers or the roof of the aircraft to reduce the possibility of in-flight contamination [21, 22, 56–58]. Similarly, it should allow the aseptic collection of samples, avoiding microbiological contamination during the process. This operation, which can be very simple in the laboratory or at ground level, becomes tremendously complicated on an airplane, since air intakes that are part of the fuselage of the aircraft are often difficult to steril- ize. It is therefore necessary to develop robust sterilization protocols. The spectacular work of DeLeon-Rodríguez of 2013 has been criticized in this aspect [40, 59]; (3) sampling time. A possible solution to the low biomass of the atmosphere is to increase sampling time, but in the case of flights, we are limited to the flight autonomy of the aircraft. Although scarce, some studies from airplanes have been conducted. The first studies that were conducted in airplanes were carried out by impaction on a petri plate with enrichment means, which allowed isolating microorganisms from the upper troposphere and even from the stratosphere [21, 57, 60]. However, advances in molecular ecology have caused the most recent studies to favor filtration [40, 58]. f gy The European Facility for Airborne Research (EUFAR) program brings together infrastructure operators of both instrumented research aircraft and remote sens- ing instruments with the scientific user community. 3.4. Airborne platforms Several studies explain and compare sampling methodologies in aerobiology, but most of them focus on the surface of the Earth (e.g., on top of mountains or build- ings) or indoors [42, 49–54]. However, small studies have been conducted at higher altitudes or in open sea areas. The use of airborne platforms (balloons, aircraft, rockets, etc.) for aerobiology sampling would allow conducting a direct study of the microbial ecology of the atmosphere. Another advantage of airborne platforms is the possibility of studying the vertical distribution of airborne microbial communi- ties. In addition, some aircraft allow us to develop studies in the upper troposphere or in the stratosphere. Unfortunately, atmospheric microbial collection instruments have not been developed enough for airborne platforms. Among the different airborne platforms, aircraft, due to their versatility and access, are particularly interesting. Some studies have been conducted, but not enough samples have been developed yet, and efficiency is still very low. As already mentioned, the efficiency of samplers in soil-level aerobiology faces a series of prob- lems (low biomass, high variability of populations, lack of standardized protocols). In the case of airplanes, in addition to these intrinsic problems associated with atmo- spheric microbial ecology, other additional ones exist: (1) the high velocity of the aircraft in relation to the relative quiescent air mass. This makes it difficult to obtain an isokinetic sampler and, therefore, one that is sufficiently efficient that would allow us to obtain a correct quantification of the incoming air [55]; (2) the sampler 5 Extremophilic Microbes and Metabolites - Diversity, Bioprospecting and Biotechnological... Figure 2. Three different samplers of airborne microorganisms. (A) Filter holder and a filter (PALL Corporation). (B) Impinger sampling of bioaerosols (BioSampler, SKC, Inc.). (C) Six-stages Andersen Cascade Impactor (Thermo Fisher Scientific). Figure 2. Three different samplers of airborne microorganisms. (A) Filter holder and a filter (PALL Corporation). (B) Impinger sampling of bioaerosols (BioSampler, SKC, Inc.). (C) Six-stages Andersen Cascade Impactor (Thermo Fisher Scientific). must be in a location on the airplane that avoids chemical contamination from the operation of the device. Previous studies have used wing-mounted air samplers or the roof of the aircraft to reduce the possibility of in-flight contamination [21, 22, 56–58]. Similarly, it should allow the aseptic collection of samples, avoiding microbiological contamination during the process. 3.4. Airborne platforms However, it lacked aircraft 6 Microbial Ecology in the Atmosphere: The Last Extreme Environment DOI: http://dx.doi.org/10.5772/intechopen.81650 prepared for microbiological sampling. The National Institute for Aerospace Technology (INTA) belonging to the Spanish Ministry of Defence has two CASA C-212-200 aircraft that were suitably modified to be used as flying research platforms. Now, these two aircraft are a unique tool for the study of atmospheric microbial diversity and the different environments of the EUFAR program Our Figure 3. Airborne microorganisms sampler installed in INTA’s CASA C-212-200 aircraft. Figure 4. Multi-sampler system tested in INTA’s CASA C-212-200 aircraft. (A) Impinger sampler, design and manufacture own. (B) Impactor sampler (Impaktor FH6, Markus Klotz GmbH). (C) Coriolis μ (Bertin Technologies SAS) a impinger biological air sampler. (D) Filter holder (PALL Corporation). (E) Six-stages Andersen Cascade Impactor (Thermo Fisher Scientific). Microbial Ecology in the Atmosphere: The Last Extreme Environment DOI: http://dx.doi.org/10.5772/intechopen.81650 Microbial Ecology in the Atmosphere: The Last Extreme Environment DOI: http://dx.doi.org/10.5772/intechopen.81650 gy p DOI: http://dx.doi.org/10.5772/intechopen.81650 Figure 3. Airborne microorganisms sampler installed in INTA’s CASA C-212-200 aircraft. 4. Microbial characterization Aerobiology studies have traditionally focused on the collection of bacterial cells and the analysis of samples by total counting and culture-based techniques. It is known that such methods capture only a small portion of the total microbial diversity [61]. The almost exclusive use, for years, of these methodologies is one of the reasons for these limitations in the knowledge of aerobiology. In addition, culture-dependent methods do not allow us to study the interactions between different species of microorganisms. Culture-independent methods have been used to assess microbial diversity, increasing the specificity of microbial identification and the sensitivity of environmental studies, especially in extreme environments. These methods have recently been applied to various areas of airborne microbiology [62–65] revealing a greater diversity of airborne microorganisms when compared to culture-dependent methods. Some good studies approach the challenges and oppor- tunities of using molecular methodologies to address airborne microbiology [20, 66]. Although molecular ecology methods allow the rapid characterization of the diver- sity of complex ecosystems, the isolation of the different components is essential for the study of their phenotypic properties in order to evaluate their role in the system and their biotechnological potential. A combination of culture-dependent and culture-independent methods is ideal to address the complete study of the system. d l d d h l f l Modern culture-independent approaches to community analysis, for example, metagenomics and individual cell genomics, have the potential to provide a much deeper understanding of the atmospheric microbiome. However, molecular ecology techniques face several particular challenges in the case of the atmospheric microbi- ome: (1) very low biomass [20]; (2) inefficient sampling methods [20]; (3) lack of standard protocols [9, 20]; (4) the composition of airborne microbes continuously changes due to meteorological, spatial, and temporal patterns [7, 62, 67–70]; and (5) avoidance of the presence of foreign DNA in the system [59]. Because these issues are not yet resolved, most of the non-culturing approaches focus on microbial diversity, where they are highly efficient. The most recurrent techniques are those based on DNA extraction, gene ampli- fication of 16S/18S rRNA, and next-generation sequencing (NGS) technologies. Often, this approach is more efficient due to the greater efficiency and sensitivity of this process, as opposed to gene cloning and Sanger sequencing; thus some authors are inclined toward metagenomics instead of amplification. Figure 3. Figure 3. Airborne microorganisms sampler installed in INTA’s CASA C-212-200 aircraft. Figure 3. Airborne microorganisms sampler installed in INTA’s CASA C-212-200 aircraft. Figure 4. Multi-sampler system tested in INTA’s CASA C-212-200 aircraft. (A) Impinger sampler, design and manufacture own. (B) Impactor sampler (Impaktor FH6, Markus Klotz GmbH). (C) Coriolis μ (Bertin Technologies SAS) a impinger biological air sampler. (D) Filter holder (PALL Corporation). (E) Six-stages Andersen Cascade Impactor (Thermo Fisher Scientific). Figure 4. g 4 Multi-sampler system tested in INTA’s CASA C-212-200 aircraft. (A) Impinger sampler, design and manufacture own. (B) Impactor sampler (Impaktor FH6, Markus Klotz GmbH). (C) Coriolis μ (Bertin Technologies SAS) a impinger biological air sampler. (D) Filter holder (PALL Corporation). (E) Six-stages Andersen Cascade Impactor (Thermo Fisher Scientific). Multi-sampler system tested in INTA’s CASA C-212-200 aircraft. (A) Impinger sampler, design and manufacture own. (B) Impactor sampler (Impaktor FH6, Markus Klotz GmbH). (C) Coriolis μ (Bertin Technologies SAS) a impinger biological air sampler. (D) Filter holder (PALL Corporation). (E) Six-stages Andersen Cascade Impactor (Thermo Fisher Scientific). prepared for microbiological sampling. The National Institute for Aerospace Technology (INTA) belonging to the Spanish Ministry of Defence has two CASA C-212-200 aircraft that were suitably modified to be used as flying research platforms. Now, these two aircraft are a unique tool for the study of atmospheric microbial diversity and the different environments of the EUFAR program. Our 7 Extremophilic Microbes and Metabolites - Diversity, Bioprospecting and Biotechnological... research group has a CASA-212 aircraft with an air intake located on the roof of the aircraft. A metal tube fits the entrance and is fitted inside the aircraft to a filter holder, a flowmeter, and a pump (Figure 3). This simple system is easy to steril- ize, and both the metal tube and the filter holder can be replaced in flight by other sterile ones if we want to take different samples. Using PM10 fiberglass filters, we can obtain isokinetic conditions and pass 1800 L of air per hour through the filter, as indicated by the flowmeter. y In a series of recent experiments, we tried to install a multi-sampler system in our aircraft, where we had five systems in parallel and connected to the same intake of the plane: one filter holder, two impingement systems, and two impactors (Figure 4). The results clearly showed that in the case of our aircraft, filtration was more efficient (data not shown). 4. Microbial characterization On this occasion, sampling was performed using a biological air sampler (Coriolis μ, Bertin Technologies SAS), where biological particles are collected and concentrated in a liq- uid (PBS). Sampling was conducted for 2 hours at ground level, pumping a total of 36,000 L of air. After this time, the sample was paraformaldehyde fixed and filtered through a 0.2 μm pore size, hydrophilic polycarbonate membrane, 13 mm diameter (GTTP, Millipore). A half sample was hybridized with the universal Bacteria domain probe, EUB338I-III [72], following a conventional protocol [73]. The second half was hybridized with the probe NON338 [74] as negative control. In this case, an average of 140 cells per liter of air was counted. Occasionally, FISH also allows to observe bacteria attached to mineral particles (Figure 5C–D). situ hybridization (FISH) [41, 47, 66, 71]. FISH is surely the best and most specific cell quantification methodology that exists. However, in the case of aerobiology, it cannot always be used. A minimum number of cells must exist so that we can observe and count them under a fluorescence microscope. Due to the variability of microbial populations in the air, this is not always achieved. In our research group, we have obtained very good results in this regard, optimizing cell concentration. y g g p g Figure 5 shows epifluorescence micrographs of bacteria from an air sample. On this occasion, sampling was performed using a biological air sampler (Coriolis μ, Bertin Technologies SAS), where biological particles are collected and concentrated in a liq- uid (PBS). Sampling was conducted for 2 hours at ground level, pumping a total of 36,000 L of air. After this time, the sample was paraformaldehyde fixed and filtered through a 0.2 μm pore size, hydrophilic polycarbonate membrane, 13 mm diameter (GTTP, Millipore). A half sample was hybridized with the universal Bacteria domain probe, EUB338I-III [72], following a conventional protocol [73]. The second half was hybridized with the probe NON338 [74] as negative control. In this case, an average of 140 cells per liter of air was counted. Occasionally, FISH also allows to observe bacteria attached to mineral particles (Figure 5C–D). p g DNA gives us much information about the diversity of the system, but if we wish to obtain information about the metabolic activity that is taking place in the ecosys- tem, metabolomic and metatranscriptomic approaches are needed [50, 66]. 4. Microbial characterization This provides more information and avoids an intermediate step, but bioinformatic processing is tedious and often only provides data in relation to diversity, making the annotation of the rest of the information very complicated [20]. These approaches can be comple- mented with quantitative methods such as qPCR, flow cytometry, or fluorescence in 8 Microbial Ecology in the Atmosphere: The Last Extreme Environment DOI: http://dx.doi.org/10.5772/intechopen.81650 Microbial Ecology in the Atmosphere: The Last Extreme Environment DOI: http://dx.doi.org/10.5772/intechopen.81650 situ hybridization (FISH) [41, 47, 66, 71]. FISH is surely the best and most specific cell quantification methodology that exists. However, in the case of aerobiology, it cannot always be used. A minimum number of cells must exist so that we can observe and count them under a fluorescence microscope. Due to the variability of microbial populations in the air, this is not always achieved. In our research group, we have obtained very good results in this regard, optimizing cell concentration. Figure 5 shows epifluorescence micrographs of bacteria from an air sample. On this occasion, sampling was performed using a biological air sampler (Coriolis μ, Bertin Technologies SAS), where biological particles are collected and concentrated in a liq- uid (PBS). Sampling was conducted for 2 hours at ground level, pumping a total of 36,000 L of air. After this time, the sample was paraformaldehyde fixed and filtered through a 0.2 μm pore size, hydrophilic polycarbonate membrane, 13 mm diameter (GTTP, Millipore). A half sample was hybridized with the universal Bacteria domain probe, EUB338I-III [72], following a conventional protocol [73]. The second half was hybridized with the probe NON338 [74] as negative control. In this case, an average of 140 cells per liter of air was counted. Occasionally, FISH also allows to observe bacteria attached to mineral particles (Figure 5C–D). situ hybridization (FISH) [41, 47, 66, 71]. FISH is surely the best and most specific cell quantification methodology that exists. However, in the case of aerobiology, it cannot always be used. A minimum number of cells must exist so that we can observe and count them under a fluorescence microscope. Due to the variability of microbial populations in the air, this is not always achieved. In our research group, we have obtained very good results in this regard, optimizing cell concentration. Figure 5 shows epifluorescence micrographs of bacteria from an air sample. 4. Microbial characterization (B) Air sample collected from C-212-200 aircraft during a Saharan dust intrusion (February 24, 2017). Filter appear completely cover of mineral particles. (B and C) Biological particles sampled using C-212-200 aircraft. (E) Diatomea sampled by C-212-200 aircraft in a fligth along the northern coast of Spain (9 March 2017). (F) Cell attached to mineral particles and organic matter. Scanning electron microscopy (SEM) also provides much information of the aerobi- ology [7]. Specifically, it allows the characterization of eukaryotic cells (e.g., diatoms) and, above all, pollens and fungal spores, from which we can obtain great information with good images alone. Figure 6A shows pine tree pollen observed via SEM in a sample obtained after a 30 minutes flight of the C-212 aircraft. 4. Microbial characterization In the case of the atmosphere, this is crucial, since we are not fully certain if the cells present are active. Some studies indicate that a part of the microorganisms in the atmosphere are developing an activity [6], but until we conduct RNA-based and metabolite-based studies, we will not have the certainty that this is the case. The big problem is that it is very difficult to carry out these studies using the current microbial capture systems. Figure 5. Epifluorescence micrographs of bacteria from an air sample. (A and C) DAPI-stained cells; (B and D) same fields a A, and C, respectively, showing cells hybridized with probes EUB338I-III (Cy3 labeled), specific for Bacteria domain. All micrographs correspond to the same hybridization process, performed with a sample obtained after 4 hours sampling at ground. C and D show microorganisms attaches to a mineral particles (arrow sign). Bars, 5 μm. Figure 5. Figure 5. Epifluorescence micrographs of bacteria from an air sample. (A and C) DAPI-stained cells; (B and D) same fields a A, and C, respectively, showing cells hybridized with probes EUB338I-III (Cy3 labeled), specific for Bacteria domain. All micrographs correspond to the same hybridization process, performed with a sample obtained after 4 hours sampling at ground. C and D show microorganisms attaches to a mineral particles (arrow sign). Bars, 5 μm. Figure 5. Epifluorescence micrographs of bacteria from an air sample. (A and C) DAPI-stained cells; (B and D) same fields a A, and C, respectively, showing cells hybridized with probes EUB338I-III (Cy3 labeled), specific for Bacteria domain. All micrographs correspond to the same hybridization process, performed with a sample obtained after 4 hours sampling at ground. C and D show microorganisms attaches to a mineral particles (arrow sign). Bars, 5 μm. 9 9 Extremophilic Microbes and Metabolites - Diversity, Bioprospecting and Biotechnological... Figure 6. SEM images of different airborne samples. (A) Pinus pollen. Ground sample after 2 hours sampling. (B) Air sample collected from C-212-200 aircraft during a Saharan dust intrusion (February 24, 2017). Filter appear completely cover of mineral particles. (B and C) Biological particles sampled using C-212-200 aircraft. (E) Diatomea sampled by C-212-200 aircraft in a fligth along the northern coast of Spain (9 March 2017). (F) Cell attached to mineral particles and organic matter. Figure 6. Figure 6. SEM images of different airborne samples. (A) Pinus pollen. Ground sample after 2 hours sampling. 5. Mechanisms of microbial survival of airborne bacteria Undoubtedly, another part of the cells will be in the form of latency and may even suffer modifications of the cell wall and slow down or stop their metabolic activity [75, 76]. These transformations can improve resistance to physical stresses, such as UV radiation [58]. On the other hand, some of the bac- teria present in the atmosphere, such as Geodermatophilus, show pigmentation that undoubtedly protects it from excessive radiation. The microorganisms that are usu- ally detected in the atmosphere originate mainly from the soil, which means they will share similar mechanisms of resistance. In some strains, metabolic adaptations have been observed to lack nutrients such as cytochrome bd biosynthesis to survive iron deprivation [77]. Deinococcus is also a recurrent genus in the atmosphere, which, like those in soil, has multiresistance mechanisms based on high DNA-repair efficiency. Bacteria that do not form spores and certain archaea, in contrast, often have genomes rich in G + C, which may increase tolerance to UV rays and overall survival [78]. y y Another strategy of resistance could be cell clustering and adhesion to particles. Several studies have confirmed the loss of viability and shielding or the reflective properties of the mineral particles as an important role for the protection of UV radiation [19, 31]. In that sense, it is very possible that many cells have mechanisms that promote aggregation. In our samples, we often find the cells adhered to each other or to minerals, which undoubtedly makes them more resistant (Figure 6). 5. Mechanisms of microbial survival of airborne bacteria As mentioned above, factors, such as the shortage of nutrients and substrates, high UV radiation, drying, changes in temperature and pH, or the presence of 10 Microbial Ecology in the Atmosphere: The Last Extreme Environment DOI: http://dx.doi.org/10.5772/intechopen.81650 Microbial Ecology in the Atmosphere: The Last Extreme Environment DOI: http://dx.doi.org/10.5772/intechopen.81650 reactive oxygen species, make the atmosphere an extreme environment. However, it is possible that the high variability of its conditions is the one characteristic that makes this environment more extreme [1, 20]. Among the cells present in the atmosphere, a considerable portion appears in the resistance forms capable of withstanding low-temperature and high-radiation conditions. This is what probably happens with fungi and gram-positive bacteria. Bacillus strains recurrently isolated from the atmosphere have characteristics and a capacity to sporulate very similar to strains isolated from the soil. Undoubtedly, another part of the cells will be in the form of latency and may even suffer modifications of the cell wall and slow down or stop their metabolic activity [75, 76]. These transformations can improve resistance to physical stresses, such as UV radiation [58]. On the other hand, some of the bac- teria present in the atmosphere, such as Geodermatophilus, show pigmentation that undoubtedly protects it from excessive radiation. The microorganisms that are usu- ally detected in the atmosphere originate mainly from the soil, which means they will share similar mechanisms of resistance. In some strains, metabolic adaptations have been observed to lack nutrients such as cytochrome bd biosynthesis to survive iron deprivation [77]. Deinococcus is also a recurrent genus in the atmosphere, which, like those in soil, has multiresistance mechanisms based on high DNA-repair efficiency. Bacteria that do not form spores and certain archaea, in contrast, often have genomes rich in G + C, which may increase tolerance to UV rays and overall survival [78]. reactive oxygen species, make the atmosphere an extreme environment. However, it is possible that the high variability of its conditions is the one characteristic that makes this environment more extreme [1, 20]. Among the cells present in the atmosphere, a considerable portion appears in the resistance forms capable of withstanding low-temperature and high-radiation conditions. This is what probably happens with fungi and gram-positive bacteria. Bacillus strains recurrently isolated from the atmosphere have characteristics and a capacity to sporulate very similar to strains isolated from the soil. 6. Emission sources Global and regional models have been used to explain bioaerosol emission, transport, and atmospheric impact [17, 18, 79–84]. Even so, it is not an easy phe- nomenon to explain, since it depends on a large number of factors. On the one hand, there are numerous sources of tropospheric aerosols, which include sea salt, volcanic dust, cosmic dust, industrial pollutants, and desert and semidesert areas [6, 85]. We must also consider the factors that make the transfer of particles pos- sible, for example, meteorological phenomena, solar radiation, temperature, tides, erosion, etc. [85]. On the other hand, anthropogenic activities can also affect dust emissions indirectly, by changing the climate and the hydrological cycle. In these aerosols, microorganisms will be included in a greater or lesser number. The degree of richness in cells of tropospheric aerosols will depend largely on the source of emission. Thus, the large wooded masses or fields of crops provide the atmosphere with a good number of microorganisms due to the effect of air or the aerosols produced by rain. Similarly, anthropogenic activity contributes large amounts of bacteria to the environment, treatment plants, and composting areas being sources of airborne microorganisms [85]. g Desert dust storms play a major role in particle emissions and with them that of microorganisms. In this way, most of the material reaching the atmosphere from the surface comes from desert and semidesert areas, which is known as desert dust. The Sahara-Sahel desert, the Middle East, central and eastern Asia, and Australia are the major sources of desert dust, although all the arid zones of the world are emis- sion sources [9, 86]. Dust storms are atmospheric events typically associated with dry lands due to the preponderance of dried and unconsolidated substrates with 11 Extremophilic Microbes and Metabolites - Diversity, Bioprospecting and Biotechnological... little vegetation cover. The strong and turbulent winds that blow on these surfaces raise fine-grained material, a large part of which consists of particles the size of silt (4–62.5 μm) and clay (<4 μm), reducing visibility to less than 1 km. The atmo- spheric concentrations of PM10 dust exceed 15,000 μg/m3 in severe events [87], although the concentrations naturally decrease with the distance from the areas of origin, extending hundreds of kilometers. The dust particles and cells associated with them are transported in this manner and will be deposited finally, by the effect of rain, snow, or other meteorological phenomena. 7. Saharan dust The Sahara-Sahel desert located in northwestern Africa is one of the major sources of windblown dust in the world [9]. This phenomenon has an impact on the Mediterranean coastline, but Saharan dust has been transported toward the north of Europe and has been found on numerous occasions in the Alps [88, 89] or blown toward the Atlantic and Caribbean [8, 90]. It has been estimated that 80–120 tons of dust are transported annually through the Mediterranean toward Europe [23, 91, 92]. In particular, dust transported by the winds can reach an elevation of up to 8 km in the atmosphere over the Mediterranean basin [93]. Because of its geographic position, the Iberian Peninsula is often affected by these dust events. Specifically, the Sahara- Bodele depression, located at the southern edge of the Sahara desert, has been described as the richest dust source reaching the Iberian Peninsula. Southern Spain is the main area affected, but dust can reach the Pyrenees and even France [43]. Different researchers have studied the mineralogical and chemical composition of Saharan dust, which has been observed to contain calcite, dolomite, quartz, different clay minerals, and feldspars as the main mineral components [94]. The intrusion of big amounts of these components is an important influence on nutrient dynamics and biogeochemical cycling in the atmosphere of the Iberian Peninsula. g y g p Despite the large number of studies on dispersion, geochemistry, and mineral- ogy of African dust, few are focused on microbiology. All these studies conclude that there are microbes associated with dust because there are higher concentra- tions of aerosolized microorganisms during dust events [43, 90, 93–96]. However, the magnitude of the concentrations and the specific microbes associated with dust events remain the subject of debate. On the other hand, the viability of these microorganisms is another big question. The United States Geological Survey (USGS) develops the Global Dust Program to investigate the viability of microor- ganisms transported in dust masses. USGS authors using DNA sequencing of the ribosomal gene were able to isolate and identify more than 200 viable bacteria and fungi in St. John’s samples in the USA [8, 36, 90]. Fungi and bacteria associated with atmospheric dust can be recovered and cultivated, but they must be gram-positive bacteria and many spore formers, which makes them resistant to the extreme condi- tions of the atmosphere. 6. Emission sources Therefore, there is a continuous transfer of mineral and biological matter through the atmosphere that moves from the air to the terrestrial environment and changes its geographical area [7, 24]. Microbial Ecology in the Atmosphere: The Last Extreme Environment DOI: http://dx.doi.org/10.5772/intechopen.81650 RNA-based methodologies. On the other hand, clinical records point to many of the viable microorganisms identified in the Saharan dust as the cause of respira- tory diseases (asthma and lung infections or allergic reactions), cardiovascular diseases, and skin infections [7, 90, 97, 98]. It is known that other microbes associated with dust in the air are pathogenic to humans, including those that cause anthrax and tuberculosis, or to livestock (such as foot and mouth disease) or plants [7, 90, 97, 98]. Characterization, quantification, and feasibility studies are vital to address these problems. p It is common to find fungal spores belonging to the genus Aspergillus, Nigrospora, Arthrinium, and Curvularia associated with Saharan dust. Bacterial taxa comprised a wide range of phyla, including Firmicutes, Proteobacteria, Actinobacteria, and Bacteroidetes. Generators of genus spores such as Clostridium and Bacillus are very common, along with other gram-positive ones such as Geodermatophilus or Streptococcus. Also, Alphaproteobacteria, a very common bac- terium class in soils (e.g., the family Sphingomonadaceae), are associated with dust [4, 9]. As regards Archaea, there are few studies of the atmosphere, in general, and of dust, in particular, that focus on this domain. Surely, reduced cases of patho- genic archaea have been studied to a lesser extent. Aeropyrum is the most detected genus of airborne archaea, but it is related to marine aerosols [11]. On the other hand, studies of pollen associated with dust are widespread. An interesting study investigated pollen transported from North Africa to Spain through Saharan dust and found that pollen from five non-native plant species was detected exclusively during dust events [99]. Lastly, viruses and virus-like particles have a great interest in the emission of dust. One study mentions virus-like particles associated with a transoceanic dust event. This report is based on epifluorescent microscopy of filters stained with a specific nucleic acid stain. An increase in the order of magnitude of virus-like particles was observed, from 104 to 2105 m−3 between the baseline condi- tion and dust conditions in the Caribbean [41]. It is speculated that free airborne viruses show worse resistance to high ultraviolet radiation and dry air associated with long-distance transport in dust events resist worse than others [9]. 7. Saharan dust Therefore, fungi and bacteria associated with dust may have been isolated from dust intrusions, but a percentage of the viable ones already remains an unanswered question. Another big question is the activity of these cells in the atmosphere. It is clear that they are resistant to extremophile conditions, but the question is whether they are developing their life cycle in this particular environ- ment. This question could be answered by molecular ecology methodologies based on the isolation and sequencing of mRNA, but low atmospheric biomass and high variability are, once again, the great problem when developing this type of 12 Microbial Ecology in the Atmosphere: The Last Extreme Environment DOI: http://dx.doi.org/10.5772/intechopen.81650 Microbial Ecology in the Atmosphere: The Last Extreme Environment DOI: http://dx.doi.org/10.5772/intechopen.81650 8. Microbial diversity study in the atmosphere of the Iberian Peninsula after a Saharan dust intrusion Four aerobiology sampling flights took place during February and March 2017 using the CASA C-212-200 aircraft from INTA. The study focused on microbial diversity in the atmosphere of the Iberian Peninsula during and after a Saharan dust intrusion. Flights took place under four different conditions: (1) during a strong Sahara dust storm that reached the north of the Iberian Peninsula, from February 22 to 24, 2017 (February 23, 2017) (Figure 7); (2) following precipitation (February 28, 2017); (3) following a dry period (March 8, 2017); and (4) along the northern coast of Spain (March 9, 2017). In each flight, samples were collected at different altitudes, and air samples were obtained simultaneously at ground level. A total of 20 samples were collected and are being analyzed. Cell presence was observed by scanning electron microscopy (SEM), and bacterial diversity is being studied by DNA extraction, 16S rRNA gene amplification, and Illumina MiSeq sequenc- ing. Results are being analyzed via bioinformatics and biostatistical software (MOTHUR, SPSS, STAMP, CANOCO, and PAST) which will allow us to compare the results between the different flows and scenarios. Although this study is not yet finished, some data can be advanced in this chapter. Figure 6 shows SEM microphotographs obtained from samples in different scenarios. In general, the samples obtained during the days of dust intrusion (flight 13 Extremophilic Microbes and Metabolites - Diversity, Bioprospecting and Biotechnological... Figure 7. Saharan dust intrusion. Dust pours off the northweat Afrincan coast and blankets the Iberian Peninsula, 23 February, 2016. NASA satelital imagen via MODIS. Figure 7. Saharan dust intrusion. Dust pours off the northweat Afrincan coast and blankets the Iberian Peninsula, 23 February, 2016. NASA satelital imagen via MODIS. of February 23) appear completely covered with mineral particles. In these cases, more biological cells were detected than in the rest of the days. In the particular case of samples from the marine coast flight, more diatoms were observed (Figure 6E). The analysis of diversity using the Shannon index showed that, in all cases, diversity was greater on days of Saharan dust intrusion, both in the samples taken from the ground and those taken at higher altitudes with the aircraft. This indicates that Saharan dust contributes microorganisms that are not present in the atmo- sphere on a daily basis. Diversity analysis showed phylum characteristics of soils, being Alpha- and Betaproteobacteria the most abundant classes. 8. Microbial diversity study in the atmosphere of the Iberian Peninsula after a Saharan dust intrusion All of the analyses performed showed that bacterial diversity detected at ground level and in-flight samples during the dust intrusion event were similar among one another. The genus taxonomic levels of Sphingomonas, Geodermatophilus, Methylobacter, Rhizobiales, Bacillus, or Clostridium were present in every sample, but their sequences were more abundant in the case of ground samples and dust intrusion samples collected dur- ing the day flight. However, sequences of the genus Flavobacterium, Streptococcus, or Cupriavidus were most abundant in the case of samples collected during flight. Preliminary conclusions show that bacterial diversity of airborne bacteria during days of dust intrusion is higher and similar to bacterial diversity commonly detected in soil samples. Further analyses are being conducted with these samples to obtain a complete description of the evolution of bacterial diversity during those days. Microbial Ecology in the Atmosphere: The Last Extreme Environment DOI: http://dx.doi.org/10.5772/intechopen.81650 Airborne platforms that allow conducting a direct study of microorganisms in the atmosphere and molecular methodologies (e.g., “omics”) could represent a major opportunity for approaching this question. Nevertheless, some challenges must yet be solved, such as low biomass, efficiency of sampling methods, the absence of standard protocols, or the high variability of the atmospheric environment. Deserts and arid lands are one of the most important sources of aerosol emis- sions. Clouds of dust generated by storms mobilize tons of mineral particles, and it is known that microorganisms remain attached to the particles being transported over long distances. The large number of mineral particles and microorganisms thus placed into the atmosphere has global implications for climate, biochemical cycling, and health. North African soils, primarily the Sahara Desert, are one of the major sources of airborne dust on Earth. Saharan dust is often transported to southern Europe and could even reach high altitudes over the Atlantic Ocean and the European continent. Again, airborne platforms could be a perfect opportunity for conducting a direct study of the microbiology of this kind of events. Acknowledgements This work has been supported by grants from the Spanish government (www. mineco.gob.es and www.csic.es) (CGL2015-69758-P and i-LINK1151). Publishing fee has been has been supported by Open Access. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Author details Ángeles Aguilera1, Graciela de Diego-Castilla1, Susana Osuna1, Rafael Bardera2, Suthyvann Sor Mendi2, Yolanda Blanco1 and Elena González-Toril1* 1 Astrobiology Institute (INTA-CSIC), National Institute for Aerospace Technology, Torrejón de Ardoz, Spain 2 Department of Aerodynamics and Propulsion, National Institute for Aerospace Technology, Torrejón de Ardoz, Spain Address all correspondence to: gonzalezte@cab.inta-csic.es © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Author details Ángeles Aguilera1, Graciela de Diego-Castilla1, Susana Osuna1, Rafael Bardera2, Suthyvann Sor Mendi2, Yolanda Blanco1 and Elena González-Toril1 1 Astrobiology Institute (INTA-CSIC), National Institute for Aerospace Technology, Torrejón de Ardoz, Spain 2 Department of Aerodynamics and Propulsion, National Institute for Aerospace Technology, Torrejón de Ardoz, Spain *Address all correspondence to: gonzalezte@cab.inta-csic.es 9. Conclusions Intense UV radiation, low pressure, lack of water and nutrients, and freezing temperatures turn the atmosphere into an extreme environment, especially its upper layers. However, it is widely known that airborne bacteria, fungal spores, pol- len, and other bioparticles exist. Numerous bacteria and fungi have been isolated and can survive even at stratospheric altitudes. Microbial survival in the atmo- sphere requires extremophilic characteristics, and therefore airborne microbiota is potentially useful for biotechnological applications. The role of airborne microbial communities is vital in the Earth, including interactions among the atmosphere, biosphere, climate, and public health. Airborne microorganisms are involved in meteorological processes and can serve as nuclei for cloud drops and ice crystals that precede precipitation, which influences the hydrological cycle and climate. Furthermore, their knowledge is essential in understanding the reproduction and propagation of organisms through various ecosystems. Furthermore, they can cause or improve human, animal, and plant diseases. 14 Microbial Ecology in the Atmosphere: The Last Extreme Environment DOI: http://dx.doi.org/10.5772/intechopen.81650 Microbial Ecology in the Atmosphere: The Last Extreme Environment DOI: http://dx.doi.org/10.5772/intechopen.81650 References dust. Trends in Ecology & Evolution. 2006;21:638-644. DOI: 10.1016/j. tree.2006.07.004 dust. Trends in Ecology & Evolution. 2006;21:638-644. 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https://openalex.org/W2904747119	https://europepmc.org/articles/pmc6378973?pdf=render	English	null	Benzene-induced mouse hematotoxicity is regulated by a protein phosphatase 2A complex that stimulates transcription of cytochrome P4502E1	Journal of biological chemistry/The Journal of biological chemistry	2,019	cc-by	11,888	This work was supported by Key Program of the National Natural Science Foundation of China Grant 81430079, Guangdong Provincial Natural Sci- ence Foundation Team Project Grant 2018B030312005, National Natural Science Foundation of China (NSFC) Grants 31401213, 81602877, 81372962, 81402715, and 81602882, the Fundamental Research Funds for the Central Universities Grant 17ykpy13, and the Natural Science Foundation of Guangdong Province Doctor Staring Program Grant 2016A030310163. The authors declare that they have no conflicts of inter- est with the contents of this article. Benzene-induced mouse hematotoxicity is regulated by a protein phosphatase 2A complex that stimulates transcription of cytochrome P4502E1 p y Received for publication,October 18, 2018, and in revised form, December 14, 2018 Published, Papers in Press, December 19 Liping Chen‡1, Ping Guo‡1, Haiyan Zhang‡, Wenxue Li§, Chen Gao‡, Zhenlie Huang¶, Junling Fan‡, Yuling Zhang, Xue Li, Xiaoling Liu‡, Fangping Wang‡, Shan Wang‡, Qingye Li‡, Zhini He¶, Huiyao Li‡, Shen Chen‡, Xiaonen Wu‡, Lizhu Ye‡, Qiong Li‡, Huanwen Tang, Qing Wang‡, Guanghui Dong‡, Yongmei Xiao‡, Wen Chen‡, and Daochuan Li‡2 From the ‡Guangdong Provincial Key Laboratory of Food, Nutrition and Health, Department of Toxicology, School of Public Health, Sun Yat-sen University, Guangzhou 510080, the §Department of Toxicology, Guangzhou Center for Disease Control and Prevention, Guangzhou 510440, the ¶Food Safety and Health Research Center, School of Public Health, Southern Medical University, Guangzhou 510515, the Institute of Mass Spectrometer and Atmospheric Environment, Jinan University, Guangzhou 510632, and the Dongguan Key Laboratory of Environmental Medicine, School of Public Health, Guangdong Medical University, Dongguan 523808, China Edited by Joel M. Gottesfeld Chronic benzene exposure is associated with hematotoxicity and the development of aplastic anemia and leukemia. However, the signaling pathways underlying benzene-induced hemato- toxicity remain to be defined. Here, we investigated the role of protein phosphatase 2A (PP2A) in the regulation of benzene- induced hematotoxicity in a murine model. Male mice with a hepatocyte-specific homozygous deletion of the Ppp2r1a gene (encoding PP2A A subunit) (HO) and matched wildtype (WT) mice were exposed to benzene via inhalation at doses of 1, 10, and 100 ppm for 28 days. Peripheral white blood cell counts and activation of bone marrow progenitors were attenuated in the HO mice, indicating that Ppp2r1a deletion protects against ben- zene-induced hematotoxicity. Moreover, elevation of urinary S-phenyl mercapturic acid, a benzene metabolite, was much greater in WT mice than in HO mice. Real-time exhalation anal- ysis revealed more exhaled benzene but fewer benzene metabo- lites in HO mice than in WT mice, possibly because of the down-regulation of Cyp2e1, encoding cytochrome P4502E1, in hepatocytes of the HO mice. Loss-of-function screening dis- closed that PP2A complexes containing the B56 subunit par- ticipate in regulating Cyp2e1 expression. Notably, PP2A–B56 suppressioninHepG2cellsresultedinpersistent-cateninphos- phorylation at Ser33-Ser37-Thr41 in response to CYP2E1 ago- nists. In parallel, nuclear translocation of -catenin was in- hibited, concomitant with a remarkable decrease of Cyp2e1 expression. ARTICLE ARTICLE Benzene-induced mouse hematotoxicity is regulated by a protein phosphatase 2A complex that stimulates transcription of cytochrome P4502E1 These findings support the notion that a regulatory cascade comprising PP2A–B56, -catenin, and Cyp2e1 is involved in benzene-induced hematotoxicity, providing critical insight into the role of PP2A in responses to the environmental chemicals. Benzene is an important industrial material and environ- mental pollutant, which is released into the environment from cigarette smoke, industrial waste, crude oil, architectural ornaments, and automobile emissions (1). The population is exposed to benzene mostly through ambient vapors by inhala- tion and polluted foods or water ingestion. Benzene has been classified as a human hematological carcinogen by the Interna- tional Agency for Research on Cancer (IARC) (2012). Epidemi- ological studies support a strong association between benzene exposure and aplastic anemia, myelodysplastic syndromes, leu- kemia, and other blood disorders (2–4). Mechanistic studies of benzene-induced toxicity have demonstrated that exposure to benzene and its metabolites resulted in chromosomal aberra- tions and gene mutations (5–7). Alterations in gene expression, oxidative stress, immune suppression, or perturbation of the fatty acid -oxidation pathway (8–10) have also been im- plicated in benzene-induced hematotoxicity. Previously, we reported that epigenetic dysregulation via hypermethylation of O6-methylguanine-DNA methyltransferase and altered H3K4me3 modification were identified in the peripheral lym- phocytes of low-dose benzene-exposed workers, resulting in perturbation of DNA damage repair-related signaling path- ways, ultimately contributing to dysfunction of hematopoietic stem/progenitor cells (11, 12). Nevertheless, the critical targets and signaling pathways underlying benzene-induced hemato- toxicity remain to be defined. Reversible protein phosphorylation mediated by protein kinase and phosphatase plays a critical role in the regulation of many cellular processes in eukaryotes. Dysregulation of protein phosphorylation can be triggered by environmental pollutants, This article contains Figs. S1–S8 and Tables S1–S3 2 Towhomcorrespondenceshouldbeaddressed:Dept.ofToxicology,School of Public Health, Sun Yat-sen University. Tel.: 86-20-87335161; Fax: 86-20- 87330446; E-mail: lidchuan@mail.sysu.edu.cn. cro cro © 2019 Chen et al. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc. g 1 Both authors contributed equally to this work. 3 Theabbreviationsusedare:PP2A,proteinphosphatase2A;AML,acutemye- loid leukemia; WBC, white blood cells; LYM, lymphocytes; RBC, red blood cell; SPMA, S-phenyl mercapturic acid; BM, bone marrow; MN, micronuclei; tt-MA, trans-, transmuconic acid; HQ, hydroquinone; MDA, malondialde- hyde; GSK3, glycogen synthase kinase 3; shRNA, short hairpin RNA; APAP, acetaminophen; CYP2E1, cytochrome P4502E1; MCV, mean corpus- cular volume; ANOVA, analysis of variance. Results Protein phosphatase 2A (PP2A)3 is a ubiquitously expressed holoenzyme that is responsible for the majority of Ser/Thr phosphatase activity in eukaryotic cells and has been implicated in the regulation of diverse signaling pathways (17). PP2A holoenzymes contain a catalytic C subunit, a structural scaf- folding A subunit, and a variable B regulatory subunit. The Ppp2r1a gene encodes the scaffolding subunit A, which is responsible for 90% of core/holoenzyme PP2A assemblies (18). The interactions between a variety of B subunits with the core AC dimer dynamically determine the target specificity and sub- cellular localization of individual PP2A holoenzymes. Perturba- tion of signaling pathways controlled by specific PP2A com- plexes has been linked to the development and progression of human diseases (19, 20). For example, the suppression of spe- cific PP2A B subunits or the inhibition of PP2A phosphatase activity has been implicated in the development of acute mye- loid leukemia (AML) (21–23). Recent studies have revealed that activation of PP2A could be a potential target for development of therapeutic drugs against AML (21, 24). Gene expression studies demonstrate that benzene exposure alters Ppp2r1a mRNA expression (25). Taken together, these observations suggest that PP2A may be a key modulator of benzene-induced hematotoxicity. Table 1 Table 1 Periphery blood cytometry in mice exposed to benzene by inhalation and oral gavage Exposure route Exposure level (mean S.D., ppm or mg/kg) 0 1 10 100 Dynamic inhalation (n 10) WBC (109/liter) 4.69 0.79 4.01 0.69a 3.67 0.62a 1.68 0.43a RBC (1012/liter) 7.43 0.58 7.04 1.16 6.97 0.78 5.08 0.84a Platelets (109/liter) 1249.07 124.90 1271.52 188.36 1256.22 83.59 1022.59 134.67a LYM (109/liter) 2.62 0.39 2.00 0.38a 1.71 0.45a 0.81 0.28a MCV (f l) 51.42 2.22 52.89 2.74 56.88 3.6a 57.45 3.30a Oral gavage (n 6) WBC (109/liter) 4.83 0.97 4.10 1.13 3.47 0.85a 1.73 0.31a RBC (1012/liter) 7.15 0.23 6.77 0.47 6.72 0.26 5.15 0.69a Platelets (109/liter) 1234.20 81.71 1200.50 70.44 1219.10 86.55 1219.87 108.16 LYM (109/liter) 2.57 0.63 1.76 0.65a 1.70 0.70a 0.77 0.22a MCV (f l) 54.87 1.68 52.66 2.36 57.68 1.51a 58.18 3.11a a p 0.05, compared with the corresponding control group, as determined by independent-sample t test or assessed with one-way ANOVA followed by Bonferroni post-test. Table 1 Periphery blood cytometry in mice exposed to benzene by inhalation and oral gavage thus mediating toxic effects (13, 14). Prior studies have shown that benzene and its metabolites may activate several key signaling pathways such as PI3K-AKT, p38 MAPK, or JNK, subsequently triggering apoptosis of marrow cells or malignant progression of human leukemia cells (15, 16), sug- gesting the involvement of protein kinase in benzene-in- duced toxicity. containing the B56 subunit suppressed Cyp2e1 expression via perturbation of the dephosphorylation of -catenin at Ser33- Ser37-Thr41. These data support a novel signaling pathway reg- ulated by PP2A B56 complexes that plays an important role in benzene metabolic activation. 2486 2486 © 2019 Chen et al. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, In PP2A regulates benzene-induced hematotoxicity Table 1 Periphery blood cytometry in mice exposed to benzene by inhalation and oral gavage Table 1 The effects of PP2A A deletion on regulation of benzene-induced hematotoxicity that reticulocyte counts, a marker for hematopoietic potency of bone marrow erythroid, dramatically declined in WT mice exposed to benzene at 1, 10, and 100 ppm compared with the respective control group (p 0.05) (Fig. 1C). In contrast, we only observed a 35.8% decrease of reticulocyte counts in the 100-ppm benzene-treated HO mice. These results were in agreement with examination of peripheral blood indicating that deletion of the PP2A A subunit led to the attenuation of benzene-induced hematopoietic toxicity. The mouse model with hepatocyte-specific deletion of the Ppp2r1a (coding PP2A A) gene was generated as described under “Experimental procedures.” Ppp2r1a deletion in the hep- atocyte had no significant impact on mouse phenotype, behav- ior, growth rate, or fertility within 6 months after birth. To explore the role of PP2A in regulation of benzene-induced hematotoxicity, male mice with hepatocyte-specific deletion of Ppp2r1a (HO) and matched wildtype (WT) mice were exposed to benzene via inhalation at doses of 0, 1, 10, or 100 ppm for 28 days. As shown in Fig. 1A, WBC counts in WT mice decreased by 23.8, 30.8, and 61.5%, respectively, in WT mice exposed to 1, 10, or 100 ppm benzene compared with the vehicle control mice (Ptrend 0.001). In HO mice, WBC counts increased by 9.6% in the 1 ppm group but declined by 16.3 and 48.8% in the 10 and 100 ppm group, suggesting that only high dose benzene treatment resulted in hematotoxicity. We noted that the decrease of WBC counts in WT mice was more profound than that in HO mice. Similarly, LYM counts in benzene-treated WT mice declined in a dose-dependent manner (Ptrend 0.001), whereas there was no significant decrease in the LYM counts in HO mice except for the 100 ppm treatment group (Fig. 1A). Moreover, the decline of RBC counts in WT and HO mice only appeared in the 100-ppm treatment group and HO mice seemed to be more sensitive to benzene treatment. Taken together, these results indicate that suppression of PP2A A seems to confer resistance to benzene-induced hematotoxicity in these mice. In addition, we also examined the degree of genetic damage and genomic instability by determining the frequencies of micronuclei formation and performing Comet assays using bone marrow cells and peripheral blood lymphocytes of mice exposed to benzene. The effects of PP2A A deletion on regulation of benzene-induced hematotoxicity As a result, we found that the frequencies of micronuclei (MN) in bone marrow cells were 4.26, 9.15, and 11.21%, respectively, in WT mice exposed to benzene via inha- lation at doses of 1, 10, and 100 ppm (Ptrend 0.001) (Fig. 1D). In contrast, we did not observe significant changes in the fre- quencies of MN in HO mice exposed to benzene at any dose (Fig. 1D), indicating that PP2A A deficiency protected bone marrow cells from DNA damage induced by benzene. These results were further confirmed by a Comet assay, the Tail Moment values of peripheral blood cells were significantly higher in WT mice compared with that in HO mice (Fig. 1e). Taken together, these observations indicate an involvement of PP2A in the regulation of benzene-induced toxicity. Table 2 Table 2 The amount of urinary SPMA in mice exposed to benzene (g/g creatinine, mean S.D., n 6) Exposure route Dose (ppm/mg/kg) Ptrend 0 1 10 100 Inhalation 0.32 0.11 1.77 1.17 31.66 9.12a 79.74 9.65a 0.0001 Oral gavage 0.40 0.10 1.80 1.40 43.63 12.06a 92.51 5.53a 0.044 a p 0.05, compared with the corresponding control group, as determined by independent-sample t test or assessed with one-way ANOVA followed by Bonferroni post-test. Comparison between inhalation and oral gavage of benzene- exposure routes To establish the benzene-exposure mouse model, we selected two independent cohorts of male FVB background mice and exposed them to benzene by inhalation or oral gavage administration. Male mice were exposed to benzene vapor at concentrations of 0, 1, 10, and 100 ppm, whereas the oral gavage group were treated with benzene at 0, 1, 10, and 100 mg/kg body weight/day, respectively, for 28 days. There was no significant change in body weight between the control and treatment groups of mice over the exposure time period. Peripheral blood cytometry was employed to assess the hematotoxicity induced by benzene. As shown in Table 1, mice in the inhalation ben- zene-exposed group displayed a significant decrease in the number of white blood cells (WBC) and lymphocytes (LYM) in a dose-dependent manner (Ptrend 0.001), indicating that WBC and LYM counts were sensitive to benzene exposure. By contrast, we did not observe a parallel decrease in the number of red blood cells (RBC) except for the 100 ppm group com- pared with the control group via inhalation. Similar results were obtained in mice administered benzene by gavage (Table 1; Table S1). In addition to blood cell counts, we also measured urine SPMA levels, an internal exposure marker of benzene. The concentration of SPMA in the benzene-exposed group via inhalation was increased by 5-, 105-, and 265-fold, respectively, compared with that in control mice (Ptrend 0.001) (Table 2). Similar results were found in benzene-ex- posed mice via oral gavage, with SPMA levels elevated by 3.5-, 108-, or 230.8-fold (Ptrend 0.044), respectively. Thus, we successfully established benzene-induced hematotoxic- ity models through both oral gavage and inhalation routes. Importantly, we clarified the dose relevance between the two exposure approaches based on the content of metabolite and the extent of hematotoxicity. In this study, we constructed a mouse model with the hepa- tocyte-specific deletion of Ppp2r1a (encoding PP2A A), gen- erating a useful tool to define the role of PP2A in the regulation of benzene-induced toxicity. We report that knockout of the PP2A A in murine hepatocytes leads to a decrease in benzene- induced hematotoxicity. Particularly, specific PP2A complexes 2487 J. Biol. Chem. (2019) 294(7) 2486–2499 PP2A regulates benzene-induced hematotoxicity PP2A regulates benzene-induced hematotoxicity s involved in regulation of benzene-induced hematotoxicity. WT and HO mice were exposed to benzene via inhalation a pectively, for 28 days. A, peripheral blood counts including WBC, RBC, MCV, and LYM (n 8). B, bone marrow cells were pla lony numbers of CFU-E, CFU-GM, CFU-G, and CFU-GEMM (n 8) were counted following a 14-day culturing. C, the number o cells (n 8) was counted and shown as mean S.D. D, the frequency of MN in 1000 bone marrow cells. , p 0.05, co ol group. E, the degree of DNA damage in benzene-exposed mice (n 6) was determined by Comet assay, and comet ta lymphocytes are shown as mean S.D. Figure 1. PP2A is involved in regulation of benzene-induced hematotoxicity. WT and HO mice were exposed to benzene via inhalation at doses of 0, 1, 10, and 100 ppm, respectively, for 28 days. A, peripheral blood counts including WBC, RBC, MCV, and LYM (n 8). B, bone marrow cells were plated in methylcel- lulose and the colony numbers of CFU-E, CFU-GM, CFU-G, and CFU-GEMM (n 8) were counted following a 14-day culturing. C, the number of reticulocytes in 1000 red blood cells (n 8) was counted and shown as mean S.D. D, the frequency of MN in 1000 bone marrow cells. , p 0.05, compared with the respective control group. E, the degree of DNA damage in benzene-exposed mice (n 6) was determined by Comet assay, and comet tail moments of 75 peripheral blood lymphocytes are shown as mean S.D. or 54.5%, respectively, compared with those in WT mice (p 0.01) (Fig. S2D), which was inversely proportional to the total amount of benzene over 210 min of observation. These findings support the idea that deficiency of the PP2A A subunit in hepatocytes leads to an inhibition of the metabolic activation of benzene. Thus, we speculate that a decline in the cytotoxicity of BM and peripheral blood cells might be present in HO mice due to lower levels of activated metabolites of benzene. To test this hypothesis, we examined the viability in HL60 cells that were cultured in medium supplemented with 2% plasma collected from WT or HO mice exposed to benzene at doses of 1, 10, and mass measurement (Table S2). As shown in Fig. PP2A A deletion leads to the suppression of the metabolic activation of benzene Metabolism of benzene to its toxic metabolites is generally thought to be a critical event in benzene-induced toxicity (26). To address whether PP2A A deletion has impact on benzene metabolism, we compared the amount of urinary SPMA in WT and HO mice upon benzene treatment. As shown in Fig. 2A, the urinary SPMA concentration increased in a dose-response manner in both WT and HO mice (Ptrend 0.05). However, the increment of increase in urinary SPMA was enhanced in WT mice compared with HO mice. Remarkably, the urinary SPMA concentration in HO mice was decreased by 42.6% compared with that in WT mice in the 100-ppm treatment group (Fig. 2A). These results implicate the involvement of the PP2A A subunit in the regulation of benzene metabolism. To further assess the effects of PP2A A on metabolic activation of ben- zene, we employed a novel system to detect the components of exhalation dynamically by using secondary nanoelectrospray ionization coupled with an ultra-high resolution MS (Sec- nanoESI-UHRMS). The molecular ions of benzene and its metabolites, including phenol, trans, transmuconic acid (tt- MA), and hydroquinone (HQ) were identified through accurate Both hematopoietic stem or progenitor cells (HS/PCs) and stromal cells in bone marrow (BM) are critical targets of ben- zene metabolites. We next assessed the proliferation capacities of BM progenitors upon benzene treatment using the colony- forming assay. By this method, a slight increase in colony num- ber of BM progenitors in WT mice was noted in the 1- and 10-ppm groups, whereas a significant reduction was evident in the 100-ppm treatment group (Fig. 1B). In particular, benzene exposure decreased the number of CFU-M, CFU-GM, and CFU-E progenitors, as well as the total colony number by 48.0, 40.6, 44.6, or 26.3% only in the 100-ppm treatment group of WT mice (p 0.05) compared with the control group. There were no significant changes with respect to CFU-G and CFU-GEMM upon benzene treatment. Notably, the colony number of CFU-E and total colony counts in HO mice exhibited a 33.5 and 13.5% decline at 100 ppm (p 0.05), whereas the colony num- ber of CFU-M, CFU-GM, CFU-G, and CFU-GEMM displayed no obvious changes. These observations indicate that PP2A A deficiency suppresses the toxicity of BM progenitor cells induced by benzene exposure. Consistently, we demonstrated 2488 J. Biol. Chem. (2019) 294(7) 2486–2499 PP2A regulates benzene-induced hematotoxicity 2B, the dynamic changes of benzene in mouse exhalation upon ben- zene exposure showed that the amount of benzene in exhala- tion achieved the highest level 15–20 min after benzene admin- istration via oral gavage and required 210 min to return to the basal level. Strikingly, the total amount of exhaled benzene was 7-fold higher in HO mice compared with the WT mice within 210 min (Fig. 2C). In parallel, we also detected the presence of three benzene metabolites in mouse exhalation, including phe- nol, tt-MA, and HQ (Fig. S2, A–C). The amount of exhaled phenol, tt-MA, and HQ in HO mice were decreased by 70, 33.3, 2489 J. Biol. Chem. (2019) 294(7) 2486–2499 2489 Figure 2. PP2A A deletion leads to suppression of the metabolic activation of benzene. A, the levels of urinary SPMA (n 6) were normalized by urinary creatinine and presented as micrograms/g of creatinine. B, WT and HO mice (n 5) were administrated 100 mg/kg of benzene. Exhaled benzene from mouse breath was measured in real-time by SE-SI-HRMS. Mass spectrum data were present as two-dimensional data with standardized m/z intensity and time. C, the level of exhaled benzene was quantified by peak area. , p 0.05 compared with WT mice, as determined by Mann-Whitney U test. HL60 cells were incubated with a medium containing 2% plasma isolated from the benzene-treated mice (n 5) for 24 h, and subjected to analyses including cytotoxicity, MDA level, and DNA damage. D, relative cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. E, MDA content was calcu- lated as micromole/g of protein. F, Comet tail moments (TM) from 150 HL60 cells counted. Data were shown as mean S.D. from three independent experiments. , p 0.05, compared with the control cells. PP2A regulates benzene-induced hematotoxicity PP2A regulates benzene-induced hematotoxicity y Figure 2. PP2A A deletion leads to suppression of the metabolic activation of benzene. A, the levels of urinary SPMA (n 6) were normalized by urinary creatinine and presented as micrograms/g of creatinine. B, WT and HO mice (n 5) were administrated 100 mg/kg of benzene. Exhaled benzene from mouse breath was measured in real-time by SE-SI-HRMS. Mass spectrum data were present as two-dimensional data with standardized m/z intensity and time. C, the level of exhaled benzene was quantified by peak area. , p 0.05 compared with WT mice, as determined by Mann-Whitney U test. HL60 cells were incubated with a medium containing 2% plasma isolated from the benzene-treated mice (n 5) for 24 h, and subjected to analyses including cytotoxicity, MDA level, and DNA damage. D, relative cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. E, MDA content was calcu- lated as micromole/g of protein. F, Comet tail moments (TM) from 150 HL60 cells counted. Data were shown as mean S.D. from three independent experiments. , p 0.05, compared with the control cells. 100 ppm. As a result, we found that the relative cell viability was decreased by 3.2, 18.6, and 38.7%, respectively, in HL60 cells treated with diluted plasma derived from WT mice exposed to benzene at 1, 10, and 100 ppm (Fig. 2D). In contrast, HL60 cells displayed a reduced relative cell viability (5.4, 36.0, and 58.9%, respectively) following treatment with diluted plasma isolated from 1, 10, and 100 ppm benzene-treated HO mice (Fig. 2D). In addition, we examined the level of malondialdehyde (MDA) to compare the degree of oxidative stress induced by benzene- treated mouse plasma from WT and HO mice. As shown in Fig. 2E, the levels of MDA significantly increased in a dose-depen- dent manner in WT mice (Ptrend 0.05). However, there was little effect on the increment of MDA in HO mice (p 0.05) (Fig. 2E). Moreover, we demonstrated that genotoxicity, indi- cated by Tail Moment values, were enhanced in HL60 cells treated with plasma from benzene-treated WT mice compared with HO mice (Fig. 2F). Taken together, these results indicate that the plasma from benzene-treated HO mice contained fewer toxic metabolites than plasma from WT mice. The atten- uation of the effects, such as oxidative stress, DNA damage, and cytotoxicity in HO mice might be attributable to the inhibition of metabolic activation of benzene. Collectively, these observa- tions demonstrate that PP2A mediates benzene-induced hema- totoxicity by regulating the metabolic activation of benzene. 100 ppm. As a result, we found that the relative cell viability was decreased by 3.2, 18.6, and 38.7%, respectively, in HL60 cells treated with diluted plasma derived from WT mice exposed to benzene at 1, 10, and 100 ppm (Fig. 2D). In contrast, HL60 cells displayed a reduced relative cell viability (5.4, 36.0, and 58.9%, respectively) following treatment with diluted plasma isolated from 1, 10, and 100 ppm benzene-treated HO mice (Fig. 2D). In addition, we examined the level of malondialdehyde (MDA) to compare the degree of oxidative stress induced by benzene- treated mouse plasma from WT and HO mice. As shown in Fig. 2E, the levels of MDA significantly increased in a dose-depen- dent manner in WT mice (Ptrend 0.05). However, there was little effect on the increment of MDA in HO mice (p 0.05) (Fig. 2E). Moreover, we demonstrated that genotoxicity, indi- cated by Tail Moment values, were enhanced in HL60 cells treated with plasma from benzene-treated WT mice compared PP2A regulates benzene-induced hematotoxicity by suppression of Cyp2e1 expression Because PP2A A subunit deficiency in the hepatocyte per- turbs the metabolism of benzene, we speculated that PP2A may regulate the expression of key enzymes involved in benzene metabolism. Thus, we examined gene expression levels of 17 metabolic enzymes (listed in Table S2) in liver tissues of mice 2490 J. Biol. Chem. (2019) 294(7) 2486–2499 Figure 3. PP2A regulated benzene-induced hematotoxicity by suppression of Cyp2e1 expression. A, immunoblotting analysis of A, -catenin, phos- phorylated -catenin, and Cyp2e1 in WT and HO mice liver treated with benzene at the indicated doses. B, the representative images are shown for liver sections stained with Cyp2e1 antibody (100 magnification, scale bar, 200 m). C, quantification of the Cyp2e1-stained area expressed as percentage of liver fields occupied (mean S.D., n 6). , p 0.05, compared with the corresponding control group. PP2A regulates benzene-induced hematotoxicity PP2A regulates benzene-induced hematotoxicity Figure 3. PP2A regulated benzene-induced hematotoxicity by suppression of Cyp2e1 expression. A, immunoblotting analysis of A, -catenin, ph Figure 3. PP2A regulated benzene-induced hematotoxicity by suppression of Cyp2e1 expression. A, immunoblotting analysis of A, -catenin, phos- phorylated -catenin, and Cyp2e1 in WT and HO mice liver treated with benzene at the indicated doses. B, the representative images are shown for liver sections stained with Cyp2e1 antibody (100 magnification, scale bar, 200 m). C, quantification of the Cyp2e1-stained area expressed as percentage of liver fields occupied (mean S.D., n 6). , p 0.05, compared with the corresponding control group. treated with different doses of benzene via inhalation. As shown in Fig. S3, the mRNA levels of Cyp2e1, gsta, and gstm4 in WT mouse liver were increased in a dose-response manner (p 0.05). However, PP2A A deletion led to an inhibition of the induction of Cyp2e1 in benzene-treated HO mice (Fig. S3), indicating that PP2A might be involved in modulating Cyp2e1 expression. Next, we performed immunoblotting analysis to examine putative changes in Cyp2e1 protein levels in mouse liver tissues resulting from benzene inhalation. We found no induction of Cyp2e1 when WT or HO mice were exposed to 1 ppm benzene. However, Cyp2e1 induction was profound in WT mice exposed to 10 or 100 ppm benzene. Conversely, Cyp2e1 induction was abolished in HO mice exposed to benzene at any dose (Fig. 3A). PP2A regulates benzene-induced hematotoxicity by suppression of Cyp2e1 expression Taken together, these findings demonstrate that PP2A A par- ticipates in the regulation of transcriptional activation of Cyp2e1 and, in turn, plays a critical role in mediating benzene- induced hematotoxicity. Prior studies have reported that the phosphorylation of -catenin at Ser33-Ser37-Thr41 is essential for ubiquitination- mediated degradation of -catenin (29, 30) and the phosphor- ylation of -catenin at Ser675 is responsible for -catenin tran- scriptional activation (31). In our study, we found that the phosphorylated -catenin at Ser33-Ser37-Thr41 decreased by 20.1, 42.6, or 65.6%, respectively, in the livers of WT mice treated with 1, 10, or 100 ppm benzene via inhalation. However, there was no difference between WT and HO mice with respect to phosphorylated -catenin at Ser675 (Fig. 3A). Importantly, we found that the phosphorylated -catenin at Ser33-Ser37- Previous studies have demonstrated that the transcriptional activation of Cyp2e1 is regulated by -catenin signaling (27, 28). We examined the levels of -catenin in liver tissues isolated from the benzene-treated WT and HO mice. As shown in Fig. 3A, benzene exposure led to a remarkable increase in -catenin expression, which correlated with the suppression of Cyp2e1. These results suggest that -catenin signaling participates in the activation of cyp2e1 transcription. In addition, we found 2491 PP2A regulates benzene-induced hematotoxicity by suppression of Cyp2e1 expression Parallel immunohistochemical examination revealed that Cyp2e1-positive staining was decreased by 53.8 and 92.3%, respectively, in HO mice exposed to 10 and 100 ppm benzene compared with WT mice (Fig. 3B). Taken together, these findings demonstrate that PP2A A par- ticipates in the regulation of transcriptional activation of Cyp2e1 and, in turn, plays a critical role in mediating benzene- induced hematotoxicity. that Wnt/-catenin-interacted components including dick- kopf-related protein 1 (DKK1), Axin, phosphorylated GSK3 at Ser9, and GSK3 were accordingly altered and might contrib- ute to activation of the Wnt/-catenin pathway in WT mice upon benzene exposure (Fig. S4), implicating a regulatory role of the Wnt/-catenin pathway in benzene-induced hematotox- icity. In contrast, this effect was greatly attenuated in HO mice. To further demonstrate the role of -catenin in benzene-in- duced hematotoxicity, we treated WT mice with 100 mg/kg of benzene and 5 mg/kg of XAV-939, a known inhibitor of -catenin inhibitor for 7 days. As shown in Fig. S5, XAV-939 treatment reversed the decline of WBC and LYM counts in WT mice exposed to benzene. Furthermore, we conducted in vitro studies in HL60 cells and revealed that XAV-939 treatment attenuated HQ-induced cytotoxicity (Fig. S6). Taken together, these results support the notion that PP2A was involved in reg- ulation of benzene-induced hematotoxicity by regulating the Wnt/-catenin pathway. treated with different doses of benzene via inhalation. As shown in Fig. S3, the mRNA levels of Cyp2e1, gsta, and gstm4 in WT mouse liver were increased in a dose-response manner (p 0.05). However, PP2A A deletion led to an inhibition of the induction of Cyp2e1 in benzene-treated HO mice (Fig. S3), indicating that PP2A might be involved in modulating Cyp2e1 expression. Next, we performed immunoblotting analysis to examine putative changes in Cyp2e1 protein levels in mouse liver tissues resulting from benzene inhalation. We found no induction of Cyp2e1 when WT or HO mice were exposed to 1 ppm benzene. However, Cyp2e1 induction was profound in WT mice exposed to 10 or 100 ppm benzene. Conversely, Cyp2e1 induction was abolished in HO mice exposed to benzene at any dose (Fig. 3A). Parallel immunohistochemical examination revealed that Cyp2e1-positive staining was decreased by 53.8 and 92.3%, respectively, in HO mice exposed to 10 and 100 ppm benzene compared with WT mice (Fig. 3B). PP2A regulates benzene-induced hematotoxicity mRNA remained unchanged upon HQ treatment, suggesting that the metabolite of benzene might up-regulate -catenin through suppression of -catenin degradation (Fig. S8, B–D). Notably, enhanced phosphorylated -catenin at Ser33-Ser37- Thr41, coupled with decreased -catenin, was also present in HepG2–SHA and HepG2–SHB56 cells following ethanol, HQ, or APAP treatment (Fig. 4C). These findings demonstrate that the increased -catenin and Cyp2e1 induction were abol- ished by suppression of PP2A A or B56 in HepG2 cells. In control experiments, we did not detect induction of Cyp2e1 in HepG2 cells expressing shB55. Moreover, a co-immunopre- cipitation assay confirmed the interaction between PP2A and -catenin (Fig. 5A), showing that the B56 subunit was in com- plex with -catenin at Ser33-Ser37-Thr41 upon APAP treatment (Fig. 5A). Consistent with these results, we visualized the -catenin translocation from the cytoplasmic membrane to the nucleus when HepG2 cells were treated with 5 M APAP, 200 mM ethanol, or 50 M HQ under a laser scan confocal micros- copy. Both the suppression of PP2A A or B56 subunit per- turbed -catenin translocation (Fig. 5B). In parallel, B56 sub- unit suppression inhibited the transcriptional activation of cyp2e1 upon these agonists’ treatments (Fig. S8, B–D). In con- trast, suppression of B55 had no effect on the translocation of -catenin in response to APAP treatment (Fig. 5B). In parallel, we revealed the cell viability was decreased by 25.3 or 27.6%, respectively, in HepG2–SHGFP or HepG2–SHB55 cells treated with 50 M HQ compared with the control cells. How- ever, it displayed on obvious effect in cells expressing shA and shB56 (Fig. 5C). Similar results were obtained when we exam- ined the MDA levels in HepG2 cells (Fig. 5D), indicating that suppression of PP2A B56 sensitized the cells to HQ-in- duced cytotoxicity and oxidative damage. Taken together, these observations demonstrate that PP2A B56 complexes were indispensable for regulating the transcriptional activa- tion of cyp2e1. In summary, we identified specific PP2A complexes containing the B56 subunit that directly con- tributes to regulating Cype21 expression through dephos- phorylation of -catenin at Ser33-Ser37-Thr41. Thr41 in benzene-treated HO mouse liver was significantly enhanced,suggestingthatPP2Amightberesponsiblefordephos- phorylation of -catenin at Ser33-Ser37-Thr41 involved in Cyp2e1 induction. These observations suggest that PP2A reg- ulates benzene-induced hematotoxicity through the -catenin pathway and, in turn, represses the transactivation of cyp2e1 expression. J. Biol. Chem. (2019) 294(7) 2486–2499 Specific PP2A complexes containing B56 participate in regulation of cyp2e1 expression To identify specific PP2A complexes participating in the reg- ulation of Cyp2e1 expression, we performed a loss-of-function screen in AML-12 (mouse liver cell line) and HepG2 cells (human liver tumor cell line) expressing shRNAs against a sub- set of PP2A regulatory subunits. Our preliminary results dem- onstrated that PP2A A deletion in mouse hepatocytes led to a remarkable decrease in protein levels of several B subunits, including B55, B56, B56, and B56. Next, we created stable AML-12 and HepG2 cell lines expressing shA through retro- viral infection and assessed the effect of A suppression on the expression of various B subunits. Two independent shRNAs that targeted different sequences of the PP2A A subunit were chosen to eliminate the off-target effects. As a result, the levels of A in AML-12–shA-1 and AML-12–shA-2 cells decreased by 54 and 90%, respectively, compared with AML-12 cells expressing a control vector (AML-12–shGFP) (Fig. 4A). Consistent with the results from mouse liver tissues, expression of B55, B56, B56, and B56 subunits were dramatically down-regulated in AML- 12–shA-1 and AML-12–shA-1 cells (Fig. 4A). Similar results were obtained in HepG2 cells expressing PP2A A shRNAs (Fig. 4B). In addition, we generated stable HepG2 cells expressing shB55,shB56,shB56,andshB56.Theeffectsofgenesuppres- sion by introduction of shRNAs were confirmed by immunoblot- ting analysis (Fig. 4B). As shown in Fig. S7, Cyp2e1 expression was profoundlydeclinedinHepG2cellsexpressingshB56,suggesting a role of PP2A B56 holoenzyme in the regulation of transcrip- tional activation of cyp2e1. Next, we assessed whether the PP2A B56 subunit mediated transcriptional activation of cyp2e1 upon treatment with known Cyp2e1 agonists. As expected, we observed a dose-de- pendent induction of Cyp2e1 expression in HepG2 cells upon treatment with ethanol, HQ, and acetaminophen (APAP) (Fig. S8A). In particular, PP2A A or B56 suppression led to a 62.5 or 78.6% decline in Cyp2e1 induction in HepG2 cells treated with 50 M HQ (Fig. 4, B and D). Similar results were obtained when HepG2 cells were treated with 200 mM ethanol or 5 M APAP (Fig. 4, C and D). As a control subunit, B55 suppression had no effect on the induction of Cyp2e1 protein expression (Fig. 4E). These results support the hypothesis that PP2A B56 holoenzyme might be responsible for the transcriptional acti- vation of Cyp2e1. 2492 J. Biol. Chem. (2019) 294(7) 2486–2499 PP2A regulates benzene-induced hematotoxicity PP2A regulates benzene-induced hematotoxicity the roles of PP2A in the regulation of dephos- i b i i ll i i ticular PP2A subunits. In this study, we gen d l i h h ifi d l i f h cationofspecificPP2AcomplexesinvolvedintheregulationofCyp2e1expression.A,AML-12cellsorHepG2cellsw shRNAs targeting GFP, or two independent shA to generate stable cell lines as indicated. The protein level was deter withtheindicatedantibodies.B,HepG2cellsstablyexpressingshB55,shB56,shB56,andshB56.Immunoblottingana tein levels in the indicated cell lines. HepG2-SHGFP cells, and (C) HepG2–SHA-1 and HepG2–SHA-2 cells, (D) Hep 2 cells, (E) HepG2–SHB55-1 and HepG2–SHB55-2 cells were treated with 200 mM ethanol, 50 M HQ, and 5 M APAP fo analysis was performed with the indicated antibodies. PP2A regulates benzene induced he Figure4.IdentificationofspecificPP2AcomplexesinvolvedintheregulationofCyp2e1expression.A,AML-12cellsorHepG2cellsweretransfectedwith vectors encoding shRNAs targeting GFP, or two independent shA to generate stable cell lines as indicated. The protein level was determined by immuno- blottinganalysiswiththeindicatedantibodies.B,HepG2cellsstablyexpressingshB55,shB56,shB56,andshB56.Immunoblottinganalysiswasperformed to detect the protein levels in the indicated cell lines. HepG2-SHGFP cells, and (C) HepG2–SHA-1 and HepG2–SHA-2 cells, (D) HepG2–SHB56-1 and HepG2–SHB56-2 cells, (E) HepG2–SHB55-1 and HepG2–SHB55-2 cells were treated with 200 mM ethanol, 50 M HQ, and 5 M APAP for 24 h, respectively. Immunoblotting analysis was performed with the indicated antibodies. Figure4.IdentificationofspecificPP2AcomplexesinvolvedintheregulationofCyp2e1expression.A,AML-12cellsorHepG2cellsweretransfectedwith vectors encoding shRNAs targeting GFP, or two independent shA to generate stable cell lines as indicated. The protein level was determined by immuno- blottinganalysiswiththeindicatedantibodies.B,HepG2cellsstablyexpressingshB55,shB56,shB56,andshB56.Immunoblottinganalysiswasperformed to detect the protein levels in the indicated cell lines. HepG2-SHGFP cells, and (C) HepG2–SHA-1 and HepG2–SHA-2 cells, (D) HepG2–SHB56-1 and HepG2–SHB56-2 cells, (E) HepG2–SHB55-1 and HepG2–SHB55-2 cells were treated with 200 mM ethanol, 50 M HQ, and 5 M APAP for 24 h, respectively. Immunoblotting analysis was performed with the indicated antibodies. (34). However, the roles of PP2A in the regulation of dephos- phorylation of a given substrate in a given cell or tissue remain largely unknown. Although many PP2A transgenic, knockout, or knock-in mice have been generated (35), few models are available for investigating the tissue-specific functions of par- ticular PP2A subunits. In this study, we generated a mouse model with a hepatocyte-specific deletion of the Ppp2r1a gene (coding PP2A A) to clarify the molecular pathways involved in regulation of benzene-induced hemototoxicity. Although the HO mice exhibit normal phenotype, behavior, growth Discussion Dysregulation of protein phosphorylation in signaling path- ways has been linked to the development of many human dis- eases (32, 33). Although several types of protein kinases have been shown to be directly involved in benzene-induced hema- totoxicity, the key events with respect to protein phosphatase remain largely unknown. In the present study, we demon- strated that the perturbation of this regulatory pathway in a murine model with hepatocyte-specific homozygous deletion of Ppp2r1a gene confers resistance to benzene-induced hema- totoxicity. We also identified specific PP2A complexes contain- ing the B56 subunit that participates in regulation of Cyp2e1 expression through direct dephosphorylation of -catenin at Ser31-Ser37-Thr41. These findings reveal a novel pathway medi- ated by protein phosphatases 2A in regulating benzene-in- duced hematotoxicity. Concurrent with Cyp2e1 induction, we found that -catenin phosphorylation at Ser33-Ser37-Thr41 significantly decreased in HepG2 cells treated with ethanol, HQ, or APAP (Fig. S8A). In contrast, up-regulation of the -catenin protein was evident in HepG2 cells treated with three agonists, indicating that -catenin phosphorylation at Ser33-Ser37-Thr41 negatively reg- ulated Cyp2e1 expression. Moreover, we found that -catenin PP2A represent a major fraction of cellular Ser/Thr phospha- tase activity in rodent and human tissues and is involved in the regulation of diverse biological and physiological processes 2492 2492 J. Biol. Chem. (2019) 294(7) 2486–2499 4 L. Chen, P. Guo, H. Zhang, W. Li, C. Gao, Z. Huang, J. Fan, Y. Zhang, S. Li, X. Liu, F. Wang, S. Wang, Q. Li, Z. He, H. Li, S. Chen, X. Wu, L. Ye, Q. Li, H. Tang, Q. Wang, G. Dong, Y. Xiao, W. Chen, and D. Li, unpublished data. 2493 J. Biol. Chem. (2019) 294(7) 2486–2499 2493 J. Biol. Chem. (2019) 294(7) 2486–2499 er functions and fertility within 3 months after birth PP2A A in the hepatocyte attenuated benzene ind PP2A B56 complexes interact with -catenin and regulate -catenin translocation. A, HepG2 cells were treated with 50 M HQ fo noprecipitation (IP) assays were performed with antibodies against -catenin and PP2A B56 and followed by immunoblotting with anti he indicated proteins. B, indicated cell lines were treated with 200 mM ethanol, 50 M HQ, and 5 M APAP for 24 h, respectively. Immunofluore as performed using an antibody against -catenin (green) and the images were visualized under the laser-scanning confocal microscopy. The e stained with 4,6-diamidino-2-phenylindole (DAPI). HepG2–SHGFP and HepG2–SHB56 cells were treated with ethanol, HQ, and APAP concentrations for 24 h, respectively, and followed by the examination, including (C) relative cell viability and (D) MDA content. Data were sh D. from three independent experiments. , p 0.05, compared with the control cells. egulates benzene-induced hematotoxicity PP2A regulates benzene-induced hematotoxicity y Figure 5. PP2A B56 complexes interact with -catenin and regulate -catenin translocation. A, HepG2 cells were treated with 50 M HQ for 24 h. Co-immunoprecipitation (IP) assays were performed with antibodies against -catenin and PP2A B56 and followed by immunoblotting with antibodies targeting the indicated proteins. B, indicated cell lines were treated with 200 mM ethanol, 50 M HQ, and 5 M APAP for 24 h, respectively. Immunofluorescence analysis was performed using an antibody against -catenin (green) and the images were visualized under the laser-scanning confocal microscopy. The nuclei (blue) were stained with 4,6-diamidino-2-phenylindole (DAPI). HepG2–SHGFP and HepG2–SHB56 cells were treated with ethanol, HQ, and APAP at the indicated concentrations for 24 h, respectively, and followed by the examination, including (C) relative cell viability and (D) MDA content. Data were shown as mean S.D. from three independent experiments. , p 0.05, compared with the control cells. rate, liver functions, and fertility within 3 months after birth, we found that these mice developed liver fibrosis at 12 months of age.4 Here, we demonstrate that depletion of PP2A A in the hepatocyte attenuated benzene-induced hematotoxicity, demonstrating a critical role of PP2A in mediating the chemical-induced hematotoxicity. Because the mouse hepatocytes are responsible for metabolic activa- tion or suppression of drugs and environmental chemicals, we focused on the effects of PP2A A depletion on benzene metabolism. 4 J. 4 L. Chen, P. Guo, H. Zhang, W. Li, C. Gao, Z. Huang, J. Fan, Y. Zhang, S. Li, X. Liu, F. Wang, S. Wang, Q. Li, Z. He, H. Li, S. Chen, X. Wu, L. Ye, Q. Li, H. Tang, Q. Wang, G. Dong, Y. Xiao, W. Chen, and D. Li, unpublished data. PP2A regulates benzene-induced hematotoxicity y We previously found that free PP2A C and B subunits were unstable in cells, and assembling of PP2A holoenzyme led to the stabilization of C and B subunits. PP2A A deficiency led to the down-regulation of B subunits including B55, B56, B56, and B56, and consequently the perturbation of a specific signaling pathway (44). In this study, we identified a novel role of PP2A B56 holoenzyme in benzene-induced hematotoxicity by reg- ulation of Cyp2e1 expression. It has been reported that the PP2A B56 subunit may function as a putative tumor suppres- sor by negatively regulating several key oncoproteins. For instance, PP2A-B56 was shown to be one component of a degradation complex for -catenin mediated by the scaffold protein Axin1 (45). Here, we demonstrated that PP2A B56 directly interacted with -catenin and controlled the degrada- tion of -catenin. Suppression of B56 markedly attenuated -catenin dephosphorylation and its translocation in cells treated with Cyp2e1 inducers. These observations suggest that the PP2A B56 holoenzyme is the key factor in regulating ben- zene-induced hematotoxicity. Previous studies support a role for the PP2A B55 complex in Drosophila Wg signaling by directly interacting with and dephosphorylating -catenin (46) and maternal PP2A:B56 was required for Wnt-dependent accumulation of -catenin protein and Xenopus dorsal devel- opment (47). A recent study reported that PP2A holoenzyme containing the B55 subunit regulated -catenin phosphoryla- tion in adenoid cystic carcinoma (48). However, in this study, we did not address whether B55, B55, or B56 subunits par- ticipated in -catenin dephosphorylation and transcriptional activation of cyp2e1. These findings indicate that regulation of the Wnt/-catenin pathway by specific PP2A B subunits varies depending on different cell types or different species. Distinct PP2A complexes might be involved in -catenin dephosphor- ylation at different residues. Previous studies reported that PP2A could modulate fetal liver erythropoiesis and maintain the survival of committed erythroid cells through the STAT5 pathway (38). This line of evidence indicates that inactivation of PP2A or dysregulation of specific pathways mediated by PP2A plays a key role in the recurrence of acute myeloid leukemia (21). PP2A-activating drugs markedly reduced survival and self-renewal of quiescent HSCs by targeting the JAK2/PP2A/-catenin pathways (39). These findings indicate that PP2A-mediated pathways might function as critical regulators for determining blood or bone marrow cells fate. PP2A regulates benzene-induced hematotoxicity PP2A regulates benzene-induced hematotoxicity Benzene toxicity is strongly dependent on biotransforma- tion. Benzene metabolism produces a variety of intermediate compounds, including benzene oxide, phenol, catechol, HQ, and benzoquinone (36). Cytochrome P4502E1 (CYP2E1) plays a critical role in metabolic activation of benzene in rodents and humans (37). Low dose benzene tends to be metabolized rap- idly and excreted primarily as conjugated metabolites. In con- trast, metabolic capacity becomes saturated or perturbed at higher doses of benzene exposure, and exhalation of unmetabo- lized benzene was thought to be the primary route of excretion (36). With the aid of new technology, we were able to examine exhaled benzene and diverse metabolites dynamically. Notably, we demonstrated that greater amounts of benzene exhaled from HO mice was due to the suppression of benzene metabolic activation in hepatocytes resulting from the depletion of the PP2A A subunit. In accordance with these observations, sev- eral benzene metabolites, including phenol, tt-MA, and HQ in the exhalation declined dramatically. This approach pro- vides a powerful tool for studying chemical biotransforma- tion. Our findings also suggest that exhalable benzene might be a feasible and noninvasive biomarker for monitoring occupational exposure. scription (31). In the current study, we found that the dephos- phorylation of -catenin at Ser33/Ser37/Thr41 in benzene- treated mouse liver led to up-regulation of -catenin and, in turn, activation of cyp2e1 transcription. PP2A A or B56 sub- units lead to abolishment of -catenin degradation. Given that suppression of PP2A A or B56 subunits lead to abolish- ment of -catenin degradation, we conclude that -catenin dephosphorylation at Ser33-Ser37-Thr41 is a prerequisite for the transcriptional activation of cyp2e1 induced by HQ or other agonists. These observations indicate that dysregulation of the -catenin pathway mediated by PP2A might contribute to ben- zene-induced hematotoxicity. scription (31). In the current study, we found that the dephos- phorylation of -catenin at Ser33/Ser37/Thr41 in benzene- treated mouse liver led to up-regulation of -catenin and, in turn, activation of cyp2e1 transcription. PP2A A or B56 sub- units lead to abolishment of -catenin degradation. Given that suppression of PP2A A or B56 subunits lead to abolish- ment of -catenin degradation, we conclude that -catenin dephosphorylation at Ser33-Ser37-Thr41 is a prerequisite for the transcriptional activation of cyp2e1 induced by HQ or other agonists. These observations indicate that dysregulation of the -catenin pathway mediated by PP2A might contribute to ben- zene-induced hematotoxicity. Biol. Chem. (2019) 294(7) 2486–2499 2494 PP2A regulates benzene-induced hematotoxicity Here, we demonstrated that PP2A was involved in the regulation of benzene-induced hematotoxicity by modulating the transcriptional activation of cyp2e1, the key enzyme of benzene metabolism. Other studies have shown that PP2A is involved in the transcriptional activation of metabolic enzymes. For example, PP2A-mediated dephosphorylation of Sp1 at Ser59 was critical in tetrachlorodibenzo-p-dioxin-in- duced CYP1A1 transcription (40). In addition, the cytoplasmic CAR–Hsp90 complex recruits PP2A upon phenobarbital treat- ment and dephosphorylates CAR at Thr38 or Ser202, leading to transactivation of metabolic enzymes (41). Our findings dem- onstrate that PP2A contributed to chemical-induced toxicity by mediating the pathways involved in regulation of metabolic enzymes. Previous studies reported that the transcriptional activation of cyp2e1 is regulated mainly by the Wnt/-catenin pathway (27). Consistent with these findings, we demonstrate that acti- vation of the Wnt/-catenin pathway contributed to benzene- induced hematotoxicity. It has been reported that regulation of -catenin is tightly associated with stress-induced post- translational modifications, including phosphorylation (42). -Catenin was sequentially phosphorylated at Ser33-Ser37- Thr41 by CKI and GSK3, leading to degradation via the ubiquitination/proteasome machinery (43). In contrast, phos- phorylation of -catenin at Ser675 promoted the nuclear accumulation of -catenin and activation of target gene tran- In summary, we identified a novel pathway and key targets involved in benzene-induced hematotoxicity. Deletion of the Ppp2r1a gene in murine hepatocytes attenuated ben- zene-induced hematotoxicity by suppressing cyp2e1 tran- scription. Specific PP2A complexes containing the B56 subunit regulated Cyp2e1 expression through dephosphor- ylation of -catenin at Ser33-Ser37-Thr41. These findings shed light on our understanding of the signaling pathway by which specific protein phosphatase regulates gene expres- sion and cellular functions in response to environmental stress. 2495 J. Biol. Chem. (2019) 294(7) 2486–2499 Stable cell line establishment and cell treatment To create stable AML-12 and HepG2 cells expressing shRNAs against PP2A A or B subunit, pLKO-shRNAs (14) were introduced into AML-12 or HepG2 cells by lentiviral infection and selected with puromycin (1 g/ml). For the induction of Cyp2e1 expression, HepG2 cells were seeded in 6-cm plates with a density of 8 105 and treated with three types of Cyp2e1 agonists for 24 h. The chemicals and their con- centrations were: ethanol (0, 100, 200, and 400 mM), acetamin- ophen (0, 1.25, 2.5, and 5 M), and hydroquinone (0, 6.25, 12.5, and 25 M). Hematopoietic colony assays The colony forming cell assay was performed to assess the ability of hematopoietic progenitors to proliferate and differen- tiate into colonies in response to benzene. Briefly, BM cells from mice femur and tibia were flushed with mPBS buffer (2% fetal bovine serum in PBS) and filtered with a 70-m cell strainer (BD Pharmingen, San Jose, CA). RBC-depleted bone marrow cells were plated at a final concentration of 5 105 cells/ml in triplicate and cultured in methylcellulose-based semisolid culture medium (R&D Systems) in an atmosphere of 37 °C with 5% CO2. 14 days after incubation, the colonies formed were viewed and scored under an inverted microscope. Hematopoietic colonies were classified as CFU-E (erythroid colonies), CFU-GM (mixed colonies of granulocytic and macrophage), CFU-G (pure granulocytic colonies), and CFU- GEMM (mixed type of colonies containing both the erythroid and myeloid cells). J. Biol. Chem. (2019) 294(7) 2486–2499 Animals Urinary SPMA was measured according to a previously described protocol, with minor modifications (49). Briefly, the urine samples were thawed, vortexed, and centrifuged at 800 g for 5 min and the supernatant was collected. Then, 0.5 ml of 10 mM sodium acetate buffer (pH 6.3) was mixed with 0.5 ml of supernatant, followed by the addition of 50 l of 10 g/liter of SPMA-d5 working solution. After solid phase extraction (Waters Oasis MAX, Milford, MA), samples were analyzed by LC/electrospray tandem MS (LC-MS/MS) with a 0.01 g/liter limit of detection. The urinary SPMA was normalized to uri- nary creatinine and expressed as g/g of creatinine. In this study, male mice with homozygous hepatocyte-spe- cific deletion of the Ppp2r1a (encoding PP2A A) gene were generated by mating Ppp2r1aflox/flox mice (purchased from the Jackson Laboratory) with Alb-Cre mice. Ppp2r1aflox/flox mice have a mixed FVB/NJ and 129S4/SvJae genomic background, whereas Alb-Cre mice have a C57BL/6J genomic background. Ppp2r1aflox/flox; Alb/ mice are hereafter termed HO mice. Ppp2r1aflox/flox; Alb / or Ppp2r1a/; Alb/ mice obtained from the same breeding were used as controls and termed wild- type (WT) mice. All animal protocols were approved by the Animal Care and Use Committee of the Model Animal Research Center of Sun Yat-sen University. Animal treatments Benzene (Sigma) was diluted in corn oil (Sigma). Male WT and HO mice (8 weeks old) were randomly divided into four groups and treated with benzene by dynamic inhalation or oral gavage. For inhalation exposure, the dose of benzene employed was 1, 10, and 100 ppm. The duration of exposure was 28 days, at 6 h/day and 6 days/week. The benzene aerosol was generated by a Permease Permeater device (PD-1B, GASTEC CORP, Ayase, Japan) connected with filter-dried air as carrier gas in a 300-liter compressed gas cylinder. The generated aerosol was imported into the whole-body inhalation chambers (Guang- zhou JIUFANG Electronics Co., Ltd., Guangzhou, China). The benzene concentrations in exposure chambers were monitored every 2 h and analyzed by GC-MS (GC-MS) (Agilent Technol- ogies, Santa Clara, CA). The daily mean concentration of ben- zene is shown in Fig. S1. In addition, WT and HO mice were administered benzene by oral gavage in a volume of 10 ml/kg/ body weight at doses of 1, 10, and 100 mg/kg, which was equiv- alent to 0, 0.108, 1.08, 10.8, and 108 ppm at an estimated inha- lation exposure level (8-h total weight average) (U.S. EPA 1999), 6 days/week for 28 days. More than 8 mice per group were used for each experiment. The body weight of mice was recorded once a week during the period of benzene exposure. To clarify the role of -catenin in benzene-induced hematotoxicity, 24 male WT mice were divided into 4 groups and treated with: 1) corn oil, 2) 100 mg/kg of benzene, 3) 5 mg/kg of XAV-939, and 4) 100 mg/kg of benzene, and 5 mg/kg of XAV-939, respec- tively, for 7 days. Author contributions—L. C., X. Li, Z. He, and W. C. funding acqui- sition; L. C. writing-original draft; L. C., X. Li, and W. C. project administration; P. G., J. F., F. W., and S. W. data curation; P. G., H. Z., W. L., C. G., J. F., Y. Z., X. Liu, F. W., S. W., Q. Li, Z. He, H. L., S. C., X. W., L. Y., and Q.Li investigation; P. G., W. L., C. G., Z. Huang, Y. Z., and X. Li methodology; J. F. formal analysis; H. T., Q. W., G. D., and D. L. conceptualization; Y. X., W. C., and D. L. supervision; W. C. writing-review and editing. Immunohistochemistry analysis Mouse livers were fixed in 4% buffered paraformaldehyde phosphate for 24 h, followed by decalcification in 10% EDTA for 2 weeks, then dehydrated, embedded in paraffin, and sec- tioned at 5-m thick slides. The slides were incubated with primary antibodies against Cyp2e1 (1:150, Proteintech Group). Quantification of fluorescence was performed using ImageJ software (National Institute of Mental Health, Bethesda, MD) as observed and depicted as percent of relative expression. PP2A regulates benzene-induced hematotoxicity prewashed 1:1 slurry of protein G-Sepharose was added to the mixture and incubated for an additional 2 h. The protein G beads–protein complexes were washed three times and eluted in 2 SDS loading buffer, followed by SDS-PAGE and immunoblotting. mRNA expression. The primers for amplifying metabolic enzyme genes are detailed in Table S2. Neutral Comet assay 5 l of whole blood was mixed with 200 l of 0.8% low- melting point agarose and spread onto a CometAssay HT 20-well slide (Trevigen). Slides were subsequently immersed in a pre-cooled lysis buffer (2 M NaCl, 30 mM Na2EDTA, 10 mM Tris-HCl, 1% Triton X-100, and 10% DMSO, pH 8.0) at 4 °C for 1 h. Next, the slides were subjected to electrophoresis, neutral- ization, dehydration, and staining, and viewed under a fluores- cence microscope (Nikon Eclipse Ti-E). 150 lymphocytes ran- domly selected per slide were scored by Comet Assay Software Project 1.2.2 (University of Wroclaw, Poland) for each sample. Olive tail moment was selected as the parameter for assessing the degree of DNA damage. Real-time breath analysis of exhaled benzene and its metabolites Exhaled benzene in mouse breath was measured according to a protocol previously described (51). In brief, 8-week-old male mice obtained from the same breeding with the same approximate body weight were treated with benzene at a dose of 100 mg/kg via oral administration and immediately placed in a 50-ml Falcon conical centrifuge tube ( 25 °C, 40% RH). The mouse exhalation was collected through PTFE tubing (4 mm inner diameter) by carrier gas (indoor air) at 1 liter/min into a device equipped with a membrane inlet single photon ionization TOF-MS(MI-SPI-TOF-MS).Massspectrumdatawereprocessed with SPIMS_V3.05 software and two-dimensional data with stan- dardized m/z intensity and time were obtained. Exhaled benzene and its metabolites were analyzed and determined by charge-to- mass ratio (m/z) and reported in Table S3. Micronucleus assay After mice were sacrificed, bone marrow smears were imme- diately made and stained with Giemsa. All slides were scored blindly. 1000 cells for each subject were examined for MN according to the criteria in the HUMN Project (50) under a microscope. Statistical analysis Data are shown as the mean S.D. All statistical analysis was performed using SPSS 20.0 statistical software (IBM, New York) and GraphPad Prism Software (La Jolla, CA). Differences of continuous variables (peripheral blood count, colony form- ing cell count, and gene expression level) between the groups were analyzed by independent-sample t test or assessed with one-way ANOVA followed by Bonferroni post-test. Kruskal- Wallis H test was used for statistical analysis of SPMA, MDA, Comet assay, and the Mann-Whitney U test was used for pair- wise comparisons. Differences were considered statistically sig- nificant at p 0.05. Ex vivo assay for measurement of MDA and cytotoxicity in HL60 or HepG2 cells 3 104 HL60 cells/well were seeded onto a 96-well plate in quadruplicate for 2 h and incubated with medium containing 2% plasma isolated from the benzene-treated mice or HQ for 24 h. Cell Proliferation kit (WST-1) was used for the cell viability assay according to the manufacturer’s protocol. MDA concentration was measured using the Lipid Peroxidation (MDA) Assay Kit (Beyotime, Nantong, China). After harvesting, the cell pellet was resuspended with ice-cold RIPA lysis buffer, followed by the addi- tionof200lofthiobarbituricacidreactionsolution.Themixture was incubated in a water bath at 100 °C for 15 min and the super- natant was collected and subjected to absorbance detection at 532 nm using a Microplate Spectrophotometer (BIOTEC, USA). MDA content was expressed as micromole/g of protein. Laser scanning confocal microscopy analysis HepG2 cells (1 105) were seeded and grown overnight on coverslips and fixed in 3.7% formaldehyde for 15 min and per- meabilized in 0.2% Triton X-100. The cells were then incubated in a blocking solution (3% FBS in PBS) for 30 min at 37 °C, followed by incubation with specific anti--catenin antibody (1:500) overnight. Alexa Fluor 488-conjugated IgG second anti- body (1:1000) was incubated for 1 h. The slides were counter- stained with 4,6-diamidino-2-phenylindole (1 g/ml) and observed under a LSM510 META laser scanning confocal microscope (Leica TCS SP5). Peripheral blood analysis Upon sacrifice, liver tissue was excised and homogenized in ice-cold RIPA lysis buffer (150 mmol/liter of NaCl, 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS, and 50 mM Tris (pH 7.4)) containing protease inhibitors. The lysates were centrifuged at 12,000 g for 20 min at 4 °C. Soluble proteins (80 g) were subjected to 4–12% gradient acrylamide gel for SDS-PAGE before immunoblotting. The following antibodies were used: mouse anti-PP2A C (1D6) (Upstate Biotechnology), rabbit anti-A, and rabbit anti--catenin, -catenin (Ser33/Ser37/ Thr41), and -catenin (Ser675) (Cell Signaling Technology), and rabbit anti-Cyp2e1 (Proteintech Group, Chicago, IL). Mice were injected with 10% pentobarbital sodium before being sacrificed. Peripheral blood was collected from the infe- rior vena cava and analyzed in a Sysmex XE-2100 automatic hematology analyzer (Sysmex, Kobe, Japan). The peripheral blood counts (RBC, hemoglobin; hematocrit; mean corpuscular volume (MCV); average concentration of hemoglobin (MCH); mean corpuscular hemoglobin concentration (MCHC); plate- lets; reticulocytes; WBC; LYM; neutrophils; monocytes; and eosinophils) were determined. After peripheral blood analysis, plasma was isolated from whole blood by centrifugation at 450 g for 10 min at room temperature and stored at 80 °C before use. For immunoprecipitation analysis, 3 mg of proteins were incubated with specific antibodies overnight at 4 °C. 100 l of 2496 J. Biol. Chem. (2019) 294(7) 2486–2499 Quantitative real-time PCR analysis Total RNA was isolated using TRIzol Reagent (Invitrogen), and reverse transcription was carried out using the Prime- ScriptTM II First Strand cDNA synthesis kit (Takara Bio, Inc., Tokyo, Japan) according to the manufacturers’ instructions. The mRNA expression levels were examined using Toyobo Real-time PCR Master Mix (TOYOBO, Osaka, Japan) and ana- lyzed using the Applied Biosystems 7500 Real-time PCR Sys- tem. Actin was used as an internal control to determine relative References 18. Zhou, J., Pham, H. T., Ruediger, R., and Walter, G. (2003) Characterization of the A and A subunit isoforms of protein phosphatase 2A: differences in expression, subunit interaction, and evolution. Biochem. J. 369, 387–398 CrossRef Medline 1. Snyder, R. (2012) Leukemia and benzene. Int. J. Environ. Res. Public Health 9, 2875–2893 CrossRef Medline 2. Vlaanderen, J., Lan, Q., Kromhout, H., Rothman, N., and Vermeulen, R. 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https://openalex.org/W4379016001	https://www.researchsquare.com/article/rs-1850734/latest.pdf	English	null	Coexistence of Tree Species Promotes the Similarity of the Elementome in Soil Profiles	Journal of soil science and plant nutrition	2,023	cc-by	9,010	Coexistence of tree species pr of elementome in soil profiles Xiaochang Wu Huayong Zhang (  rceens@ncepu.edu.cn ) Tousheng Huang Chengfeng Yu Shijia Zhang Yonglan Tian Research Article License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License License:   This work is licensed under a Creative Commons Attribution 4.0 International License. R d F ll Li Page 1/21 Page 1/21 Abstract Purpose: The soil elementome has been recently proposed as a promising novel approach for describing the response of soil bioelemental composition to tree species. Using bioelement stoichiometry, we explored the effects of soil biogeochemical processes and tree species coexistence on soil elementome. Methods: Soil bioelements were analyzed at three soil layers (A, B and C horizons) in four forests (Picea asperata (PA), Larix principis-rupprechtii (LP), Betula Platyphylla (BP), and Betula Platyphylla - Larix principis-rupprechtii (L-B) mixed forest) in Chongli District, Zhangjiakou City, Hebei Province, China., The soil elementomes of 11 bioelements (C, N, P, O, S, K, Ca, Na, Mg, Mn and Cr) were analyzed by principal component analysis (PCA) based on soil C:N:P stoichiometry. We calculated elementome distance (ED) to examine differences between soil horizons and forests. Results: We found that soil elementomes in the soil layers decreased with depth and that ED between the B and C horizons was larger than that between A and B horizons. Moreover, differences in soil elementomes were smaller for tree species that often coexist compared to those that rarely live together. Conclusions: Our results suggest that tree species coexistence promotes similarity in soil elementomes probably due to creation of similar soil conditions. The findings of this research provide a new understanding about the relationship between tree species coexistence and soil bioelemental composition or stoichiometry. Introduction Concentrations and ratios of soil C, N, and P in terrestrial ecosystems were used to indicate plant physiology, nutrient cycles, and nutrient limitations in ecosystem productivity (Deng et al. 2015; Kirkby et al. 2011; Xu et al. 2015); for instance, C:N in soil or litter was a quality indicator of organic matter (Ostrowska and Porębska 2015), and N:P was related to nutrient constraints in ecosystems (Bui and Henderson 2013). In recent decades, the soil ecological stoichiometry of forest ecosystems has also attracted much attention. Aponte et al. (2010) investigated the stoichiometry of C, N, and P in the soil of Mediterranean forests and found that vegetation and soil depth simultaneously regulated C:N:P stoichiometry. Tian et al. (2010) reported that the soil C:N, C:P, and N:P ratios in China were 11.9, 61, and 5.2, respectively, and that the ratios of C:N:P and especially the C:N ratios were relatively consistent in the topsoil (0–10 cm). Fan et al. (2015) found that soil C and P decreased with the age of trees, and the plant N:P ratio was strongly related to the soil N:P ratios in subtropical plantations in Fujian Province, China. However, an elementome based on C, N, and P only may miss key information that could be offered by additional elements (Kaspari 2021). Beyond C, N, and P, bioelements such as potassium (K), sodium (Na), magnesium (Mg), calcium (Ca), manganese (Mn), and chromium (Cr) have specific functions even though they are normally present at low concentrations in the environment. For instance, the compositions and ratios of K are related to drought resistance, Mg to the light environment, and K, Ca, Mg, and S to the levels of N and S deposition (Rivas-Ubach et al. 2012; Sardans et al. 2016; Sardans et al. 2011). Introducing additional elements in the analysis of the elementome can effectively improve the prediction of ecosystem functioning (Hofmann et al. 2021; Huang et al. 2019) and should be considered as an entirety in ecological stoichiometric studies. One efficient way to deal with the entirety of bioelements was to perform a dimensionality reduction analysis such as principal components analysis (PCA) (Peñuelas et al. 2019; Sardans et al. 2021). The “biogeochemical niche” (BN) concept, which is an elementome defined as the content of all (or most) bioelements has been recently proposed (Peñuelas et al. 2019). Introduction All living beings are made of various bioelements in constant ratios (Zhang et al. 2012). Carbon (C), hydrogen (H), and oxygen (O) are the three bioelements that constitute the skeleton of organic molecules. Nitrogen (N), phosphorus (P), and sulfur (S) are the main bioelements involved in biochemical reactions. The stable concentrations and ratios of bioelements in organisms are important indicators for understanding the balance of multiple chemical elements in ecological interactions (Elser et al. 2000a; Elser et al. 2000b; Sardans et al. 2014). In 1958, Redfield (1960) reported that planktonic biomass contains C, N, and P in a comparatively steady atomic ratio of 106:16:1, similar to the proportions of C, N, and P in marine water. The well-known “Redfield ratio” provides promising insight into the nutrient limitation of ocean C storage and contributes to the knowledge of the biogeochemical cycling of N and P in the world’s oceans (Cooper et al. 1996). Afterwards, the study of ecological stoichiometry, which focuses on the balance of multiple chemical elements in ecological interactions, has become a fundamental feature of understanding biogeochemical cycles (Sterner and Elser 2002). The elegant simplicity of the Redfield ratio prompted the research on searching for similar patterns and relationships in terrestrial ecosystems (Elser and Hassett 1994). McGroddy et al. (2004) reported that C:N:P in plant rootlets at a global scale was 1157:24:1, and the ratios of tree leaves and litters were highly variable. Conversely, Han et al. (2005) studied 753 terrestrial plant species in China and found that the N:P of grass leaves was stable at approximately 14.4. Parallel interactions existed between the terrestrial Page 2/21 Page 2/21 environment and vegetation, resulting in similar ratios of C:N:P in soils and plants (McGroddy et al. 2004; Reich and Oleksyn 2004). Cleveland and Liptzin (2007) found that C:N:P ratios in soils and biomass were 186:13:1 and 60:7:1 at the global scale and concluded that a relationship existed between C, N and P in soils, which was similar to the concept of the Redfield ratio. environment and vegetation, resulting in similar ratios of C:N:P in soils and plants (McGroddy et al. 2004; Reich and Oleksyn 2004). Cleveland and Liptzin (2007) found that C:N:P ratios in soils and biomass were 186:13:1 and 60:7:1 at the global scale and concluded that a relationship existed between C, N and P in soils, which was similar to the concept of the Redfield ratio. Introduction The objectives of this study were (1) determining the distribution of C:N:P ratio and concentrations of bioelements C, N, O, S, P, K, Ca, Na, Mg, Mn and Cr, and (2) investigating the soil profiles of different horizons and forests types and their relationships with soil elementomes. We suppose that: (1) the extent soil elementome was affected by biological and chemical processes decreased with the soil depth, and (2) coexisting of tree species reduced the differences in the soil elementome of the forests. Study area The study area is located in Chongli District, Zhangjiakou City, Hebei Province, P.R. China. The latitudes and longitudes of Chongli are 40°47' N to 41°17' N and 114°17'E to 115°34' E. The altitude extends from 814 to 2174 meters. The climate is classified as continental monsoon with average annual temperatures of 3.7 - 19°C and annual precipitation of 483.3 mm. Eighty percent of the territory in Chongli is mountainous, and the forest coverage rate reaches 67% in 2021 The main tree species are Picea asperata, Larix principis-rupprechtii, and Betula platyphylla, among which Picea asperata and Larix principis-rupprechtii are artificially planted. Introduction It is effective in measuring ecological stoichiometry and can be regarded as an extension of the ecological niche concept. Peñuelas et al. (2019) investigated tree species in a holm-oak evergreen Mediterranean forest distribution and found that species with more overlapping ecological niches had greater differences in their BNs. Plants growing in diverse communities tended to change their elemental compositions to either reduce or enhance N and P concentrations depending on the species compared to monocultures (Dehuang et al. 2020; Guiz et al. 2018). Fernández-Martínez et al. (2021) further revealed that pairwise differences in elementomes between species were large as the possibility of coexistence increased. Based on these empirical approaches, it was hypothesized that “at equilibrium, coexisting species tend to have distinct elementomes to minimize competitive pressure” (Sardans et al. 2021). This BN hypothesis suggested that each species would have a specific need for certain bioelement to avoid nutritional competition with Page 3/21 Page 3/21 other species. In a forest, the fierce competition between species under similar soil properties would resulting in remarkable differences in the plant elementome with their likelihood of coexistence (Bai et al. 2018; Fernández-Martínez et al. 2021). Considering that coexisting species are able to adjust ecological strategies by competing for soil resources, it can be further hypothesized that the similarity of the soil elementome leads to elementome segregation for coexisting species in competition. In this study, we assessed the soil stoichiometry of different soil horizons in four different forests, i.e. Picea asperata, Larix principis-rupprechtii, Betula platyphylla and a mixture of Betula platyphylla-Larix principis-rupprechtii in Hebei Province, China. The objectives of this study were (1) determining the distribution of C:N:P ratio and concentrations of bioelements C, N, O, S, P, K, Ca, Na, Mg, Mn and Cr, and (2) investigating the soil profiles of different horizons and forests types and their relationships with soil elementomes. We suppose that: (1) the extent soil elementome was affected by biological and chemical processes decreased with the soil depth, and (2) coexisting of tree species reduced the differences in the soil elementome of the forests. In this study, we assessed the soil stoichiometry of different soil horizons in four different forests, i.e. Picea asperata, Larix principis-rupprechtii, Betula platyphylla and a mixture of Betula platyphylla-Larix principis-rupprechtii in Hebei Province, China. Soil sample collection and chemical analyses In July 2019, four different sampling plots in the forests of Picea asperata (PA), Larix principis- rupprechtii (LP), Betula platyphylla (BP) and the mixed forest of Betula platyphylla and Larix principis- rupprechtii (B-L) were selected in the study area. The four plots were all on mountainous slopes, and the size of each plot was set as 100 m × 100 m. In each plot, three quadrate subplots (20 m × 20 m) were uniformly arranged from the bottom to the top of the slope. One sampling point was set in the center of each subplot. The steps of sample collection were as follows: first, the surface coverages of litter and other sundry were removed from the sampling points; second, a vertical soil profile of approximately 1 m depth was dug using shovels, and the soil profile was found to be three soil formation layers (A, B and C horizons) according to the soil textures; finally, in each soil layer, two samples (each approximately 100 cm3) were collected with a ring knife to analyze the soil physicochemical properties and bioelements. Samples were put into sealed bags and brought to the laboratory for analysis. according to the soil textures; finally, in each soil layer, two samples (each approximately 100 cm3) were collected with a ring knife to analyze the soil physicochemical properties and bioelements. Samples were put into sealed bags and brought to the laboratory for analysis. Page 4/21 Page 4/21 Page 4/21 Soil pH was measured using a pH meter (type:PHS-3Cby China) with the soil and water ratio as 2.5:1. Soil bulk density (BD) was measured using the ring knife method. Soil organic matter (SOM) was determined by the external heating method of potassium dichromate and concentrated sulfuric acid (Liu et al. 1996). Purging and trapping techniques were used to determine O, N, and S concentration by an elemental analyzer (type: Elementar Vario Macro cube by Germany). The total concentrations of several nutrients (P, K, Ca, Na, Mg, Mn, and Cr) in soils were determined by inductively coupled plasma‒optical emission spectrometry (Bremner. 1996). (type: Agilent 5110 ICP‒OES by the USA). Table 1 summarizes the results of the soil chemical and physical properties. Soil sample collection and chemical analyses hemical and physical properties Forest Horizon Bulk density Specific gravity pH(H2O) SOM TN g/cm3 % g/kg PA A 0.85 2.14 6.74 70.7 3.60 B 1.02 2.08 6.65 67.0 3.34 C 1.28 2.22 6.72 49.0 2.46 LP A 1.09 2.05 6.83 53.7 2.85 B 1.12 2.09 7.00 47.3 2.53 C 1.32 2.26 7.01 40.0 2.07 BP A 1.05 2.18 6.84 45.9 2.61 B 1.19 2.26 6.91 31.4 1.78 C 1.65 2.42 7.00 13.0 0.71 B-L A 0.79 2.05 6.40 85.9 4.35 B 0.87 2.04 6.63 68.2 3.41 C 1.13 2.16 6.34 43.2 2.35 anic matter; TN: total nitrogen; PA: Picea asperata; LP: Larix principis-rupprech B: Betula Platyphylla - Larix principis-rupprechtii mixed forest Table 1. Soil chemical and physical properties Table 1. Soil chemical and physical properties SOM: soil organic matter; TN: total nitrogen; PA: Picea asperata; LP: Larix principis-rupprechtii; BP: Betula Platyphylla; L-B: Betula Platyphylla - Larix principis-rupprechtii mixed forest Statistical analysis Statistical analysis All data in this study were described by the mean and standard deviation. SOM and elemental concentrations were described by mass content, the values of C:N, C:P, and N:P were molar ratios. A significance level of p<0.05 was specified in this study. Analysis of variance (ANOVA) and Least significant difference (LSD) at a 5% level of significance were used to compare the difference among Page 5/21 horizon and forest.PCA was performed on elemental concentrations to estimate elementomes. Elementome Euclidean distance (ED) was used to quantitatively indicate the difference between elementomes. p elementomes. Elementome Euclidean distance (ED) was used to quantitatively indicate the difference between elementomes. elementomes. Elementome Euclidean distance (ED) was used to quantitatively indicate the difference between elementomes. All statistical analyses mentioned above were implemented using SPSS 25.0 (IBM, Armonk, New York, NY, USA). Related graphs were drawn by Origin 2021b (Hampton, MA, USA). Biological and chemical processes within the soil profile (Figure 3) were generated in BioRender with authorization. Soil C, N, P stoichiometry of different forests The soil chemical and physical properties, which varied greatly with soil depth across all sampling plots. BD was significantly influenced by soil horizons, whereas pH was not significantly affected. SOM and N concentrations were the highest in the A horizon (P < 0.05) and declined with soil depth. Comparing the data in different forests, it was found that have SOMB-L＞SOMBP＞SOMLP＞SOMPA and the same trend for the N concentration. il C:N, N:P, and C:P in different forest types of this study Table 2. Soil C:N, N:P, and C:P in different forest types of this study Forest Horizon C:N C:P N:P C:N:P PA A 12.15±1.04Ba 134.77±17.98Ba 11.26±2.36ABa 137:11:1 B 12.12±1.47Ba 97.85±22.29Bb 8.14±1.80Bb 99:08:1 C 12.55±1.24Aa 54.66±18.20Cc 4.32±1.24Cc 54:04:1 LP A 12.74±0.42ABa 127.64±25.50Ba 9.99±1.71Ba 127:10:1 B 12.67±0.44ABa 103.53±7.33Bb 8.18±0.55Bb 104:08:1 C 13.39±1.42Aa 96.65±15.26Bb 7.32±1.50Bb 98:07:1 BP A 13.32±0.38Aa 166.86±13.18Aa 12.55±1.8Aa 167:13:1 B 13.50±0.23Aa 146.47±17.01Ab 10.85±1.24Ab 146:11:1 C 13.50±0.39Aa 148.73±8.99Ab 11.03±0.91Ab 149:11:1 B-L A 13.08±0.53Aab 123.51±18.59Ba 9.43±1.17Ba 123:9:1 B 12.73±0.67ABb 103.48±16.42Ba 8.10±1.00Ba 103:8:1 C 13.64±0.59Aa 115.97±39.24Ba 8.46±2.75Ba 115:8:1 e: Different uppercases mean the significant differences between forests (p <0.05); different ercases mean the significant difference between soil horizons p <0.05. Note: Different uppercases mean the significant differences between forests (p <0.05); different lowercases mean the significant difference between soil horizons p <0.05. Note: Different uppercases mean the significant differences between forests (p <0.05); different lowercases mean the significant difference between soil horizons p <0.05. The C:N ratios did not differ significantly between the PA, LP, and BP forests during the three horizons (Table 2) and were significantly lower in the soil A and B horizons than in the C horizon of the B-L mixed forest. All forests showed no significant differences in the C:N of the C horizon. A lower C:N ratio was observed in PA surface soil than that in BP and B-L mixed forests. As the soil depth increased, the C:P and N:P ratios decreased. BP forest obtained highest C:P and N:P ratios. There was no significant differences in the C:P and N:P ratios between the A and B horizons of other three forests. PA forests had the lowest ratios of C:P and N:P at the C horizon. Overall, the SOM in broad-leafed forests (BP) was higher than that in coniferous forests (PA and LP). Soil C, N, P stoichiometry of different forests In addition, the soil C:N, C:P and N:P ratios were higher in the B-L mixed forest than in the Larix principis-rupprechtii monoculture, indicating that mixed forest can effectively enhance soil organic matter quality in Larix forest. Compared to the average N:P and C:N ratios in China (13.83 and 8.43), all four forests had lower soil N:P ratios (4.32 - 12.55) and higher C:N ratios (12.12 - 13.50) (Tian et al. 2010). In general, when N:P was less than 14:1, plant growth was more restricted by N; when N:P was higher than 16:1, plant productivity was more restricted by P; and when N:P was in the middle, plant growth was restricted by both nitrogen and phosphorus (Olde Venterink et al. 2003). Our study found that soil N in BP, LP, PA, and B-L mixed forests was all N-limited for plant growth. Soil elementome distribution from PCA The distribution of elementomes analyzed by PCA method are shown in Figure 2. Three principal components was able to explain a total of 86.05% of the variance. According to the results, loading values and explained variance were mapped to each component after PCA. PC1 accounted for 52.46% of the total variance and was significantly correlated to C, N, O, S, and P contents. Thus, PC1 remarkable described the biological elements, i.e. C, N, O, S, and P, which are indispensable nutrients for the growth and development of all plants in forest ecosystems. PC2 explained 22.17% of the variance in the original data, with K, Ca, Na, and Mg having the major loadings. These elements are nutrient cations that are subjected to biological activity and chemical activity to maintain their normal growth. Accounting for 11.42% of the variance, PC3 substantially described the contents of Mn and Cr in the study area. This component can be described as soil bedrock which is the main influencer of these elements. Soil elementome differences between horizons and forests Soil elementome differences between horizons and forests The elementome distances (ED) between horizons in four forests (Picea asperata, Larix principis- rupprechtii, Betula platyphylla and Betula platyphylla - Larix principis-rupprechtii mixed forest) in this study were calculated and shown in Figure 3. Page 7/21 Page 7/21 As with the genome, the soil elementomes can represent the state of soil development. The soil elementomes were defined as the element concentrations in soil (Fernández-Martínez et al. 2019). In all four forests, elementomes decreased along with the depth of the soil. The elementome distances (ED) between the B and C horizons were larger than the ED between A and B horizons (Figure 3), among which EDBC accounted for 61%~91% of the entire soil profile. In comparison with EDBC, the proportion of EDAB was as low as 9%~39%, which showed a larger difference in the bottom two horizons. Soil is formed by the interaction of geological and biological cycles (Chen et al. 2014). Biological and chemical processes take place throughout the soil profile, interacting at a wide range of temporal scales and together driving the elemental cycle of the soil profile (Kirkby 2018). Our results show that the biological cycle was more vigorous than the chemical cycle, and soil elementomes were more affected by the biological activity rather than the bedrock. Organisms played an important role in soil ecosystem balance and stability as the most active factors in soil formation. We found that soils of different forest had different elementomes. In mixed forests of Betula platyphylla and Larix principis-rupprechtii, the soil elementomes were higher than those in pure forests (Figure 4A). Among all forests, Picea asperata had the lowest soil elementomes. Successive plantation planting can degrade forest soil fertility, and nutrient accumulation can be effectively increased by mixed needle and broad-leaved planting. Based on the forest survey, we can obtain the distribution and coexistence situation of tree species. We found that species rarely living together show larger differences in soil elementomes than those that frequently coexist (Figure 4B).The highest elementome distance (ED) value, 1.69, appeared between Picea asperata and Betula Platyphyllaplatyphylla, and the lowest ED value, 0.53, appeared between Picea asperata and Larix principis-rupprechtii. Effects Of Forests On Soil C, N, P Stoichiometry The proportional relationship between C, N, and P is an important indicator of soil nutrient status (Wang et al. 2021). The C:N and C:P ratios of soil determined the decomposition of SOM, whereas the N:P ratio reflected the element restriction of ecosystem (Hui et al. 2021). In this study, we found that the SOM in the broad-leaved forest was better than that in the coniferous forest. According to previous studies, dissolved organic matter in forest soil was mainly formed by litter decomposition and plant root exudates (Goller et al. 2006; Huang and Schoenau 1998). Therefore, a high level of SOM was associated with broad-leaved trees which suggested a high litter decomposition rate. Some research found that keratin prevented microorganisms from adhering and invading leaves with high keratin content, thus causing the slow decomposition of leaves with high keratin content (Garnier and Laurent 1994). As a consequence, in the present study, the litter decomposition rate of Betula platyphylla was significantly higher than that of pure Larix principis-rupprechtii. Page 8/21 Soil C:N:P stoichiometry in the B-L mixed forest was higher than in the LP monoculture. More studies found that a mixed forest composed of multiple tree species was stronger in soil nutrient protection, which was mainly based on three theories, namely, the natural enemy hypothesis (R. 1973), resource- concentration hypothesis (Freney 1986) and associational resistance hypothesis (Hambäck et al. 2000). In mixed forests, chemical differences in litter, the transfer of nutrients and secondary metabolites between litter, and variations in the microhabitat of decomposers led to the accelerated decomposition of mixed litters (Gartner and Cardon 2004; Song et al. 2010). Our study provided evidence that stand conversion from BP to B-L mixed culture substantially improved soil quality. Soil C:N:P stoichiometry in the B-L mixed forest was higher than in the LP monoculture. More studies found that a mixed forest composed of multiple tree species was stronger in soil nutrient protection, which was mainly based on three theories, namely, the natural enemy hypothesis (R. 1973), resource- concentration hypothesis (Freney 1986) and associational resistance hypothesis (Hambäck et al. 2000). In mixed forests, chemical differences in litter, the transfer of nutrients and secondary metabolites between litter, and variations in the microhabitat of decomposers led to the accelerated decomposition of mixed litters (Gartner and Cardon 2004; Song et al. 2010). Our study provided evidence that stand conversion from BP to B-L mixed culture substantially improved soil quality. Effects Of Forests On Soil C, N, P Stoichiometry In our results, the stoichiometric ratios of soil C, N, and P varied dramatically from forest to forest. As reported by Xu (2012), soil C:N:P ratios ranged from 64:5:1 to 1347:72:1, with an average of 287:17:1. According to Tian (2010), the average C:N:P ratio of China’s soils was 60:5:1. The results we obtained were within these reported ranges. We found that the C:N:P ratios decreased with soil depth, consistent with some previous reports such as Bing et al. (2015) reported that C:N:P ratios decreased from 343:16:1 in the A horizon to 63:3:1 in the C horizon. Elementomes Between Horizons And Mechanisms Of Soil Formation The adsorption, analysis, decomposition, and aggregation of various elements in soil constitute the biogeochemical cycle of the soil environment (Chen et al. 2014). The biological process is mainly composed of two parts: biological residue is decomposed into inorganic compounds via humification and mineralization by soil microorganisms, and living organisms absorb soil elements. In geological processes, leaching and diagenesis fix soil elements into bedrock while weathering releases them (Banfield et al. 1999; Waroszewski et al. 2019). The study of soil formation and classification has made significant advances since the mid-1800s, evolving from conceptual frameworks to descriptive studies and finally to more quantitative approaches (Hartemink and Bockheim 2013). Schaetzl et al. (2013) proposed that soil formation was directly affected by the nature and direction of parent materials. By analyzing the elemental composition and weathering rate of Zr and Ti particles, Anda et al. (2009) concluded that soil profiles can be specifically characterized by different parent materials due to their varying weathering processes and rates. Jackson and Sheldon (1949) addressed the role of tree roots in limestone disintegration. Almeida (1994) examined the ability of higher plants to promote weathering. Plants also changed the weathering process and impacted the nutrient characteristics of the profile (Hasenmueller et al. 2017). examined the ability of higher plants to promote weathering. Plants also changed the weathering process and impacted the nutrient characteristics of the profile (Hasenmueller et al. 2017). Soil geochemical properties are important parameters of soil development. Soil C, N, O, S, and P are major structural components in living organisms and also participate in many biochemical organisms (Melvlle et al. 1971). Moreover, K, Ca, Na and Mg, are essential elements for plant growth. Therefore, the changes in the main mineral elements in the ecosystem and the mechanism of their recycling are important Page 9/21 Page 9/21 contents of the primary succession theory since they represent the main functional process of an ecosystem and determine its pattern. Healthy ecosystems depend heavily on the normal circulation of mineral elements that related to their stability and sustainability (Diaz et al. 2016; Reich and Oleksyn 2004). In addition, manganese (Mn) and chromium (Cr) are also required for normal plant growth and development which cannot be decomposed by soil microorganisms, so they are easy to accumulate. Nevertheless, excessive concentrations of Mn and Cr would be detrimental to plant growth. (Guo et al. 2020; Zemunik et al. 2020). Elementomes Between Horizons And Mechanisms Of Soil Formation The elements in this study were significantly different between soil layers and were decreased with soil depth, in particular, a more significant difference in the bottom two horizons. Soils in different forests showed quite different environments. Based on our results, soil elementomes under different forest types were mainly affected by biological processes. Soil Elementomes Controlled By The Coexistence Of Tree Species Plant species controlled the composition of soil elements (Zederer et al. 2017). Tree species created soil environments that improved their competitive abilities, thus increasing their fitness (Cools et al. 2014). The nutrient content of tree species determined leaf-fall decomposition, nutrient return, and nutrient release into the soil in forests, affecting soil fertility. In many studies, differences in litter lignin and nutrient content were found to influence microbial decomposition, i.e, litters with higher lignin forms decomposed slower, which subsequently affected the soil elements of the forest floor (Hansson et al. 2011; Hobbie et al. 2006; Lovett et al. 2002; Vesterdal et al. 2012). Each species generated soil conditions that reflected the environmental conditions where it dominated, at a local level, with its life history and nutritional strategies (Pérez-Ramos and Marañón 2011; Vivanco and Austin 2008). According to Aponte et al. (2013), tree species-induced variations in soil conditions created positive feedback through niche partitioning that enabled the coexistence of tree species. Species were unique genetic pools and products of long-term evolutionary processes. The genotypic elements shaped coexistence and accounted for a large part of foliar element composition (Sardans et al. 2021). Tree species-induced varaitions in soil nutrient contents influenced elementomes, enabled the separation of biogeochemical niches and maintained their coexistence. According to our results, elementome distances (ED) between PA and BP/LP were more significant than those between BP and LP. Differences in soil elementomes were minor for tree species that often coexist. Numerous studies have demonstrated that elementomes differed more for coexisting species and individuals than for noncoexisting ones (Fernández-Martínez et al. 2021). Additionally, there was evidence that species would compete for resources under similar soil elementomes, causing niche partitioning (Loreau and de Mazancourt 2013) with the likelihood of coexistence. A limited amount of research has focused on applying soil elementomes, and we attempted to analyze soil biogeochemistry through soil elementomes. Page 10/21 Page 10/21 The evolution and bioelemental composition of ecosystems were bidirectional because nutrient supply could affect evolutionary processes and the effects of evolution on nutrient supply (Durston and El- Sabaawi 2017). We can understand the processes underlying species shifts in bioelemental composition by studying their responses to environmental changes (Leal et al. 2017; Yamamichi et al. 2015) and, therefore, the effects of organisms on ecosystem functioning and services (Leal et al. 2017). Soil Elementomes Controlled By The Coexistence Of Tree Species In this way, elementomes constitute a quantifiable tool for detecting, quantifying, and understanding the mechanisms and processes underlying community evolution and species turnover (Peñuelas et al. 2019). Under global change, the study of ecosystem functioning should be based on an elementomes approach. Conclusion In this study, we investigated the soil elementomes in four forests to reveal the effects of species coexistence on soil biogeochemistry. The following results were obtained: (1) The SOM in the broad-leaved forest was better than the coniferous forest. (1) The SOM in the broad-leaved forest was better than the coniferous forest. (2) Soil C, N, and P stoichiometry was higher in the B-L mixed forest than in the Larix principis-rupprechtii monoculture. In mixed forests, chemical differences, transfer of nutrients, and secondary metabolites in litters led to accelerated decomposition of mixed litters. (2) Soil C, N, and P stoichiometry was higher in the B-L mixed forest than in the Larix principis-rupprechtii monoculture. In mixed forests, chemical differences, transfer of nutrients, and secondary metabolites in litters led to accelerated decomposition of mixed litters. (3) Elementome distances (ED) between the B and C horizons were larger than ED between A and B, which indicated that soil elementomes were more affected by biological activity. (4) Differences in soil elementomes were smaller for tree species that often coexist compared to those that rarely live together. Tree species-induced changes in soil nutrient content affected the elementomes and created a soil condition that allowed for biogeochemical niche separation and sustained their coexistence. (4) Differences in soil elementomes were smaller for tree species that often coexist compared to those that rarely live together. Tree species-induced changes in soil nutrient content affected the elementomes and created a soil condition that allowed for biogeochemical niche separation and sustained their coexistence. The results provide implications for understanding of the processes underlying species shifts in soil bioelemental composition and the responses of organisms to environmental changes and, in turn, the effects of organisms on ecosystem functioning and services. Elementomes constitute a quantifiable tool to detect, quantify and thus better comprehend the mechanisms and processes underlying community evolution and species turnover. Further studies are warranted to discern the ecological and evolutionary processes based on an elementomes approach involved in all types of species, habitats, and ecosystems. Acknowledgments We are grateful to Dr. Edmond Sanganyado from Northumbria University for helping polishing the language. 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J Soils Sediments 12:1309–1315. doi: 10.1007/s11368-012-0551-8 Page 17/21 Page 17/21 Figures Figures Figures Figure 1 Study area and soil sampling plots Figure 1 Study area and soil sampling plots Page 18/21 Figure 2 Distribution of elementomes analyzed by PCA method. Biplots showing loadings and mean ± SE scores. Red arrows indicate factor loadings, and blue dots indicate the mean ± SE scores per sample. Figure 2 Distribution of elementomes analyzed by PCA method. Biplots showing loadings and mean ± SE scores. Red arrows indicate factor loadings, and blue dots indicate the mean ± SE scores per sample. Distribution of elementomes analyzed by PCA method. Biplots showing loadings and mean ± SE scores. Red arrows indicate factor loadings, and blue dots indicate the mean ± SE scores per sample. Page 19/21 Page 19/21 Figure 3 Elementome distances between horizons Figure 4 Figure 3 Figure 3 Numbers represent 1, no coexistence; 2, occasional coexistence; 3, frequent coexistence. Based on Wen et al. (2017), Liu et al. (2011) and Kang (2013). Figure 5 Biological and chemical processes within the soil profile Figure 5 Figure 3 Elementome distances between horizons Elementome distances between horizons Figure 4 Figure 4 Page 20/21 Soil elementome segregation and distances among forest species. (A) Soil elementome segregation among forest species. We plotted the soil scores for the first two principal components of the principal component analysis (PCA) conducted with C, N, O, P, S, K, Ca, Na, Mg, Mn, and Cr concentrations as variables. (B) Score distances s for PC1 and PC2 of the PCA of the soil stoichiometry in 3 species, dominant species as a function of the frequency of species coexistence. Numbers represent 1, no coexistence; 2, occasional coexistence; 3, frequent coexistence. Based on Wen et al. (2017), Liu et al. (2011) and Kang (2013). Soil elementome segregation and distances among forest species. (A) Soil elementome segregation among forest species. We plotted the soil scores for the first two principal components of the principal component analysis (PCA) conducted with C, N, O, P, S, K, Ca, Na, Mg, Mn, and Cr concentrations as variables. (B) Score distances s for PC1 and PC2 of the PCA of the soil stoichiometry in 3 species, dominant species as a function of the frequency of species coexistence. Numbers represent 1, no coexistence; 2, occasional coexistence; 3, frequent coexistence. Based on Wen et al. (2017), Liu et al. (2011) and Kang (2013). Soil elementome segregation and distances among forest species. (A) Soil elementome segregation among forest species. We plotted the soil scores for the first two principal components of the principal component analysis (PCA) conducted with C, N, O, P, S, K, Ca, Na, Mg, Mn, and Cr concentrations as variables. (B) Score distances s for PC1 and PC2 of the PCA of the soil stoichiometry in 3 species, dominant species as a function of the frequency of species coexistence. Numbers represent 1, no coexistence; 2, occasional coexistence; 3, frequent coexistence. Based on Wen et al. (2017), Liu et al. (2011) and Kang (2013). Soil elementome segregation and distances among forest species. (A) Soil elementome segregation among forest species. We plotted the soil scores for the first two principal components of the principal component analysis (PCA) conducted with C, N, O, P, S, K, Ca, Na, Mg, Mn, and Cr concentrations as variables. (B) Score distances s for PC1 and PC2 of the PCA of the soil stoichiometry in 3 species, dominant species as a function of the frequency of species coexistence. Figure 5 Biological and chemical processes within the soil profile Page 21/21 Page 21/21
W4242938216.txt	https://alpineentomology.pensoft.net/article/21937/download/pdf/	de		Dr. Gerhard Bächli, Redaktor der Mitteilungen der Schweizerischen Entomologischen Gesellschaft von 1993–2001 und 2005–2016	Alpine Entomology	2,017	cc-by	452	Alpine Entomology 1 2017, 3–4 \| DOI 10.3897/alpento.1.21937 Dr. Gerhard Bächli, Redaktor der Mitteilungen der Schweizerischen Entomologischen Gesellschaft von 1993–2001 und 2005–2016 Daniel Burckhardt1 1 Naturhistorisches Museum, Augustinergasse 2, 4001 Basel, Switzerland http://zoobank.org/73BCE790-6DDB-4F49-BDC8-4C124DDEE001 Corresponding author: Daniel Burckhardt (daniel.burckhardt@bs.ch) Received 30 October 2017 Accepted 7 November 2017 Published 20 November 2017 Academic editor: Thibault Lachat Foto: Florin Rutschmann Dr. Gerhard Bächli, der als langjähriger Redaktor un sere „Mitteilungen“ wie kein anderer geprägt hat, ist auf Ende des letzten Jahres als Redaktor zurückgetreten. Dies ist eine gute Gelegenheit, seine Arbeit zu würdigen und ihm für das Geleistete ganz herzlich zu danken. Gerhard Bächli trat 1970 der SEG bei und arbeitete seit 1993 im Vorstand mit, von 1993–2001 und 2005– 2016 als Redaktor der „Mitteilungen“, von 2002–2005 als Präsident und von 2005–2008 als Vizepräsident. In Anerkennung seiner Verdienste um die Gesellschaft wurde ihm deshalb 2005 die Ehrenmitgliedschaft verlie hen (Merz 2005). Unter der Redaktion von Gerhard Bächli kamen 20 Jahrgänge der Mitteilungen heraus, die auf 7420 Seit en 584 wissenschaftliche Arbeiten, 142 Buchbespre chungen und 46 diverse Beiträge sowie die Berichte der Jahresversammlungen und die Jahresberichte der Sektionen enthalten, eine wahrhafte Titanenarbeit. Die Hefte erschienen immer pünktlich, Hefte 1/2 am 30. Juni und Hefte 3/4 am 31. Dezember; dies ist für wis senschaftliche Zeitschriften nicht selbstverständlich. Im Gegensatz zu früheren Redaktoren erstellte er auch das Layout der einzelnen Hefte selbst, eine sehr zeitauf wendige Arbeit. Die Dokumente, die er der Druckerei übergab, waren druckfertig, was nicht nur die Druck kosten beträchtlich senkte, sondern auch viel Zeit er sparte. Kurz nachdem eine Arbeit angenommen wurde, bekam der Autor auch schon seinen Text im Layout zur Korrektur. Regelmässig hat Gerhard Bächli auch eigene interessante Arbeiten über Drosophiliden und andere Dipteren in den „Mitteilungen“ publiziert, was manch mal half, ein Heft zu füllen, etwa wenn zu wenige Ar beiten vorhanden waren. Als Leser der „Mitteilungen“ durften wir in den letz ten zwei Jahrzehnten qualitativ hochstehende, thema tisch vielfältige und interessante Arbeiten lesen, wofür wir Gerhard Bächli ganz herzlich danken möchten. Copyright Daniel Burckhardt. 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. 4 Burckhardt, D.: Dr. Gerhard Bächli, Redaktor der Mitteilungen der Schweizerischen Entomologischen... Meinen persönlichen Dank möchte ich ihm auch für die sehr angenehme und gute Zusammenarbeit im Vorstand aussprechen, wo er immer hilfsbereit, kompetent und gut vorbereitet war, und bei heiklen Fragen seine grosse Er fahrung einbrachte. alpineentomology.pensoft.net Wir wünschen Ihnen, dass Sie die wohlverdiente Musse beim Studium Ihrer geliebten Fliegen geniessen können. Merz B (2005) Laudatio für Dr. Gerhard Bächli. Mitteilungen der Schweizerischen Entomologischen Gesellschaft 68: 227–228.
https://openalex.org/W4287854877	https://aclanthology.org/2022.semeval-1.216.pdf	English	null	NamedEntityRangers at SemEval-2022 Task 11: Transformer-based Approaches for Multilingual Complex Named Entity Recognition	Proceedings of the 16th International Workshop on Semantic Evaluation (SemEval-2022)	2,022	cc-by	4,070	Proceedings of the 16th International Workshop on Semantic Evaluation (SemEval-2022), pages 1570 - 1575 July 14-15, 2022 ©2022 Association for Computational Linguistics Abstract it allows to test for transfer learning across lan- guages within a single pre-trained model. The dataset, proposed in the shared task, has several unique features previously neglected in NER eval- uation: syntactically complex entities, ambiguous entities, divers, and low-frequency (aka long-tail) entities. This makes the shared task setup closer to real-life settings, where datasets are way noisier and nonuniformly distributed. This paper presents the two submissions of NamedEntityRangers Team to the Multi- CoNER Shared Task, hosted at SemEval-2022. We evaluate two state-of-the-art approaches, of which both utilize pre-trained multi-lingual lan- guage models differently. The first approach follows the token classification schema, in which each token is assigned with a tag. The second approach follows a recent template- free paradigm (Ma et al., 2021), in which an encoder-decoder model translates the input se- quence of words to a special output, encoding named entities with predefined labels. We uti- lize RemBERT and mT5 as backbone models for these two approaches, respectively. Our re- sults show that the oldie but goodie token classi- fication outperforms the template-free method by a wide margin. Our code is available at: https://github.com/Abiks/MultiCoNER. Our solution consists of two state-of-the-art ap- proaches adopted to the task. First, we use a main- stream NER technique, token classification. Under this approach, the model is trained to assign a la- bel to each input token. The second approach falls into the group of prompt-based techniques, at the core of which are the capabilities of auto-regressive language models to memorize and reproduce input texts. In this case, we train an encoder-decoder model to replace entities in the input sentence with predefined labels. We utilize RemBERT (Chung et al., 2021) and mT5 (Xue et al., 2021) as back- bone models for these two approaches, respectively. These two models provide state-of-the-art results for the common test-beds of cross-lingual experi- ments (Hu et al., 2020). NamedEntityRangers at SemEval-2022 Task 11: Transformer-based Approaches for Multilingual Complex Named Entity Recognition Amina Miftahova Innopolis University, Russia noteisenheim@gmail.com Alexander Pugachev HSE University, Russia avpugachev@edu.hse.ru Tatiana Batura Moscow State University Novosibirsk State University, Russia t.batura@g.nsu.ru Vladimir Ivanov Innopolis University Kazan Federal University, Russia v.ivanov@innopolis.ru Tatiana Batura Moscow State University Novosibirsk State University, Russia t.batura@g.nsu.ru Ekaterina Artemova HSE University Huawei Noah’s Ark lab, Russia elartemova@hse.ru Pavel Braslavski HSE University Ural Federal University, Russia pbras@yandex.ru Vladimir Ivanov Innopolis University Kazan Federal University, Russia v.ivanov@innopolis.ru 2 Related Work Our baseline is to treat the NER task as a token clas- sification problem. The base model is a pre-trained RemBERT (Chung et al., 2021) with a linear layer on top of the hidden-states output. Named Entity Recognition is one of the central tasks in Natural Language Processing, which at- tracts a lot of research efforts. Yang et al. (2016) encode morphology and context information via character and word embeddings. Recent studies (Ghaddar and Langlais, 2018; Jie and Lu, 2019; Liu et al., 2019; Meng et al., 2021) employ syntac- tic dependencies, lexical similarity, gazetteers, etc. in the word representations before feeding them to context encoding layers. The authors show that ad- ditional information may lead to improvements in NER performance. However, NER still faces mul- tiple challenges (Li et al., 2022) such as detection of fine-grained and nested named entities (Kim and Kim, 2021; Ringland et al., 2019; Loukachevitch et al., 2021), NER in domain-specific areas (Weber et al., 2021), NER from noisy data (Derczynski et al., 2017) and code-mixed data (Fetahu et al., 2021). RemBERT is based on a multilingual BERT bi- directional transformer architecture. It uses de- coupled embeddings, which allows changing the size of input and output embeddings. The input embeddings are reduced in size, thus making the fine-tuning process faster without performance loss compared to BERT.i In the first experiment on a tokenization step the original label is propagated to all of the word to- kens. We fine-tuned the model on multilingual data for three epochs to predict the labels in BIO for- mat. The batch size is 32, Adam optimizer is used with the learning rate of 10−5 and the scheduler decreases the learning rate by 0.1 each epoch. The metrics obtained on the development set for this approach are presented in Table 1. In the following experiment we investigated how the performance changes if we combine the models into an ensemble. The ensemble consists of three models trained with different seeds as described above. The final predictions are made based on a hard or soft voting scheme. The models perform very similarly; the differences in evaluation scores are insignificant. Recent research in this area considers not only standard types of entities (person, location, or- ganization) but also semantically ambiguous and complex entities (Hanselowski et al., 2018). 1 Introduction This paper describes two submissions to the Multi- lingual Complex Named Entity Recognition (Mul- tiCoNER) Shared Task, held by SemEval-2022 (Malmasi et al., 2022b). This shared task aims at recognizing named entities with the ambitious end goal of building systems that support up to 11 languages. Multilingual setups are complicated when the dataset mixes languages from different groups. This way, the MultiCoNER dataset com- prises 11 languages from different families, and multiple scripts (Malmasi et al., 2022a). This setup hayj s become increasingly popular recently since In other words, we explore the following ques- tions: ‘How do pre-trained transformer-based mod- els perform in the Multilingual Complex Named Entity Recognition task?’ and ‘Which of the two approaches perform better?’. Our results show that plain fine-tuning of the above-mentioned state-of-the-art multilingual transformer-based models can give moderate re- 1570 sults. Results are within 10% margin of the top so- lution for the multilingual NER task. We analyzed the errors of both models. As expected, for two novel ‘complex’ entity types (‘Creative Work’ and ‘Product’) error rate is higher than for ‘common’ types (‘Group’, ‘Location’, ‘Person’). However, some of the errors might be caused by inconsis- tencies in the labeling of the MultiCoNER dataset. The performance of the Template-free approach suffers from such inconsistencies more than the performance of the Token classification approach. tem for multiple languages. The results of a recent Multilingual Named Entity Challenge in six Slavic languages (Piskorski et al., 2021) have also con- firmed the complexity and significance of the task. Training competitive multilingual NER systems re- quires either manually labeled text collections or large automatically annotated datasets (Nothman et al., 2013). 2 Related Work For example, a system has to recognize the titles of movies, books, or songs, which may contain verbs, adverbs, prepositions, etc. Cui et al. (2021) propose a template-based method, treating NER as a lan- guage model ranking problem in a seq2seq manner. Original sentences and statement templates filled by a candidate named entity span are the source sequence and the target sequence, respectively. Ma et al. (2021) induces a language model to predict label words at entity positions during fine-tuning. This method demonstrates the effectiveness under the few-shot setting. The confusion matrix in Figure 1 is built for the multilingual model. The largest ratio of erro- neously assigned O labels is PROD (Product) or CW (Creative work). Almost for all individual lan- guages, the confusion matrix is very similar to the aggregated one. The picture differs a bit for Farsi and Russian languages: the number of mislabelled entities as O is higher for each entity tag. This might be due to the difference in the language struc- ture. For the Chinese language, 23% of the GRP (Group) entities were classified as CORP (Corpo- ration). This behavior is unique to the Chinese The SemEval-2020 shared task MultiCoNER (Malmasi et al., 2022a) focuses on a more exciting and challenging problem of building a NER sys- 1571 unique label words mapping for each language. O CORP CW GRP LOC PER PROD Predicted label O CORP CW GRP LOC PER PROD True label 0.99 0.00 0.00 0.00 0.00 0.00 0.00 0.09 0.82 0.02 0.04 0.01 0.01 0.02 0.12 0.00 0.82 0.01 0.01 0.02 0.02 0.07 0.03 0.02 0.83 0.03 0.02 0.00 0.08 0.00 0.00 0.01 0.90 0.01 0.00 0.05 0.00 0.01 0.00 0.00 0.93 0.00 0.19 0.01 0.02 0.00 0.01 0.00 0.77 Figure 1: Confusion matrix for fine-tuned RemBERT O CORP CW GRP LOC PER PROD Predicted label O CORP CW GRP LOC PER PROD True label 0.99 0.00 0.00 0.00 0.00 0.00 0.00 0.09 0.82 0.02 0.04 0.01 0.01 0.02 0.12 0.00 0.82 0.01 0.01 0.02 0.02 0.07 0.03 0.02 0.83 0.03 0.02 0.00 0.08 0.00 0.00 0.01 0.90 0.01 0.00 0.05 0.00 0.01 0.00 0.00 0.93 0.00 0.19 0.01 0.02 0.00 0.01 0.00 0.77 Figure 1: Confusion matrix for fine-tuned RemBERT 0.2 0.4 0.6 0.8 As the backbone model for the Template-free approach, we considered the mT5 pre-trained lan- guage model (Xue et al., 2021). language. The ensemble model does not introduce much improvement. However, training models with dif- ferent seed values converge almost to the exact predictions. On the dev set, only one model’s pre- diction out of three base models differs in 10% of the cases, and all three models have different predictions in 1% of the cases. O CORP CW GRP LOC PER PROD Predicted label O CORP CW GRP LOC PER PROD True label 0.99 0.00 0.00 0.00 0.00 0.00 0.00 0.08 0.84 0.02 0.03 0.01 0.01 0.01 0.13 0.01 0.82 0.01 0.00 0.02 0.01 0.09 0.04 0.01 0.83 0.02 0.01 0.00 0.09 0.00 0.01 0.01 0.89 0.00 0.00 0.08 0.00 0.01 0.00 0.01 0.90 0.00 0.18 0.02 0.01 0.00 0.00 0.00 0.78 Figure 2: Confusion matrix for Template-free mT5-XL 2 Related Work mT5 is a multi- lingual variant of the T5 model that is pre-trained on a new Common Crawl-based dataset covering 101 languages. We chose two variants of the mT5 model for our experiments: mT5-Large and mT5- XL; the latter model showed better results. Due to the computational complexity, we could not run similar experiments with the mT5-XXL language model. Both models were trained with batch size equal to 8 and optimizer AdamW with learning rate 5 · 10−5. During the experiments with the Template- free approach, we encountered several challenges. Firstly, if the input phrase has two or more consec- utive entity spans with a token length of more than one, it was impossible to reconstruct these spans unambiguously based on the sequence of predicted label words. In this case, we assigned the first k −1 predicted label words to k −1 input tokens, and the last label word was assigned to the rest of the to- kens. Secondly, because of the punctuation issues occurring in some languages described in more de- tail in Section 4, it was difficult to generalize the reconstruction rules for all languages. Figure 1: Confusion matrix for fine-tuned RemBERT 3.2 Template-free Approach The second approach which we considered was Template-free (Ma et al., 2021). This approach showed decent results on CoNLL03 (Sang and Meulder, 2003) and MIT-Movie (Liu et al., 2013) datasets, so we decided to apply it to the multilin- gual data. The language model is trained to substi- tute named entity text spans with several predefined label words. For instance, given the sentence “its headquarters are in sandy springs, united states of america” we require the model to replace “sandy springs” and “united states of america” text spans with a predefined label word “germany” since these spans are considered as LOC (location) entities. In this case, the target for this example would be: “its headquarters are in germany, germany”. After the model has substituted several text spans with the la- bel words, we need to reconstruct these spans based on surrounding tokens to match each initial token with its predicted label. The label word for a spe- cific entity class was chosen as the most frequent token of this class in the training data. Since we were working with multilingual data, we created a Figure 2: Confusion matrix for Template-free mT5-XL The token-level confusion matrix for the Template-free approach on the development set is shown on Figure 2. The results for the mT5- XL fine-tuned model on the dev set is presented in Table 1. Based on Figure 2 we can mention that the most 1572 Table 1: Results for Token Classification and Template- free approaches on dev and test sets Approach Dev Test P R F1 P R F1 Token Classification 0.81 0.84 0.82 0.83 0.80 0.81 Template-free 0.82 0.77 0.79 0.58 0.51 0.54 Table 1: Results for Token Classification and Template- free approaches on dev and test sets and consistent labeling might be crucial for de- veloping high-performing models for complex entity recognition. Another example is coun- try names. In one sentence a country name can be considered as a named entity, but in another sentence a country name is annotated as O to- ken. For instance, in the sentence2 “spain has an embassy ...” the token “spain” has O label, but in the sentence “in madrid, spain ...” the same token is considered as a LOC (location) entity despite the fact that in both contexts this token refers to country name. 1The example sentence ID: 2e9d398c-a956-4419-a48d- f53790d2d237 (file en_train.conll) 3.2 Template-free Approach Despite, we call such cases “mislabeled-as-O entities”, not all of them can be considered wrong la- bels. As far as we have noticed, this problem is very common for the Farsi language. To be precise, we calculated the average number of mislabeled-as-O entities for each language (see Table 2). We suppose that this issue can influence the performance of both models. common model’s error is predicting the O-tag for tokens which are actually considered as parts of the entities. In other words, the model more often fails to recognize a named entity than confuses two dif- ferent entities. We can also mention that in terms of languages the fine-tuned model shows the best re- sults for English, Dutch and German and the worst results for Farsi and Russian according to F1 score. The dramatic performance downgrade on the test set compared to the dev set for the Template-free model may occur due to the significant distribution shift in the test data. For instance, the average sen- tence length in terms of tokens in the train and dev data is approximately equal to 16.4. However, the average length in the test data is equal to 9.6, we assume that the Template-free model could not be resistant for such changes. Language Mislabeled-as-O BN-Bangla 0.691 DE-German 0.474 EN-English 1.002 ES-Spanish 3.061 FA-Farsi 20.234 HI-Hindi 1.995 KO-Korean 5.489 NL-Dutch 3.269 RU-Russian 1.606 TR-Turkish 3.494 ZH-Chinese 1.094 Table 2: Mean number of mislabeled-as-O entities 2The example sentence IDs are df4360c4-a483-493b-bd93- 87814db0104c and 475fb6b2-b9aa-4ec8-8b58-443f5e2774e8 (file en_train.conll) 3 3The example sentence IDs are be16705b-7f6e-4c28-b086- 5eabf5950d29 and ea916f1b-b9a5-4959-9537-aeb875c1faf1 (file en_train.conll) 4 Discussion We provide the performance analysis of the mod- els that we developed. The confusion matrices for both approaches demonstrate similar commonly occurring errors. Moreover, while working on ex- periments, we noticed several dataset issues which we suppose are worth mentioning. The problems potentially contribute to the low performance of the Template-free approach. Table 2: Mean number of mislabeled-as-O entities Table 2: Mean number of mislabeled-as-O entities 2. The second issue is that we noticed while working on the Template-free approach is punctuation labeling. For example, in the sen- tence3 “thomas earnshaw, inventor of ...” the comma is presented as a separate token. How- ever, in the sentence “... museum of fine arts, houston ...” the comma is part of token “arts,”. This issue was crucial on the reconstruction entity spans step since the tokenizer always considers any punctuation token as a separate one. This issue is common for MultiCoNER data in many languages, especially in German and Bangla. 2. The second issue is that we noticed while working on the Template-free approach is punctuation labeling. For example, in the sen- tence3 “thomas earnshaw, inventor of ...” the comma is presented as a separate token. How- ever, in the sentence “... museum of fine arts, houston ...” the comma is part of token “arts,”. This issue was crucial on the reconstruction entity spans step since the tokenizer always considers any punctuation token as a separate one. This issue is common for MultiCoNER data in many languages, especially in German and Bangla. 1. Classification decisions for tokens from PROD and CW entity types are more often confused with the O labeled tokens (Fig. 1, 2). These entities are examples of complex entities, which the competition was focused on. At the same time, the labeling for the two entity types in the provided dataset shows low consistency. For example, in the sen- tence1 “rice - long, medium, or short-grain white; also popcorn rice” the first occurrence of “rice” is labelled as PROD. However, “pop- corn rice” is not labeled as a named entity. The precise definitions of the complex entities 1573 3. For the purposes of more detailed perfor- mance analysis we considered the dependence between prediction accuracy and entity length (in number of tokens). The Figure 3 shows this dependence for both Template-free and Token Classification approaches considering the dev dataset. References Hyung Won Chung, Thibault Fevry, Henry Tsai, Melvin Johnson, and Sebastian Ruder. 2021. Rethinking em- bedding coupling in pre-trained language models. In International Conference on Learning Representa- tions. 1 2 3 4 5 6 7 8 9 10 Named Entity Length 0.65 0.70 0.75 0.80 0.85 Fraction of Correct Predictions Template-free Token Classification Figure 3: The dependence of prediction accuracy on the entity lengths Leyang Cui, Yu Wu, Jian Liu, Sen Yang, and Yue Zhang. 2021. Template-based named entity recognition us- ing BART. In Findings of the Association for Com- putational Linguistics: ACL-IJCNLP 2021, pages 1835–1845. Association for Computational Linguis- tics. Leon Derczynski, Eric Nichols, Marieke van Erp, and Nut Limsopatham. 2017. Results of the wnut2017 shared task on novel and emerging entity recognition. 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This could be the reason why the Token Classifi- cation method outperforms the Template free approach on the test set since the average sen- tence length in the test data is dramatically less than the average sentence length in the train and dev datasets. model, but this issue needs further investigation. Evaluation of systems for multilingual NER with complex entity types is still challenging. Our study analyzed the dataset and found several issues that can be addressed in future versions of MultiCoNER Track or in similar competitions. Acknowledgements The project is supported by the Russian Sci- ence Foundation, grant # 20-11-20166. This re- search was supported in part through computa- tional resources of HPC facilities at HSE University (Kostenetskiy et al., 2021). 5 Conclusion In the paper, we implemented and evaluated two straightforward yet different approaches to the mul- tilingual NER subtask with complex types of en- tities (MultiCoNER). 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https://openalex.org/W4285808599	https://pure.uva.nl/ws/files/110885883/Constraints_on_Higgs_boson_properties.pdf	English	null	Constraints on Higgs boson properties using $$WW^{*}(\rightarrow e\nu \mu \nu )jj$$ production in $$36.1\,\mathrm{fb}^{-1}$$ of $$\sqrt{s}=13$$ TeV pp collisions with the ATLAS detector	European physical journal. C, Particles and fields	2,022	cc-by	32,932	UvA-DARE (Digital Academic Repository) ATLAS Collaboration DOI 10.1140/epjc/s10052-022-10366-1 Publication date 2022 Document Version Final published version Published in European Physical Journal C License CC BY Link to publication Citation for published version (APA): ATLAS Collaboration (2022). Constraints on Higgs boson properties using WW(→ eνμν )jj production in 36.1fb–1 of √s = 13 TeV pp collisions with the ATLAS detector. European Physical Journal C, 82(7), Article 622. https://doi.org/10.1140/epjc/s10052-022-10366-1 Citation for published version (APA): ATLAS Collaboration (2022). Constraints on Higgs boson properties using WW(→ eνμν )jj production in 36.1fb–1 of √s = 13 TeV pp collisions with the ATLAS detector. European Physical Journal C, 82(7), Article 622. https://doi.org/10.1140/epjc/s10052-022-10366-1 Constraints on Higgs boson properties using WW∗(→eνμν) j j production in 36.1 fb−1 of √s = 13 TeV pp collisions with the ATLAS detector ATLAS Collaboration⋆ CERN,1211 Geneva 23, Switzerland Received: 29 September 2021 / Accepted: 24 April 2022 / Published online: 18 July 2022 © CERN for the beneﬁt of the ATLAS collaboration 2022 ATLAS Collaboration⋆ CERN,1211 Geneva 23, Switzerland Received: 29 September 2021 / Accepted: 24 April 2022 / Published online: 18 July 2022 © CERN for the beneﬁt of the ATLAS collaboration 2022 Abstract This article presents the results of two studies of Higgs boson properties using the W W ∗(→eνμν) j j ﬁnal state, based on a dataset corresponding to 36.1fb−1 of √s = 13 TeV proton–proton collisions recorded by the ATLAS experiment at the Large Hadron Collider. The ﬁrst study targets Higgs boson production via gluon–gluon fusion and constrains the CP properties of the effective Higgs–gluon interaction. Using angular distributions and the overall rate, a value of tan(α) = 0.0 ± 0.4(stat.) ± 0.3(syst.) is obtained for the tangent of the mixing angle for CP-even and CP-odd contributions. The second study exploits the vector-boson fusion production mechanism to probe the Higgs boson couplings to longitudinally and transversely polarised W and Z bosons in both the production and the decay of the Higgs boson; these couplings have not been directly con- strained previously. The polarisation-dependent coupling- strength scale factors are deﬁned as the ratios of the measured polarisation-dependent coupling strengths to those predicted by the Standard Model, and are determined using rate and kinematicinformationtobeaL = 0.91+0.10 −0.18(stat.)+0.09 −0.17(syst.) and aT = 1.2 ± 0.4(stat.)+0.2 −0.3(syst.). These coupling stren- gths are translated into pseudo-observables, resulting in κV V = 0.91+0.10 −0.18(stat.)+0.09 −0.17(syst.) and ϵV V = 0.13+0.28 −0.20 (stat.)+0.08 −0.10(syst.). All results are consistent with the Stan- dard Model predictions. and CMS experiments to be m H = 125.09 ± 0.21(stat.) ± 0.11(syst.) GeV [3] using √s = 7 and 8 TeV pp collision data, and there are strong indications that the spin and parity states are J P = 0+ [4–6]. By probing the ﬁnal-state parti- cles in H →W W ∗, H →Z Z∗, and H →γ γ decays, a pure CP-odd Higgs boson has been excluded at a conﬁdence level above 99.9%. In addition, a potential CP-odd contribu- tion to the HW W and H Z Z couplings has been signiﬁcantly constrained [7–9]. Constraints on Higgs boson properties using WW∗(→eνμν) j j production in 36.1 fb−1 of √s = 13 TeV pp collisions with the ATLAS detector Recently, studies of the CP properties of the top-quark Yukawa coupling using Htt events, and the τ-lepton Yukawa coupling using H →ττ decays have been published [10–12]. The gluon–gluon fusion (ggF) process probes a differ- ent kinematic regime than Htt production, and the proper- ties of the effective ggH interaction itself may differ from SM predictions if there is a loop contribution from pre- viously unobserved particles. The CP nature of the effec- tive coupling between the Higgs boson and gluons, ggH, in the gluon–gluon fusion production mode [13] has been studied in Refs. [14–16]. This article presents new anal- ysis techniques for enhancing the sensitivity to interfer- ence effects between the CP-even and CP-odd contributions to the Higgs–gluon coupling, and provides constraints on the mixing angle between CP-even and CP-odd contribu- tions. The approach is therefore complementary to those used in previous studies of the CP properties of the Higgs boson. The vector-boson fusion (VBF) Higgs boson production process has been measured by ATLAS and CMS in numer- ous channels [7,8,17–20]. While measurements of σVBF · BH→W W are consistent with the SM, individual polarisation- dependent Higgs boson couplings to the massive electroweak gauge bosons have so far not been studied directly. Longitu- dinally polarised electroweak bosons emerge from massless degrees of freedom of the Higgs boson and are therefore closely related to the mechanism of electroweak symme- try breaking. The strength of the Higgs boson coupling to longitudinally polarised W bosons ensures the unitarity of General rights I i i General rights It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulations If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: https://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. Disclaimer/Complaints regulations UvA-DARE is a service provided by the library of the University of Amsterdam (https://dare.uva.nl) Download date:24 Oct 2024 Eur. Phys. J. C (2022) 82:622 https://doi.org/10.1140/epjc/s10052-022-10366-1 Regular Article - Experimental Physics ⋆e-mail: atlas.publications@cern.ch 1 ATLAS uses a right-handed coordinate system with its origin at the nominal interaction point (IP) in the centre of the detector and the z- axis along the beam pipe. The x-axis points from the IP to the centre of the LHC ring, and the y-axis points upwards. Cylindrical coordinates (r, φ) are used in the transverse plane, φ being the azimuthal angle around the beam pipe. The pseudorapidity is deﬁned in terms of the polar angle θ as η = −ln tan(θ/2). Angular distance is measured in units of R ≡ ( η)2 + ( φ)2. 1 Introduction The Higgs boson (H) discovery at the Large Hadron Collider (LHC) was a great success of the ATLAS and CMS Collabo- rations [1,2]. Since the discovery, multiple studies have been performed to establish whether the Higgs boson has the prop- erties predicted by the Standard Model (SM), or instead is a particle of an as yet unobserved sector beyond the SM. The mass of the Higgs boson has been measured by the ATLAS 12 3 Eur. Phys. J. C (2022) 82 :622 622 Page 2 of 33 ith ng M 2] on M selected H + 2-jets candidate events to test for deviations from the SM expectations. The angular difference is deﬁned as j j = φ j1 −φ j2 if η j1 > η j2, and j j = φ j2 − φ j1 otherwise, where j1 is the highest-pT jet and j2 is the next-highest-pT jet in the event. The distribution of j j is probed in various disjoint kinematic regions, optimised for each analysis speciﬁcally. sociated with selected H + 2 jets candidate events to test for deviations les of Feynman ibuting to the Higgs boson in h two jets via the luons or two V ) at leading The presented examples for the qq →Hqq, b d c gg →Hgg, vector-boson and the ay of the Higgs vector bosons Fig. 1 Examples of Feynman diagrams contributing to the production of a Higgs boson in association with two jets via the fusion of two gluons or two vector bosons (V ) at leading order in QCD. The presented diagrams show examples for the subprocesses a qq →Hqq, b qg →Hqg and c gg →Hgg, as well as d the vector-boson fusion process and the subsequent decay of the Higgs boson into two vector bosons the SM. However, if the Higgs ﬁeld is not associated with a fundamental scalar particle but is an effective ﬁeld arising from new dynamics, the coupling may deviate from its SM value. For example, Higgs compositeness models [21,22] predict more degrees of freedom, allowing the Higgs boson couplings to electroweak bosons to deviate from their SM values. selected H + 2-jets candidate events to test for deviations from the SM expectations. 1 Introduction The angular difference is deﬁned as j j = φ j1 −φ j2 if η j1 > η j2, and j j = φ j2 − φ j1 otherwise, where j1 is the highest-pT jet and j2 is the next-highest-pT jet in the event. The distribution of j j is probed in various disjoint kinematic regions, optimised for each analysis speciﬁcally. selected H + 2-jets candidate events to test for deviations from the SM expectations. The angular difference is deﬁned as j j = φ j1 −φ j2 if η j1 > η j2, and j j = φ j2 − φ j1 otherwise, where j1 is the highest-pT jet and j2 is the next-highest-pT jet in the event. The distribution of j j is probed in various disjoint kinematic regions, optimised for each analysis speciﬁcally. This article presents the results of two analyses study- ing the properties of the Higgs boson using its decay into W W ∗→eνμν and its production in association with two jets ( j j). The ﬁrst analysis targets gluon–gluon fusion Higgs boson production and is sensitive to the CP properties of the ggH effective coupling and the top-quark Yukawa coupling. The second analysis constrains Higgs boson couplings to longitudinally and transversely polarised W and Z bosons in the VBF production mode, assuming a CP-even Higgs boson. Both studies are based on proton-proton collision data corre- sponding to an integrated luminosity of 36.1 fb−1 collected with the ATLAS detector at √s = 13 TeV in the years 2015 and 2016. Representative leading-order diagrams for the ggF + 2 jets and VBF production modes are depicted in Fig. 1a–c and d, respectively. 1 The structure of this article is as follows. Section 2 gives a short summary of the theoretical frameworks used to study the CP properties of the Higgs boson’s coupling to top quarks and gluons, as well as its coupling to polarised elec- troweak bosons. The ATLAS detector and the Monte Carlo and data samples used in these studies are discussed in Sects. 3 and 4, respectively. The event selection and cate- gorisation requirements are presented in Sect. 5, while the estimation of the various background processes is detailed in Sect. 6. The theoretical and experimental uncertainties are presented in Sect. 7. Finally, the results are discussed in Sect. 8. Fig. 1 Examples of Feynman diagrams contributing to the production of a Higgs boson in association with two jets via the fusion of two gluons or two vector bosons (V ) at leading order in QCD. The presented diagrams show examples for the subprocesses a qq →Hqq, b qg →Hqg and c gg →Hgg, as well as d the vector-boson fusion process and the subsequent decay of the Higgs boson into two vector bosons 2 Theoretical framework and methodology Interference between CP- even and CP-odd contributions affects the shape of the signed j j distribution, but has no impact on the cross section of the ggF production mode, which is a function of κ2 gg cos2(α) and κ2 gg sin2(α) only. Three benchmark scenarios with differ- ent CP properties are deﬁned in Table 1, and the distribution of the signed j j observable is shown in Fig. 2a for these parameter choices. The POs are related to the coupling-strength scale factors aL and aT via the following equations: κV V = aL − L(q1, q2)εV V , εV V = aT −aL T(q1, q2) − L(q1, q2) . The analysis targeting HV V couplings in Higgs boson production and decay uses polarisation-dependent coupling- strength scale factors deﬁned in Ref. [27] as The functions L(q1, q2) and T(q1, q2) depend on the momenta of electroweak bosons q1 and q2 (either in the pro- duction or in the decay) according to: L = m2 H 2m2 W 4q2 1q2 2 m2 H(m2 H −q2 1 −q2 2) , T = m2 H 2m2 W m2 H −q2 1 −q2 2 m2 H . aL = gHVLVL gHV V , aT = gHVTVT gHV V , (2) (2) where gHV V is the SM HV V coupling strength and gHVLVL and gHVTVT are the measured polarisation-dependent cou- plings. Based on the simulations of the VBF signal discussed in Sect. 4, L(q1, q2) = 0 and T(q1, q2) = 2 are found to be good approximations, leading to the mapping The polarisations of the vector bosons in Eq. (2) are deﬁned in the Higgs boson rest frame so that mixed- polarisation couplings HVLVT do not contribute to σVBF · BH→W W. Other BSM effects are not considered. Within the SM (aL = aT = 1), the HV V couplings are insensitive to the polarisations. κV V ≃aL , εV V ≃0.5 · (aT −aL) . The above POs description focusses on VBF production and thus differs from the one used in Refs. [30,31], in which couplings to leptons (εL, εR) and to Z bosons (κZ Z) are con- strained using Higgs boson decays into four leptons, inclu- sively with respect to the production channels. The above POs description focusses on VBF production and thus differs from the one used in Refs. 2 Theoretical framework and methodology Both analyses use the shape of the signed azimuthal angle1 difference j j between the two leading hadronic jets in the For the studies targeting beyond-the-SM (BSM) contribu- tions to the top-quark Yukawa coupling and the effective Higgs–gluon coupling, an effective ﬁeld theory (EFT) frame- work is chosen to parameterise possible deviations from the SM. The EFT operators probed in this article are provided by the Higgs Characterisation (HC) model [23], which is imple- mented in the MadGraph5_aMC@NLO generator [24,25]. In the large top-quark-mass limit, mtop →∞, the CP struc- ture of the top-quark Yukawa coupling is inherited by the effective Higgs–gluon interaction [26]. Thus, constraints on 12 3 Eur. Phys. J. C (2022) 82 :622 Page 3 of 33 622 article appear as κV V and εV V in the effective Lagrangian BSM contributions will be directly set on the CP-even and CP-odd coupling strength modiﬁers of the effective Higgs– gluon interaction. The effective Lagrangian that describes the Higgs–gluon interaction is expressed as L = κV V 2m2 W v HW + μ W −μ + m2 Z v H ZμZμ −εV V 2v 2HW + μνW −μν + H Zμν Zμν + H Aμν Aμν , Lloop 0 = − gHgg 4 κgg cos(α)Ga μνGa,μν +κgg sin(α)Ga μν ˜Ga,μν H, (1) where in the SM κV V = 1 and εV V = 0. The universality of where in the SM κV V = 1 and εV V = 0. The universality of Higgs boson interactions with longitudinal W and Z bosons followsfromassumingcustodialsymmetry(seeEqs.(33)and (35) in Ref. [29]), no new physics in the boson–fermion cou- plings W f f and Z f f , and a CP-even Higgs boson with CP- conserving interactions with vector bosons. Since the study does not probe H Zγ and Hγ γ interactions, for simplicity a common coupling factor is assumed for Higgs-boson inter- actions with transversely polarised W and Z bosons, and photons. (1) where Ga μν is the gluon ﬁeld strength tensor, ˜Ga,μν = Ga ρσ εμνρσ/2 is the dual tensor, gHgg is the effective coupling f h S C i i i h li ρ gg for the SM CP-even ggH interaction, κgg is the coupling- strength scale factor for the effective Higgs–gluon interac- tion and α is the CP-mixing angle. 2 Theoretical framework and methodology [30,31], in which couplings to leptons (εL, εR) and to Z bosons (κZ Z) are con- strained using Higgs boson decays into four leptons, inclu- sively with respect to the production channels. Since the polarisations depend on the measurement frame, the above description is not Lorentz invariant and as such cannot be described in the Lagrangian framework. Instead, the coupling strength modiﬁers aL and aT can be related to pseudo-observables (POs) [28]. The POs considered in this This article consists of two studies. The ﬁrst one places constraints on tan(α) in the effective Hgg coupling in ggF + 2 jets Higgs boson production, assuming standard HV V couplings. The second study constrains the HV V parame- ters (aL, aT) and (κV V , εV V ), assuming a pure CP-even Higgs state with standard Hgg coupling (κgg cos(α) = 1). A non- SM ggH coupling would negligibly impact the quantities determined in the VBF analysis and vice versa. The con- straints are derived from the rates of each production pro- cess as well as the distribution of signed j j, whose shape dependence on the coupling modiﬁers is shown in Fig. 2. The distribution of j j is displayed in the full range [0, 2π] to Table 1 Deﬁnition of the three benchmark scenarios used in the ggF + 2 jets analysis. The parameter settings correspond to a CP-even (i.e. the SM hypothesis), a CP-odd, and a CP-mixed scenario Scenario Parameters CP-even (SM) κgg = 1, cos(α) = 1 CP-odd κgg = 1, cos(α) = 0 CP-mixed κgg = 1, cos(α) = 1 √ 2 12 3 622 Page 4 of 33 622 Page 4 of 33 Eur. Phys. J. C (2022) 82 :622 (a) (b) Fig. 2 Distributions of the signed j j observable shown for a CP- even, CP-odd and CP-mixed benchmark models of the ggF + 2 jets production mode, and b various conﬁgurations of the aL and aT param- eters in VBF events. These comparisons are performed at the generator level using the predictions of the MadGraph5_aMC@NLO 2.4.2 + Pythia 8.212 [32] generators (a) Fig. 2 Distributions of the signed j j observable shown for a CP- even, CP-odd and CP-mixed benchmark models of the ggF + 2 jets production mode, and b various conﬁgurations of the aL and aT param- (b) eters in VBF events. 4 Datasets and Monte Carlo predictions Candidateeventsindataareselectedfromthecombined2015 and 2016 ATLAS √s = 13 TeV pp collision dataset in which all ATLAS subdetectors were fully operational [34]. The corresponding total integrated luminosity [35] is 36.1 ± 0.8 fb−1. 2 Theoretical framework and methodology These comparisons are performed at the generator level using the predictions of the MadGraph5_aMC@NLO 2.4.2 + Pythia 8.212 [32] generators (b) (a) Fig. 2 Distributions of the signed j j observable shown for a CP- even, CP-odd and CP-mixed benchmark models of the ggF + 2 jets production mode, and b various conﬁgurations of the aL and aT param- eters in VBF events. These comparisons are performed at the generator level using the predictions of the MadGraph5_aMC@NLO 2.4.2 + Pythia 8.212 [32] generators The ATLAS detector has a two-level trigger system to select events for further analysis. capture the asymmetry for mixed CP interactions (shown in Fig. 2a) resulting from the interference between CP-even and CP-odd contributions.2 Fig. 2b shows the j j distribution for several choices of aL and aT. 2 As the CP-even amplitude is positive under the transformation j j −π →− j j −π and the CP-odd amplitude is nega- tive under this transformation, their interference is asymmetric whereas the individual squared amplitudes are positive. 3 ATLAS detector For the studies of ggF + 2 jets production, the VBF pro- duction of the Higgs boson is considered as a background and an additional sample was generated to model the SM prediction for this process. In this case, Powheg-Box v2 [39] was used to model the matrix element at NLO in QCD with the NNPDF3.0 NLO PDF set, while Pythia8.212 was used to model parton shower effects. The q ¯q →W H and q ¯q →Z H processes were gen- erated with Powheg-Box v2 MiNLO [40] interfaced to Pythia8.186, with the AZNLO set of tuned parameters [41] for the underlying event, showering and hadronisation. The gg →Z H process was simulated with Powheg-Box v2 + Pythia8.186, with the AZNLO set of tuned parameters for the underlying event, showering and hadronisation. For all V H samples, the PDF4LHC15 PDF set [42] was used. The cross sections of the V H production modes were ﬁxed to SM predictions. The V H processes are considered as back- grounds to the studies of the ggF + 2 jets and VBF production modes. The diboson production processes with q ¯q and qg ini- tial states and leptonic ﬁnal states were simulated using Sherpa 2.2.2 [52,53]. The cross-section calculation is based on the NNPDF3.0 NNLO PDF set and includes all relevant off-shell components. Diagrams with up to one additional emission were calculated with NLO precision in QCD, while diagrams with two or three parton emissions were calcu- latedwithLOaccuracy[54].Thevariousjet-multiplicityﬁnal states were merged using the MEPS@NLO formalism [55] and a merging scale of Qcut = 20 GeV. Loop-induced dibo- son processes that are initiated via the gg production mode were simulated at NLO in QCD using OpenLoops [56] in Sherpa 2.1.1 and the CT10 NLO PDF set. The production of dibosons with semileptonic decays, as well as the elec- troweak production of dibosons in association with two jets, was modelled using Sherpa 2.1.1 and the CT10 PDF set. Other production and decay modes of the Higgs boson were either ﬁxed to SM predictions (for H →ττ decay) or neglected (for t ¯t H and b ¯bH associated production) due to their insigniﬁcant contributions to the phase-space regions probed by the studies presented in this article. 3 A common correction factor is applied to all VBF signal samples to account for the difference between the branching fractions predicted by HDECAY and those calculated by MadGraph5_aMC@NLO for the SM scenario. 3 ATLAS detector The helic- ity amplitudes used in the matrix element generation of the Higgs boson production and decay were modiﬁed to account for different Higgs coupling strengths in the Higgs boson rest frame, following the prescription in Ref. [27]. Signal sam- ples were produced for the following benchmark scenarios: (aL, aT) ∈{(1, 1), (1, 1.3), (1.3, 0.7), (1.3, 1), (0.7, 1.3)}. Parton shower effects were simulated with the Pythia8.212 generator using the A14 set of tuned parameters. The renor- malisation and factorisation scales were both set to the sum of the transverse momenta of the jets, and the shower scale was set to 0.25 times the maximum pT of the radiated par- tons. The SM (CP-even) sample described above was used for simulating the gluon-fusion Higgs boson production in association with two jets. cross-section predictions at next-to-next-to-next-to-leading- order (NNNLO) precision in QCD [44]. The VBF sam- ples were normalised to match cross-section predictions at next-to-next-to-leading-order (NNLO) precision in QCD with NLO electroweak (EW) corrections, using the programs VBF@NNLO [45] and HAWK [46]. The main sources of background include events from the production of top quarks (t ¯t and Wt), dibosons (W W, W Z/W Z(∗), Z Z, Wγ (∗), Zγ ) and single vector-bosons (W, Z/γ ∗) plus jets. The production of a top-quark pair (t ¯t) was modelled using the Powheg-Box v2 generator interfaced to Pythia8.210 with the A14 set of tuned parton shower param- eters [36]. The matrix elements were calculated at NLO pre- cision in QCD assuming a top-quark mass of 172.5 GeV. The NNPDF3.0 NLO PDF set was used and the hdamp parameter [47] was set to 1.5 times the top-quark mass. The t ¯t produc- tion cross section was normalised to the predictions calcu- lated with the Top++2.0 program to NNLO in perturbative QCD, including soft-gluon resummation calculated to the next-to-next-to-leading logarithm (NNLL) [48]. The associ- ated production of a single top quark and a W boson (Wt) was generated with Powheg-Box interfaced to Pythia6.428 for parton showering, using the Perugia 2012 tune [49]. The matrix elements were calculated at NLO precision in QCD. The cross-section calculation is based on the CT10 [50] PDF set. The diagram removal (DR) scheme [51] was used to remove overlaps with the top-quark pair production process that occur at NLO in QCD. 3 ATLAS detector The ATLAS detector [33] is a general-purpose particle detec- tor used to investigate a broad range of physics processes. It includes an inner tracking detector (ID) surrounded by a thin superconducting solenoid, electromagnetic and hadronic calorimeters, and a muon spectrometer (MS) incorporating three large superconducting toroid magnets with eight coils each. The ID consists of ﬁne-granularity silicon pixel and microstrip detectors, and a straw-tube tracker. It is immersed in a 2 T axial magnetic ﬁeld produced by the solenoid and provides precision tracking for charged particles in the range \|η\| < 2.5, where η is the pseudorapidity of the particle. The straw-tube detector also provides transition radiation mea- surements for electron identiﬁcation. The calorimeter system covers the pseudorapidity range \|η\| < 4.9. It is composed of sampling calorimeters with either liquid argon (LAr) or scin- tillator tiles as the active medium, and lead, steel, copper, or tungsten as the absorber material. The MS provides muon identiﬁcation and momentum measurements for \|η\| < 2.7. The modelling of the gluon-induced production of Higgs bosons in association with jets was realised using the Mad- Graph5_aMC@NLO 2.4.2 generator [24,25], which pro- vides a calculation of the matrix element at next-to-leading- order (NLO) precision for ggF events with up to two addi- tional partons in the ﬁnal state. The calculations of the matrix element are based on the predictions of the HC model, while the parton shower, hadronisation and underlying-event activ- ity were simulated with the Pythia8.212 [32] generator using the A14 set of tuned parameters [36]. The cross-section calculation is based on the NNPDF3.0 [37] NLO parton dis- tribution function (PDF) sets. In total, three different Monte Carlo samples were produced, corresponding to a CP-even, a CP-odd or a CP-mixed coupling between Higgs bosons and gluons, following the recommendations from Ref. [26] and using the FeynRules model HC_NLO_X0_UFO-heft [38]. The decay H →W W ∗→eνμν was modelled according to the SM. For the measurement of the Higgs boson couplings to longitudinally and transversely polarised W and Z bosons, 12 3 Eur. Phys. J. C (2022) 82 :622 Page 5 of 33 62 622 the VBF production of the Higgs boson and its subsequent decays into W bosons were simulated at leading order (LO) in QCD using MadGraph5_aMC@NLO 2.4.2. 3 ATLAS detector The branching fractions for Higgs boson decays, cal- culated for a Higgs boson mass m H = 125 GeV, were taken from the HDECAY program [43], except for the signal samples used in the polarisation studies, for which the branching fractions were taken from the predictions of the MadGraph5_aMC@NLO generator.3 The inclusive ggF samples were normalised to match the state-of-the-art The production of a Z/γ ∗boson in association with jets was modelled by Sherpa 2.2.1 with the NNPDF3.0 NNLO PDF set. Diagrams with up to two additional parton emis- sions were simulated with NLO precision in QCD, and those with three or four additional parton emissions to LO accu- racy. Matrix elements were merged with the Sherpa parton shower using the MEPS@NLO formalism with a merging scale of Qcut = 20 GeV and the ﬁve-ﬂavour numbering scheme (5FNS). Contributions from the electroweak pro- 12 3 622 Page 6 of 33 622 Page 6 of 33 Eur. Phys. J. C (2022) 82 :622 conﬁgurations marked with (⋆⋆) are used as background in the ggF + 2 jets analysis. Alternative event generators and conﬁgurations used to estimate systematic uncertainties are shown in parentheses. Further information about the alternative event generators and their conﬁgura- tions is given in Sect. 7, where MG5 is again used as an abbreviation for MadGraph5 Table 2 Overview of the simulation tools used to generate signal and background processes, and to model the underlying event and parton shower. The PDF sets are also summarised. The perturbative accuracy (in QCD and if relevant in EW corrections) of the total cross section is stated for each process. The conﬁguration marked with (⋆) is used for the signal samples in the studies of the Higgs boson couplings to longitudinally and transversely polarised W and Z bosons, while the up conditions4 observed in the ATLAS data recorded during the 2015 and 2016 runs of the LHC. up conditions4 observed in the ATLAS data recorded during the 2015 and 2016 runs of the LHC. duction of q ¯q →Zq ¯q events were considered, in which the matrix element was generated with up to one additional emis- sion beyond the ﬁrst two partons using Sherpa 2.1.1 and the CT10 [50] PDF set. Matrix elements were merged with the Sherpa parton shower using the MEPS@NLO formalism with the merging scale set to Qcut = 15 GeV. 3 ATLAS detector Contributions due to processes containing a single W boson produced in association with jets are estimated in a purely data-driven approach. 4 An average of 13 (21) interactions per bunch crossing were observed during the 2015 (2016) run. 5 Event selection and categorisations In the VBF analysis, an ‘outside lepton veto’ is applied, which requires the two lep- tons to be within the rapidity gap spanned by the two leading jets, and a ‘central jet veto’ rejects events with additional jets with pT > 20 GeV in the rapidity gap between the two lead- ing jets. Table 3 summarises the selection requirements used to deﬁne the SRs of the ggF + 2 jets and VBF analyses. In order to be considered for the ﬁnal analysis, candidate events must contain exactly two prompt leptons with oppo- site electrical charge and different lepton ﬂavour, with the higher-pT (leading) lepton having pT > 22 GeV and the subleading lepton having pT > 15 GeV. The invariant mass of the dilepton system, mℓℓ, must exceed 10 GeV. At least one of the leptons must be matched to an object that triggered the recording of the event. In the case that a dilepton trigger is solely responsible for the recording of an event, each lepton must be matched to one of the trigger-level objects. In addi- tion, events must contain at least two jets passing all selec- tion requirements. To reduce backgrounds from top-quark production, events are vetoed if they contain a b-tagged jet with a pT larger than 20 GeV (Nb-jet,pT>20 GeV = 0). The Z(→ττ) + jets background is reduced by introducing an mττ < 66 GeV selection requirement. The observable mττ is calculated from the four-vectors of the two charged leptons and the ⃗Emiss T vector using the collinear approximation [73]. Variousidentiﬁcationrequirementsincludingcalorimeter- and track-based isolation criteria [62,63] are used to reduce the number of hadrons and soft leptons that arise from heavy- ﬂavour decays and that are misidentiﬁed as prompt lep- tons. The prompt-electron identiﬁcation efﬁciencies range between 88% and 94% depending on the electron pT and \|η\|, while the prompt-muon identiﬁcation efﬁciency is close to 95% over the full instrumented pseudorapidity range. Jets are reconstructed from noise-suppressed topological clusters [64] of energy deposits in the calorimeter system using the anti-kt algorithm [65,66] with a radius parameter of R = 0.4. The jet four-momentum is corrected using pT- and η-dependent scale factors, which account for the calorime- ter’s non-compensating response, signal losses due to noise threshold effects, energy lost in passive materials, and contri- butions from pile-up interactions [67]. 5 Event selection and categorisations Candidate events consistent with the ﬁnal state H (→W W ∗→eνμν) + 2 jets are selected. Events are retained for further analysis using single-lepton and dilepton triggers. The transverse momentum (pT) thresholds range between 24 GeV and 26 GeV for single-electron triggers and between 20 GeV and 26 GeV for single-muon triggers, depending on the run period [59–61], while the dilepton trig- ger requires an electron with pT > 17 GeV and a muon with pT > 14 GeV. The combined efﬁciency of the single-lepton TheMCgenerators,PDFsets,andprogramsusedtomodel the underlying event and parton shower (UEPS) are sum- marised in Table 2. The order of the perturbative prediction for each sample is also reported. All simulated events were generated at a centre-of-mass energy of √s = 13 TeV and then passed through the full ATLAS detector simulation [57,58]. The simulated events were overlaid with additional inelastic pp interactions that were generated with Pythia8.153 in order to match the pile- 123 Eur. Phys. J. C (2022) 82 :622 Page 7 of 33 622 jets, as well as all tracks compatible with the primary vertex but not associated with any of these objects [72]. and dilepton triggers is 95% in the ﬁducial regions used in these analyses. Candidate events are required to have at least one vertex having at least two associated tracks with pT > 400 MeV. The vertex with the highest p2 T of the associated tracks is taken as the primary vertex. Ambiguities from overlapping reconstructed jet and lep- ton candidates are resolved as follows. If a reconstructed muon shares an ID track with a reconstructed electron, the electron is removed. Reconstructed jets are discarded if they are within a cone of size R = 0.2 around an electron can- didate or if they have fewer than three associated tracks and are within a cone of size R = 0.2 around a muon can- didate. Electrons and muons are removed if they are within R = min(0.4, 0.04 + 10 GeV/pT) of the axis of any sur- viving jet. Electron candidates are reconstructed from tracks in the ID matched to clusters [62] of energy deposits in the electro- magnetic (EM) calorimeter system. Electrons are required to satisfy \|η\| < 2.47, excluding the transition region between calorimeters,1.37 < \|η\| < 1.52.Muoncandidatesarerecon- structed from combined tracks using information from both the MS and the ID [63]. 5 Event selection and categorisations The absolute value of the pseudorapidity of each jet is required to be lower than 4.5 and the transverse momentum has to be at least 30 GeV. To reduce the contamination from jets originating from pile-up vertices, selection requirements on two independent multi- variate classiﬁers are applied to the selected jets. The ﬁrst classiﬁer is based on calorimeter and tracking information and is applied to jets with pT < 60 GeV and \|η\| < 2.4 [68], while the second classiﬁer is based on jet shapes and topo- logical jet correlations in pile-up interactions and is applied to jets with pT < 50 GeV and \|η\| > 2.4 [69]. and the ET vector using the collinear approximation [73]. Further selection requirements speciﬁc to the topolo- gies of the ggF + 2 jets and VBF signal processes are used to deﬁne the two signal regions (SRs). In the ggF + 2 jets analysis, the angular distance between the two leading jets, R j j, is required to be larger than 1.0, the transverse momentum of the dilepton system, pT,ℓℓ, has to exceed 20 GeV, and mℓℓhas to be below 90 GeV. In addition, the transverse mass mT of the Higgs boson can- didate must be below 150 GeV. This transverse mass is deﬁned as mT = (Eℓℓ+ Emiss T )2 −\| ⃗pT,ℓℓ+ ⃗Emiss T \| 2 with Eℓℓ= \| ⃗pT,ℓℓ\|2 + m2 ℓℓand ⃗pT,ℓℓthe combined dilepton momentum vector in the transverse plane. The selection requirement placed on pT,ℓℓreduces contributions from the Z + jets background, while the requirements on mℓℓand mT decrease the top-quark background. In the VBF analysis, an ‘outside lepton veto’ is applied, which requires the two lep- tons to be within the rapidity gap spanned by the two leading jets, and a ‘central jet veto’ rejects events with additional jets with pT > 20 GeV in the rapidity gap between the two lead- ing jets. Table 3 summarises the selection requirements used to deﬁne the SRs of the ggF + 2 jets and VBF analyses. Further selection requirements speciﬁc to the topolo- gies of the ggF + 2 jets and VBF signal processes are used to deﬁne the two signal regions (SRs). 5 Event selection and categorisations This combination is based on an overall ﬁt using the hits of the ID track, the energy loss in the calorimeter, and the hits in the muon spectrometer. The absolute pseudorapidity of the muon candidate is required to be lower than 2.5. viving jet. In order to be considered for the ﬁnal analysis, candidate events must contain exactly two prompt leptons with oppo- site electrical charge and different lepton ﬂavour, with the higher-pT (leading) lepton having pT > 22 GeV and the subleading lepton having pT > 15 GeV. The invariant mass of the dilepton system, mℓℓ, must exceed 10 GeV. At least one of the leptons must be matched to an object that triggered the recording of the event. In the case that a dilepton trigger is solely responsible for the recording of an event, each lepton must be matched to one of the trigger-level objects. In addi- tion, events must contain at least two jets passing all selec- tion requirements. To reduce backgrounds from top-quark production, events are vetoed if they contain a b-tagged jet with a pT larger than 20 GeV (Nb-jet,pT>20 GeV = 0). The Z(→ττ) + jets background is reduced by introducing an mττ < 66 GeV selection requirement. The observable mττ is calculated from the four-vectors of the two charged leptons and the ⃗Emiss T vector using the collinear approximation [73]. Further selection requirements speciﬁc to the topolo- gies of the ggF + 2 jets and VBF signal processes are used to deﬁne the two signal regions (SRs). In the ggF + 2 jets analysis, the angular distance between the two leading jets, R j j, is required to be larger than 1.0, the transverse momentum of the dilepton system, pT,ℓℓ, has to exceed 20 GeV, and mℓℓhas to be below 90 GeV. In addition, the transverse mass mT of the Higgs boson can- didate must be below 150 GeV. This transverse mass is deﬁned as mT = (Eℓℓ+ Emiss T )2 −\| ⃗pT,ℓℓ+ ⃗Emiss T \| 2 with Eℓℓ= \| ⃗pT,ℓℓ\|2 + m2 ℓℓand ⃗pT,ℓℓthe combined dilepton momentum vector in the transverse plane. The selection requirement placed on pT,ℓℓreduces contributions from the Z + jets background, while the requirements on mℓℓand mT decrease the top-quark background. 5 Event selection and categorisations In the ggF + 2 jets analysis, the angular distance between the two leading jets, R j j, is required to be larger than 1.0, the transverse momentum of the dilepton system, pT,ℓℓ, has to exceed 20 GeV, and mℓℓhas to be below 90 GeV. In addition, the transverse mass mT of the Higgs boson can- didate must be below 150 GeV. This transverse mass is deﬁned as mT = (Eℓℓ+ Emiss T )2 −\| ⃗pT,ℓℓ+ ⃗Emiss T \| 2 with Jets containing b-hadrons are identiﬁed using the MV2c10 b-tagging algorithm [70,71] with an operating point that has an overall efﬁciency of 85%, evaluated in simulated t ¯t events. The corresponding overall rejection rate for jets orig- inating from light-ﬂavour hadrons or gluons is 34, while the overall rejection rate for jets containing c-hadrons is approximately 3. ⃗ The missing transverse momentum ⃗Emiss T is deﬁned as the negative vector sum of the ⃗pT of all the selected leptons and 12 3 3 Eur. Phys. J. C (2022) 82 :622 622 Page 8 of 33 Table 3 Event selection criteria used to deﬁne the signal regions for the ggF + 2 jets and VBF event categories Table 3 Event selection criteria used to deﬁne the signal regions for the ggF + 2 jets and VBF event categories (t ¯t), single top quarks (Wt), non-resonant dibosons (W W, W Z/γ ∗, Z Z/γ ∗, Wγ ,or Zγ ),andDrell–Yan Z/γ ∗(primar- ily in the decay Z →ττ). Other background contributions arise from W + jets and multijet production with misiden- tiﬁed leptons, which originate either from decays of heavy- ﬂavour hadrons or from jets mimicking prompt-lepton signa- tures in the detector. A small but non-negligible contribution to the signal region arises from V H production processes and events including H →ττ decays. These two sources are summarised as one component referred to as “Other Higgs”, which is treated as a background to the studies of the ggF + 2 jets and VBF production modes. Boosted decision trees (BDTs) are used in both analy- ses to further distinguish between the signal processes and the most dominant background processes. 5 Event selection and categorisations In the ggF + 2 jets analysis, the discriminating observables used by the BDT are mℓℓ, mT, pT,ℓℓ, and the azimuthal angle φℓℓbetween the two leptons, as well as the observables min R(ℓ1, ji) and min R(ℓ2, ji), deﬁned as the minimum distance between the leading or subleading lepton and the two tagging jets. The mℓℓand mT observables provide the best separation between the signal and background processes, and are therefore the most important inputs to the BDT. The sample used to train the BDT consists of the sum of simulated ggF + 2 jets events stemmingfromthethreebenchmarksmodelsdeﬁnedinTable 1 as signal, and the sum of the top quark, diboson and Z + jets processes as background. Neither the input observables nor the BDT response show any signiﬁcant separation between the three CP benchmark scenarios. In the VBF analysis, the same BDT is used as described in Ref. [20], where mℓℓ, φℓℓ, and mT are used in addition to the dijet invariant mass m j j, the rapidity difference y j j between the two leading jets, the lepton centrality ( ℓCℓwhere Cℓ= \|2ηℓ− j η j\|/ η j j quantiﬁes the position of a lepton relative to the two lead- ing jets), the sum of the invariant masses of all four possible lepton–jet pairs, ℓ, j mℓ, j, and the total transverse momen- tum ptot T , deﬁned as the magnitude of the vectorial sum of all selected objects. The most discriminating observables used by the BDT of the VBF analysis are m j j and y j j. Dedicated control regions (CRs), which do not overlap with the signal region, are used to constrain the normalisation of the most dominant background processes. A top CR is used to correct the normalisation of the combined t ¯t and Wt backgrounds. A CR is employed to adjust the normalisation of the Z(→ττ) + 2 jets production. In the ggF + 2 jets analysis, an additional W W CR is used. The selection criteria used to deﬁne the various CRs are detailed in Table 4. Diboson backgrounds other than W W are estimated using MC simulations, while the contributions from background processes containingmisidentiﬁedleptons areestimatedwith a data-driven technique [20]. For this purpose, a control sam- ple is deﬁned using events with one identiﬁed lepton and one lepton failing the nominal object deﬁnition requirements but passing looser requirements (referred to as “anti-identiﬁed”). 5 Event selection and categorisations The contribution of the misidentiﬁed-lepton background to the signal region is estimated by scaling the control sam- ple via pT- and η-dependent extrapolation factors, which are deﬁned as the ratio of the identiﬁed leptons to anti-identiﬁed Eur. Phys. J. C (2022) 82 :622 Table 4 Event selection criteria used to deﬁne the various control regions for the ggF + 2 jets and VBF event categories. Only the changes relative to the signal region deﬁnitions (see Table 3) are stated Table 4 Event selection criteria used to deﬁne the various control regions for the ggF + 2 jets and VBF event categories. Only the changes relative to the signal region deﬁnitions (see Table 3) are stated leptons and are determined using Z + jets and multijet data samples. Table 5 Post-ﬁt NFs and their uncertainties for the Z + jets, top and W W backgrounds. Both sets of normalisation factors differ slightly depending on which (B)SM model is tested, but are consistent within their total uncertainties Table 5 Post-ﬁt NFs and their uncertainties for the Z + jets, top and W W backgrounds. Both sets of normalisation factors differ slightly depending on which (B)SM model is tested, but are consistent within their total uncertainties Phase space NFZ+jets NFtop NFW W ggF + 2 jets 0.85+0.09 −0.09 1.05+0.06 −0.05 1.0+0.7 −0.4 VBF 0.94+0.21 −0.18 1.00+0.06 −0.05 – The composition of the signal and control regions is detailed in Table 6 for the ggF + 2 jets analysis and in Table 8 for the VBF analysis. For both analyses, the largest back- groundcontributiontothesignalregionstemsfromtop-quark pair production with around 50% of all selected events. The next leading contributions to the signal regions are from W W and Z + jets production, which contribute each with roughly 20% of all selected events. Phase space NFZ+jets NFtop NFW W ggF + 2 jets 0.85+0.09 −0.09 1.05+0.06 −0.05 1.0+0.7 −0.4 VBF 0.94+0.21 −0.18 1.00+0.06 −0.05 – of the misidentiﬁed-lepton background [20]. Smaller uncer- tainties are due to the lepton momentum scale and resolution, the lepton identiﬁcation and isolation criteria [62,63,75], the measurement of missing transverse momentum [72], and the luminosity measurement [76]. The luminosity uncertainty is only applied to those processes that are normalised to theo- retical predictions. The procedure described in Sect. 8 is used to obtain the normalisation factors (NFs) of the dominant background pro- cesses from a combined SR + CR ﬁt to data, where each background normalisation is correlated across all regions. In the ggF + 2 jets analysis, the normalisations of the top quark, Z + jets and W W backgrounds are determined simultane- ously. Eur. Phys. J. C (2022) 82 :622 The W W CR has approximately equal contributions from W W and top processes, so there is a moderate anti- correlation between the NFs of the W W and top-quark back- grounds. The W W CR nonetheless constrains the sizeable W W modelling uncertainty. In the VBF analysis, the nor- malisations of the top quark and Z + jets backgrounds are determined from a simultaneous ﬁt to their dedicated CRs. The normalisation factors obtained from the combined SR + CR ﬁt to data are summarised in Table 5 separately for the ggF + 2 jets and VBF analyses. Theoretical uncertainties are assessed by comparing nom- inal and alternative event generators and UEPS models, as indicated in Table 2. In general, the perturbative precision and the PDF sets used in these alternative conﬁgurations match those of the nominal generators (unless explicitely stated). Uncertainties due to the PDF set parameterisation are evaluated using replica sets, and uncertainties due to miss- ing higher orders are evaluated by varying appropriate scale parameters, as described below. The uncertainties in modelling the t ¯t background are derived as follows. To assess potential differences in the matching between the matrix element and parton shower, the predictions of the nominal generator set-up are compared with those of the Sherpa 2.2.1 generator. The cross-section calculation of this alternative generator set-up is based on the NNPDF3.0 NNLO PDF set and includes diagrams with up to one additional emission at NLO precision in QCD, while dia- grams with two, three or four parton emissions are described at LO accuracy. Parton shower modelling uncertainties are derived by replacing Pythia8.210 by Herwig 7.0.1 [77] (which uses the H7-UE-MMHT set of tuned parameters [78]) and comparing the corresponding yields and shapes with those from the nominal set-up. The uncertainty due to neglected higher orders in QCD is estimated by simul- taneously increasing (decreasing) the renormalisation and factorisation scales μr and μf by a factor of 2 (0.5), and by setting the hdamp parameter to 1.0 (3.0) times the top- 6 Background estimation The background contamination in the SRs originates from various processes including the production of top-quark pairs 3 Page 9 of 33 622 Page 9 of 33 622 Eur. Phys. J. C (2022) 82 :622 Table 4 Event selection criteria used to deﬁne the various control regions for the ggF + 2 jets and VBF event categories. Only the changes relative to the signal region deﬁnitions (see Table 3) are stated 7 Systematic uncertainties Its impact on the ﬁnal results is small with respect to the statistical uncertainties. In the VBF analysis, parton shower model uncertainties on the VBF signal are calculated by setting the Pythia shower scale factor to 2 or 1/2. quark mass. For Wt production, uncertainties in the match- ing between the matrix element and parton shower are evalu- ated by comparing the predictions of Powheg-Box v2 + Herwig++ [79] with those of MG5_aMC@NLO 2.2.2 + Herwig++, while the parton shower model uncertainties are estimated by comparing Powheg-Box v2 + Pythia6.428 with Powheg-Box v2 + Herwig++. In these samples, the UE-EE-5-CTEQ6L1 set of tuned parameters [79] is used to conﬁgure the Herwig++ generator. In addition, the nominal conﬁguration for the Wt process is compared with an alter- native approach in which the diagram subtraction scheme [80] is used instead of the DR scheme. The uncertainty due to neglected higher orders in QCD is estimated by increasing (decreasing) the renormalisation and factorisation scales by a factor of 2 (0.5) relative to their nominal value. The uncertainties in modelling the production of dibosons with jets are evaluated by comparing the predictions of the Sherpa 2.2.2 and MG5_aMC@NLO 2.3.3 + Pythia8.212 generators, where the latter provides NLO precision in QCD for the simulation of production modes with up to one parton in addition to the diboson system [54]. The cross-section cal- culation is based on the NNPDF3.0 PDF set, and the A14 set of tuned parameters is used for the simulation of the parton shower. In this generator set-up, the FxFx merging scheme [81] is used to remove overlaps between the partonic conﬁgu- ration produced during the simulation of the matrix element and the parton shower, using a merging scale of 20 GeV. For the predictions of the W Z/γ ∗, Z Z/γ ∗, and W W pro- cesses, variations of the matching scale are also considered, where the nominal value, 20 GeV, is changed to 30 GeV and 15 GeV. In addition, the effects of QCD factorisation and renormalisation scale variations are considered by indi- vidually varying μr and μf by a factor of 2 or 0.5. Six com- binations are considered: (μr, μf) = (0.5, 0.5), (0.5, 1.0), (1.0, 0.5), (1.0, 2.0), (2.0, 1.0), and (2.0, 2.0) times their nominal value. 7 Systematic uncertainties The effects of the systematic uncertainties on the expected signal and background yields in the various signal and con- trol regions are evaluated following the procedures in Ref. [20]. In addition, the effects of the uncertainties on the shapes of the j j and BDT response distributions are considered. They are evaluated by individually comparing the nominal distribution with those corresponding to up and down varia- tions of a particular uncertainty. The sources of uncertainty are grouped into two cate- gories: experimental and theoretical. The dominant exper- imental uncertainties for both analyses are related to the b- tagging efﬁciency [74], the jet energy scale and resolution [67], the modelling of pile-up activity, and the estimation 12 3 3 622 Page 10 of 33 Eur. Phys. J. C (2022) 82 :622 622 Page 10 of 33 tions of the nominal generator conﬁguration with those of the MG5_aMC@NLO 2.4.2 + Herwig 7.0.1 generators, where the H7-UE-MMHT set of tuned parameters is used for the simulation of the parton shower. The effects of parton shower model uncertainties on the VBF signal are calculated by vary- ing the shower scale up and down by a factor of 2 in Pythia8. The effects of QCD scale variations are determined for the ggF +2jetsandVBFprocessesinthesamewayasforthevec- tor boson plus jets backgrounds. Uncertainties in the ggF and VBF production cross sections and jet bin migration effects for the ggF process are accounted for following the recom- mendations in Ref. [44]. In the ggF + 2 jets analysis, further uncertainties are considered for the VBF background. Uncer- tainties due to the matching of the matrix element and the parton shower are evaluated by comparing the predictions of Powheg-Box v2 + Pythia8.212 with the predictions of MG5_aMC@NLO 2.3.3 + Pythia8.212, while uncertainties due to the parton shower model are derived by comparing the predictions of Powheg-Box v2 + Pythia8.212 with those of Powheg-Box v2 + Herwig 7.0.1, where the H7-UE- MMHT set of tuned parameters is used for the simulation of the parton shower. In the VBF study, an additional shape uncertainty is applied to the j j and BDT response dis- tributions of all backgrounds to account for a non-closure between data and the simulations in the dijet invariant mass spectrum. This non-closure is on the order of a few percent at low m j j values and ranges up to 15% for m j j values above 1 TeV. 123 6 BSM effects can have signiﬁcant contributions to the production rates of the signal process. For example, the production cross section of the CP-odd benchmark scenario deﬁned in Table 1 is enhanced by a factor of around 2.3 with respect to the SM expectations, while the CP-mixed benchmark scenario has a cross section of around 1.6 times the SM expectation. 5 The split into BDT score regions aims to maximise the signal to background ratio, while the split into \| η j j\| regions is motivated by the fact that the separation between the various CP hypotheses for the signed j j distribution increases with higher \| η j j\| values. 7 Systematic uncertainties Finally, the uncertainty due to the QCD scales is obtained as the largest effect of these six variations of μr and μf from their nominal value. Uncertainties due to the modelling of PDFs are evaluated for the signal processes and all relevant backgrounds by com- paring the predictions of the nominal PDF set with those of the CT14 and MMHT2014 PDF sets and then comparing the maximum difference with the difference from the root-mean- square spread of the NNPDF3.0 replica sets. The larger of the two is taken as the uncertainty. The uncertainties in the signal processes are evaluated for the SM hypotheses and are extrapolated to the various BSM scenarios while assum- ing that QCD scale, PDF and parton shower model effects factorise with the modulations of the j j shape and cross sections due to BSM contributions. These modelling uncer- tainties are treated as fully correlated between the SM and BSM hypotheses. For the Z + jets background, matrix element and parton shower uncertainties are both accounted for by comparing the predictions of the Sherpa 2.2.1 generator with those of MG5_aMC@NLO 2.2.2 + Pythia8.186, which provides matrix elements at LO accuracy with up to four ﬁnal-state partons. The alternative event generator set-up is based on the NNPDF2.3 LO PDF set for the cross-section calcula- tions and the A14 set of tuned parameters for the simulation of the parton shower. The effect of QCD scale variations on the Z + jets background are determined in the same way as for the diboson plus jets backgrounds. The most signiﬁcant theoretical uncertainties are related to the modelling of the top-quark and W W backgrounds and the ggF process. Tables 7 and 11 rank the various uncer- tainties and show their impact on the studies of the ggF + 2 jets and VBF processes. Both studies are dominated by sta- tistical uncertainties. Some systematic uncertainties have a signiﬁcant impact on the determination of the post-ﬁt nor- malisation factors (presented in Table 5). In the ggF + 2 jets The uncertainties in modelling the ggF and VBF produc- tion modes of the Higgs boson are evaluated as follows. Uncertainties due to the parton shower model for the ggF + 2 jets process are determined by comparing the predic- 123 123 Page 11 of 33 Eur. Phys. J. 8 Results A maximum likelihood (ML) ﬁt is used for the statistical interpretation of the results from both the ggF + 2 jets and VBF analyses. Fits are simultaneously performed on all con- sidered SRs and CRs of each analysis independently in order to constrain the normalisation of backgrounds and the nui- sance parameters (NPs) describing the systematic uncertain- ties. Each systematic variation enters the ﬁt as an individual nuisance parameter. Correlations between systematic uncer- tainties are maintained across processes and channels. Due to the presence of bins with low event yields, all the NPs describing the systematic uncertainties are incorporated into the ﬁt using a log-normal constraint, such that expected event counts remain positive for all values of the corresponding NPs. Asimov datasets [82] are used to study the expected performance of each ﬁt. Four different ﬁts are performed in the ggF + 2 jets event category: • The signal strength parameter μggF+2jets for the ggF + 2 jets signal process is determined. This parameter is deﬁned as the ratio of the measured signal yield to that predicted by the SM. • In order to constrain BSM effects in the effective Higgs– gluon coupling, tan(α) is scanned. Two different conﬁg- urations are used in the ML procedure: Parameter morphing [83,84] is used to interpolate and extrapolate from a small set of κgg cos(α) and κgg sin(α) (or aL and aT) coupling benchmarks to a large variety of coupling scenarios. The input distributions to the morphing are normalised to their expected cross sections. – The normalisation of the signal process is uncon- strained such that the analysis only exploits the shape information of the ﬁt input distribution to distinguish between the different CP scenarios. The ﬁnal results are obtained by applying the maximum- likelihood procedure individually to each coupling parameter hypothesis, where the background prediction is only affected by changes to nuisance parameters in the minimisation. A negative log-likelihood (NLL) curve is constructed as a func- tion of the relevant coupling parameters. The best estimate for the parameter of interest is obtained at the point where the NLL curve reaches its minimum. In addition, central conﬁ- denceintervalsaredeterminedfromtheappropriatedeviation of the NLL from its minimum. – The signal normalisation is constrained to the model predictions. Thus both the shape and rate information are considered for each CP scenario. 7 Systematic uncertainties C (2022) 82 :622 622 vals.5 The bin boundaries for the BDT score and \| η j j\| intervals that deﬁne the categories are [0.1, 0.4, 0.7, 1.0] and [0.0, 1.0, 2.0, 3.0, ∞], respectively. Finally, the event yields from the top, Z →ττ and W W CRs, as well as the event yields within the low-BDT-score intervals wBDT ∈ [−1.0, −0.3] and wBDT ∈[−0.3, 0.1] in each \| η j j\| region are included in the ML ﬁt. These regions provide constraints on the normalisations of the top quark, Z + jets and W W + jets backgrounds, which are free to ﬂoat in the ﬁt, and help to reduce the impact of the b-tagging uncertainties. All other background contributions are set to their respective SM pre- dictions, but are allowed to vary within their uncertainties. analysis, there are signiﬁcant anti-correlations between the normalisation of the top-quark background and the uncer- tainty in the b-tagging efﬁciency. The normalisation factor of the W W background is signiﬁcantly anti-correlated with both the uncertainty in the b-tagging efﬁciency and the effect of the QCD scale uncertainty on the W W backgrounds, while the normalisation factor of the Z + jets background is sig- niﬁcantly anti-correlated with the jet energy scale uncertain- ties. In the VBF analysis, no signiﬁcant anti-correlations are observed between the normalisation factors and the various uncertainties. For the measurement of the signal strength parameter for ggF + 2 jets events, μggF+2jets, the ML ﬁt uses the same ﬁve BDT-score and four \| η j j\| intervals as those used for the studies of the effective Higgs–gluon coupling. In addition to these intervals, the event yields from the top, Z →ττ, and W W CRs are included in the ﬁt. The normalisations of the top quark, Z + jets, and W W + jets backgrounds are free to ﬂoat in the ﬁt, while all other background contributions are set to their respective SM predictions, but are allowed to vary within their uncertainties. 8 Results Using rate information in addition to the shape in the ﬁt increases the sensitivity to distinguish between the vari- ous benchmark points.6 However, the rate can be affected 8.1 ggF + 2 jets category 3 Post-ﬁt distribution of the BDT response observable presented in the four \| η j j\| categories of the ggF + 2 jets signal region, with signal and background yields ﬁxed from the ﬁt for μggF+2jets. Data-to- simulation ratios are shown at the bottom of the plot. The shaded areas depict the total uncertainty. The distributions of the ggF + 2 jets and VBF processes are overlaid with their respective contributions multiplied by 50 The post-ﬁt distribution of the inputs to the signal strength parameter μggF+2jets determination is depicted in Fig. 3. The normalised j j distribution in the various event categories of the ggF + 2 jets signal region is depicted in Fig. 4. Events are weighted by ln(1 + NS/NB), where NS (NB) is the post- ﬁt signal (background) event yield for each event category. This distribution is presented for the minimum of the NLL curve from the ﬁt conﬁguration that exploits both shape and rate information. The post-ﬁt event yields in the signal region and the various control regions are presented in Table 6. Fig. 4 The weighted j j post-ﬁt distribution in the ggF + 2 jets sig- nal region, with signal and background yields ﬁxed from the ﬁt to tan(α) using shape and rate information. Data-to-simulation ratios are shown at the bottom of the plot. The shaded areas depict the total uncertainty The signal strength parameter of the ggF + 2 jets pro- cess is found to be μggF+2jets = 0.5 ± 0.4(stat.)+0.7 −0.6(syst.), which is consistent with the SM prediction. The observed and expected likelihood curves corresponding to scans over tan(α) are presented in Fig. 5. The values of the NLL are evaluated in steps of (tan(α)) = 0.2, and the minima of all ﬁts are consistent with zero, i.e. the SM hypothesis. If only the shape information is used, the data are not sensi- tive enough to provide 68% conﬁdence level (CL) intervals on tan(α). The ML ﬁt conﬁguration that uses both shape Fig. 4 The weighted j j post-ﬁt distribution in the ggF + 2 jets sig- nal region, with signal and background yields ﬁxed from the ﬁt to tan(α) using shape and rate information. Data-to-simulation ratios are shown at the bottom of the plot. 8.1 ggF + 2 jets category The ML ﬁts that constrain BSM effects in the effective Higgs–gluon coupling use the distribution of the signed j j observable as input, divided into 12 categories. These 12 different categories are obtained by splitting the signal region into three BDT score intervals times four \| η j j\| inter- 12 3 622 Page 12 of 33 Eur. Phys. J. C (2022) 82 :622 Fig. 3 Post-ﬁt distribution of the BDT response observable presented in the four \| η j j\| categories of the ggF + 2 jets signal region, with signal and background yields ﬁxed from the ﬁt for μggF+2jets. Data-to- simulation ratios are shown at the bottom of the plot. The shaded areas depict the total uncertainty. The distributions of the ggF + 2 jets and VBF processes are overlaid with their respective contributions multiplied by 50 by multiple BSM effects, while the shape isolates CP- dependent variations. by multiple BSM effects, while the shape isolates CP- dependent variations. • A simultaneous ﬁt of the coupling-strength scale fac- tors κgg cos(α) and κgg sin(α) is performed. This study exploits both shape and rate information. The inclusive ggF production process is split into two components, onedescribingcontributions fromggF +0/1jets and the other ggF + 2 or more jets, where only the latter one is sensitive to the CP nature of the effective Higgs–gluon cou- pling. The treatment of these two components varies between the different ﬁt types. For scans over tan(α) where only the shape is used, the normalisation of the ggF + 2 jets compo- nent ﬂoats freely in the ﬁt, while the normalisation of the ggF + 0/1 jets component may vary taking into account its uncer- tainties. The normalisations of both components are scaled consistently to the model predictions when rates are used in the ﬁt. Fig. 3 Post-ﬁt distribution of the BDT response observable presented in the four \| η j j\| categories of the ggF + 2 jets signal region, with signal and background yields ﬁxed from the ﬁt for μggF+2jets. Data-to- simulation ratios are shown at the bottom of the plot. The shaded areas depict the total uncertainty. The distributions of the ggF + 2 jets and VBF processes are overlaid with their respective contributions multiplied by 50 Fig. Table 6 Post-ﬁt event yields in the signal and control regions obtained from the study of the signal strength parameter μggF+2jets. The quoted uncertainties include those from theoretical and experimental systematic sources and those due to sample statistics. The ﬁt constrains the total expected yield to the observed yield. The diboson background is split into W W and non-W W contributions 7 Precise measurements of the inclusive ggF cross section give tighter constraints on the individual parameters [85], due to its dependence on κ2 gg cos2(α) and κ2 gg sin2(α). 8.1 ggF + 2 jets category The shaded areas depict the total uncertainty Table 6 Post-ﬁt event yields in the signal and control regions obtained from the study of the signal strength parameter μggF+2jets. The quoted uncertainties include those from theoretical and experimental systematic sources and those due to sample statistics. The ﬁt constrains the total expected yield to the observed yield. The diboson background is split into W W and non-W W contributions Process Top CR W W CR Z →ττ CR SR ggF + 2 jets 20 ± 20 < 0.1 10 ± 10 60 ± 80 ggF + 0/1 jets 4 ± 1 < 0.1 3 ± 1 40 ± 20 VBF 8 ± 1 < 0.1 7 ± 1 70 ± 10 Other Higgs 6 ± 3 2 ± 1 20 ± 10 30 ± 10 t ¯t, Wt 17800 ± 200 3100 ± 500 390 ± 60 2300 ± 300 W W 180 ± 80 1400 ± 500 200 ± 70 1200 ± 400 Z + jets 220 ± 30 16 ± 3 1960± 70 1000 ± 100 W + jets 600 ± 200 140 ± 30 90 ± 20 390 ± 80 Non-W W dibosons 40 ± 30 100 ± 30 120 ± 50 240 ± 80 Observed 18886 4778 2800 5209 12 3 Page 13 of 33 622 Page 13 of 33 622 Eur. Phys. J. C (2022) 82 :622 622 (a) (b) Fig. 5 Expected and observed likelihood curves for scans a over tan(α) where only the shape is taken into account in the ﬁt, and b over tan(α) when both shape and normalisation are used (a) (b) (b) (a) Fig. 5 Expected and observed likelihood curves for scans a over tan(α) where only the shape is taken into account in the ﬁt, and b over tan(α) when both shape and normalisation are used Fig. 6 68% and 95% CL two-dimensional likelihood contours of the CP-even and CP-odd coupling parameters κgg cos(α) and κgg sin(α). The minima are represented by black stars, while the SM point is shown as a red star Table 7 Breakdown of the main contributions to the total uncertainty in tan(α) based on the ﬁt that exploits both shape and rate information. Individual sources of systematic uncertainty are grouped into either the theoretical or the experimental uncertainty. 8.1 ggF + 2 jets category The sum in quadrature of the individual components differs from the total uncertainty due to correlations between the components correlations between the components Source (tan(α)) Total data statistical uncertainty 0.4 SR statistical uncertainty 0.33 CR statistical uncertainty 0.10 MC statistical uncertainty 0.14 Total systematic uncertainty 0.28 Theoretical uncertainty 0.23 Top-quark bkg. 0.15 ggF signal 0.14 W Z, Z Z, Wγ , Zγ bkg. 0.06 W W bkg. 0.06 Z/γ ∗bkg. 0.016 VBF bkg. 0.015 Experimental uncertainty 0.21 b-tagging 0.16 Modelling of pile-up 0.10 Jets 0.07 Misidentiﬁed leptons 0.04 Luminosity 0.034 Total 0.5 Fig. 6 68% and 95% CL two-dimensional likelihood contours of the CP-even and CP-odd coupling parameters κgg cos(α) and κgg sin(α). The minima are represented by black stars, while the SM point is shown as a red star val. The relative impact of the main uncertainties on tan(α) is presented in Table 7. The 68% and 95% CL two-dimensional likelihood con- tours of the simultaneous scan over the CP-even and CP-odd coupling strength parameters κgg cos(α) and κgg sin(α) are presented in Fig. 6. Two minima are found, as the signal rate depends on the square of both couplings. The best-ﬁt values observed in the data are consistent with the SM predictions within the 68% CL, while \|κgg cos(α)\| values above 1.6 and \|κgg sin(α)\| values above 1.1 are excluded at 95% CL.7 8.2 VBF category and rate information provides a best-ﬁt value of tan(α) = 0.0 ± 0.4(stat.) ± 0.3(syst.) for both the ﬁts to data and to Asimov data. The observed sensitivity is slightly worse than the expected sensitivity due to a lower than expected signal yield (consistent with a signal strength value below unity). Hence, the ﬁt to this dataset does not provide a 95% CL inter- and rate information provides a best-ﬁt value of tan(α) = 0.0 ± 0.4(stat.) ± 0.3(syst.) for both the ﬁts to data and to Asimov data. The observed sensitivity is slightly worse than the expected sensitivity due to a lower than expected signal yield (consistent with a signal strength value below unity). Hence, the ﬁt to this dataset does not provide a 95% CL inter- To constrain the polarisation-dependent coupling-strength scale factors in the VBF production process, the signal region 12 3 622 Page 14 of 33 Eur. Phys. J. C (2022) 82 :622 Fig. pendently in order to probe them in a model-independent way. Figure 7 depicts the weighted j j distribution in all categories of the VBF signal region. Events are weighted by ln(1 + NS/NB) in their corresponding event category. The post-ﬁt event yields in the signal and the control regions are presented in Table 8. The results of the likelihood scans are given in Fig. 8. The scan over aL (aT) is shown in the upper (lower) panel. Both scans have been performed in two con- ﬁgurations: the LLH curves shown in Fig. 8b and c are the results of the ﬁt in which both the shape and normalisation of the signal are taken into account, while the LLH curves in Fig. 8a are obtained using only the shape information. The largest sensitivity to aL stems from the rate information. The asymmetry of the curves results from the asymmetric behaviour of the cross section with respect to the changes in the parameter values (see Ref. [27]). The expected sensitiv- ity to aT comes predominantly from the shape information, as the likelihood ratio increases only slightly when adding the normalisation information. The resulting best-ﬁt values and their uncertainties computed at 68% CL are presented in Table 9. All measurements are consistent with the SM expec- tations. The dominant sources of uncertainty are related to the limited data yields and to the uncertainties in modelling the top-quark and W W backgrounds. Figure 7 depicts the weighted j j distribution in all categories of the VBF signal region. Events are weighted by ln(1 + NS/NB) in their corresponding event category. The post-ﬁt event yields in the signal and the control regions are presented in Table 8. The results of the likelihood scans are given in Fig. 8. The scan over aL (aT) is shown in the upper (lower) panel. Both scans have been performed in two con- ﬁgurations: the LLH curves shown in Fig. 8b and c are the results of the ﬁt in which both the shape and normalisation of the signal are taken into account, while the LLH curves in Fig. 8a are obtained using only the shape information. The largest sensitivity to aL stems from the rate information. 8 A proﬁled parameter is ﬁxed to its unconditional maximum likelihood value for each value of the parameter of interest (POI). Then, the POI is ﬁxed to its conditional maximum likelihood value. 8.1 ggF + 2 jets category 7 The weighted j j distribution in the VBF signal region, with signal and background yields ﬁxed from the ﬁt for εV V using shape and rate information. Data-to-simulation ratios are shown at the bottom of the plot. The shaded areas depict the total uncertainty Table 8 Post-ﬁt event yields in the signal and control regions obtained from a scan over εV V exploiting both shape and rate information. The quoted uncertainties include those from theoretical and experimental systematic sources and those due to sample statistics. The ﬁt constrains the total expected yields to the observed yields. The diboson background is split into W W and non-W W contributions Process Top CR Z →ττ CR SR VBF 3.2 ± 2.2 2.6 ± 1.8 34 ± 22 ggF 3.9 ± 1.7 2.4 ± 1.0 28 ± 12 Other Higgs 1.5 ± 0.7 6.2 ± 3.1 6.0 ± 3.0 t ¯t, Wt 7400 ± 100 53 ± 7 1220 ± 100 W W 51 ± 6 21.8 ± 2.9 360 ± 70 Z + jets 54 ± 10 370 ± 24 320 ± 70 W + jets 190 ± 40 23.0 ± 2.4 115 ± 27 Non-W W dibosons 14.3 ± 1.8 20.8 ± 3.3 83 ± 11 Observed 7668 501 2164 Fig. 7 The weighted j j distribution in the VBF signal region, with signal and background yields ﬁxed from the ﬁt for εV V using shape and rate information. Data-to-simulation ratios are shown at the bottom of the plot. The shaded areas depict the total uncertainty and background control regions deﬁned in Tables 3 and 4 are used as inputs to the ML ﬁt. In the signal region, the distribution of the signed j j observable is used in the four categories deﬁned by the BDT-score intervals [−1, 0.26, 0.61, 0.86, 1], following Ref. [20]. The ﬁrst (last) interval corresponds to the enhanced background (signal) phase-space region. No shape information is used in the con- trol regions. pendently in order to probe them in a model-independent way. Several types of ﬁts are performed in the VBF event cate- gory with both (aL, aT) and (κV V , εV V ) parameterisations: • One-dimensional ﬁts are performed – using only the dependence of the input distribution’s shape on the selected parameter of interest, with the other parameter ﬁxed to its SM value; – exploiting both shape and rate information, ﬁxing one parameter to its SM value. The parameters aL and κV V dominantly affect the total event yields because the longitudinal polarisation vec- tors of the massive gauge bosons are proportional to the energy and can give large enhancements to the total cross section.Theseparametersareonlyweaklyconstrainedby the shape of the j j distribution. The parameters aT and εV V , on the other hand, affect the shape of the j j distribution and can be constrained from both types of ﬁts. The kinematic distributions of the two tagging jets are related to the intrinsic structure of the Higgs boson production vertex and carry information about the polar- isation of the fusing gauge bosons. The asymmetry of the curves results from the asymmetric behaviour of the cross section with respect to the changes in the parameter values (see Ref. [27]). The expected sensitiv- ity to aT comes predominantly from the shape information, as the likelihood ratio increases only slightly when adding the normalisation information. The resulting best-ﬁt values and their uncertainties computed at 68% CL are presented in Table 9. All measurements are consistent with the SM expec- tations. The dominant sources of uncertainty are related to the limited data yields and to the uncertainties in modelling the top-quark and W W backgrounds. The results of the ML scans of pseudo-observables are shown in Table 10, with the uncertainty breakdown given in Table 11. Figure 9 shows the expected and observed like- lihood curves for scans over one pseudo-observable with the other one proﬁled. Both shape and rate information are employed. • Fits to one parameter are performed with the other being proﬁled.8 In these ﬁts, both parameters may vary inde- 8 A proﬁled parameter is ﬁxed to its unconditional maximum likelihood value for each value of the parameter of interest (POI). Then, the POI is ﬁxed to its conditional maximum likelihood value. 8 A proﬁled parameter is ﬁxed to its unconditional maximum likelihood value for each value of the parameter of interest (POI). pendently in order to probe them in a model-independent way. Results of ﬁts to one parameter with the other one ﬁxed or proﬁled are presented Type Expected Observed εV V shape-only ﬁt (κV V = 1 ) 0.00+0.23 −0.25(stat.)+0.14 −0.17(syst.) 0.14+0.39 −0.22(stat.)+0.16 −0.12(syst.) κV V shape + rate ﬁt (εV V = 0) 1.00+0.08 −0.10(stat.)+0.08 −0.13(syst.) 0.91+0.09 −0.12(stat.)+0.09 −0.18(syst.) εV V shape + rate ﬁt (κV V = 1 ) 0.00+0.18 −0.24(stat.)+0.08 −0.13(syst.) 0.09+0.13 −0.16(stat.)+0.06 −0.07(syst.) κV V shape + rate ﬁt (εV V proﬁled) 1.00+0.08 −0.10(stat.)+0.08 −0.13(syst.) 0.91+0.10 −0.18(stat.)+0.09 −0.17(syst.) εV V shape + rate ﬁt (κV V proﬁled) 0.00+0.22 −0.24(stat.)+0.11 −0.15(syst.) 0.13+0.28 −0.20(stat.)+0.08 −0.10(syst.) for aL and aT are shown. Results of ﬁts to one parameter with the other one ﬁxed or proﬁled are presented Table 9 Best-ﬁt values and their uncertainties as obtained from the shape-only and shape-plus-rate likelihood ﬁts to the Asimov dataset and to ATLAS data. Results of both the shape-only and shape+rate ﬁts Table 9 Best-ﬁt values and their uncertainties as obtained from the shape-only and shape-plus-rate likelihood ﬁts to the Asimov dataset and to ATLAS data. Results of both the shape-only and shape+rate ﬁts and to ATLAS data. Results of both the shape-only and shape+rate ﬁts Type Expected Observed aT shape-only ﬁt (aL = 1) 1.0 ± 0.5(stat.)+0.3 −0.4(syst.) 1.3+0.8 −0.4(stat.)+0.3 −0.2(syst.) aL shape + rate ﬁt (aT = 1) 1.00+0.08 −0.10(stat.)+0.07 −0.13(syst.) 0.90+0.09 −0.13(stat.)+0.08 −0.18(syst.) aT shape + rate ﬁt (aL = 1) 1.00+0.36 −0.49(stat.)+0.19 −0.27(syst.) 1.19+0.27 −0.32(stat.)+0.12 −0.14(syst.) aL shape + rate ﬁt (aT proﬁled) 1.00+0.08 −0.10(stat.)+0.08 −0.13(syst.) 0.91+0.10 −0.18(stat.)+0.09 −0.17(syst.) aT shape + rate ﬁt (aL proﬁled) 1.0+0.4 −0.5(stat.)+0.2 −0.4(syst.) 1.2 ± 0.4(stat.)+0.2 −0.3(syst.) Table 10 Best-ﬁt values and their uncertainties as obtained from the shape-only and shape-plus-rate likelihood ﬁts to the Asimov dataset and to ATLAS data. Results of both shape-only and shape+rate ﬁts for εV V Table 10 Best-ﬁt values and their uncertainties as obtained from the shape-only and shape-plus-rate likelihood ﬁts to the Asimov dataset and to ATLAS data. Results of both shape-only and shape+rate ﬁts for εV V and κV V are shown. Results of ﬁts to one parameter with the other one ﬁxed or proﬁled are presented shape-only and shape-plus-rate likelihood ﬁts to the Asimov dataset and to ATLAS data. pendently in order to probe them in a model-independent way. Then, the POI is ﬁxed to its conditional maximum likelihood value. 12 3 Eur. Phys. J. C (2022) 82 :622 Page 15 of 33 622 Page 15 of 33 622 622 (a) (b) (c) Fig. 8 Likelihood scans over the transversely (a, b) and longitudinally (c) polarised couplings. Fits using shape-only (a) and shape+rate (b, c) information are shown. All relevant experimental and modelling systematic uncertainties are considered in the ﬁts (a) (b) (a) (c) Fig. 8 Likelihood scans over the transversely (a, b) and longitudinally (c) polarised couplings. Fits using shape-only (a) and shape+rate (b, information are shown. All relevant experimental and modelling systematic uncertainties are considered in the ﬁts (c) (c) (c) Fig. 8 Likelihood scans over the transversely (a, b) and longitudinally (c) polarised couplings. Fits using shape-only (a) and shape+rate (b, c) information are shown. All relevant experimental and modelling systematic uncertainties are considered in the ﬁts Fig. 8 Likelihood scans over the transversely (a, b) and longitudinally (c) polarised couplings. Fits using shape-only (a) and shape+rate (b, c) information are shown. All relevant experimental and modelling systematic uncertainties are considered in the ﬁts Table 9 Best-ﬁt values and their uncertainties as obtained from the shape-only and shape-plus-rate likelihood ﬁts to the Asimov dataset and to ATLAS data. Results of both the shape-only and shape+rate ﬁts for aL and aT are shown. Results of ﬁts to one parameter with the other one ﬁxed or proﬁled are presented Type Expected Observed aT shape-only ﬁt (aL = 1) 1.0 ± 0.5(stat.)+0.3 −0.4(syst.) 1.3+0.8 −0.4(stat.)+0.3 −0.2(syst.) aL shape + rate ﬁt (aT = 1) 1.00+0.08 −0.10(stat.)+0.07 −0.13(syst.) 0.90+0.09 −0.13(stat.)+0.08 −0.18(syst.) aT shape + rate ﬁt (aL = 1) 1.00+0.36 −0.49(stat.)+0.19 −0.27(syst.) 1.19+0.27 −0.32(stat.)+0.12 −0.14(syst.) aL shape + rate ﬁt (aT proﬁled) 1.00+0.08 −0.10(stat.)+0.08 −0.13(syst.) 0.91+0.10 −0.18(stat.)+0.09 −0.17(syst.) aT shape + rate ﬁt (aL proﬁled) 1.0+0.4 −0.5(stat.)+0.2 −0.4(syst.) 1.2 ± 0.4(stat.)+0.2 −0.3(syst.) Table 10 Best-ﬁt values and their uncertainties as obtained from the shape-only and shape-plus-rate likelihood ﬁts to the Asimov dataset and to ATLAS data. Results of both shape-only and shape+rate ﬁts for εV V and κV V are shown. pendently in order to probe them in a model-independent way. The theoretical and experimental uncertainties are subdivided further into categories Table 11 The contributions of the leading individual systematic uncer- tainties together with the data statistical uncertainties, in the one- dimensional ﬁt for the pseudo-observables κV V (a) and εV V (b) for (b) (a) (b) Fig. 9 Likelihood scans over a κV V and b εV V , with the other parameter proﬁled. The ﬁts are performed using both shape and rate information. All relevant experimental and theoretical systematic uncertainties are considered in the ﬁts (b) (a) Fig. 9 Likelihood scans over a κV V and b εV V , with the other parameter proﬁled. The ﬁts are performed using both shape and rate information. All relevant experimental and theoretical systematic uncertainties are considered in the ﬁts pendently in order to probe them in a model-independent way. Results of both shape-only and shape+rate ﬁts for εV V ﬁxed or proﬁled are presented Type Expected Observed εV V shape-only ﬁt (κV V = 1 ) 0.00+0.23 −0.25(stat.)+0.14 −0.17(syst.) 0.14+0.39 −0.22(stat.)+0.16 −0.12(syst.) κV V shape + rate ﬁt (εV V = 0) 1.00+0.08 −0.10(stat.)+0.08 −0.13(syst.) 0.91+0.09 −0.12(stat.)+0.09 −0.18(syst.) εV V shape + rate ﬁt (κV V = 1 ) 0.00+0.18 −0.24(stat.)+0.08 −0.13(syst.) 0.09+0.13 −0.16(stat.)+0.06 −0.07(syst.) κV V shape + rate ﬁt (εV V proﬁled) 1.00+0.08 −0.10(stat.)+0.08 −0.13(syst.) 0.91+0.10 −0.18(stat.)+0.09 −0.17(syst.) εV V shape + rate ﬁt (κV V proﬁled) 0.00+0.22 −0.24(stat.)+0.11 −0.15(syst.) 0.13+0.28 −0.20(stat.)+0.08 −0.10(syst.) 123 622 Page 16 of 33 Eur. Phys. J. C (2022) 82 :622 6 age 6 o 33 u . ys. J. C ( 0 ) 8 :6 Table 11 The contributions of the leading individual systematic uncer- tainties together with the data statistical uncertainties, in the one- dimensional ﬁt for the pseudo-observables κV V (a) and εV V (b) for electroweak-boson polarisation in the VBF H →W W channel. Both the shape and rate information is exploited in the ﬁt. The theoretical and experimental uncertainties are subdivided further into categories (a) κV V ﬁt, εV V = 0 (b) εV V ﬁt, κV V = 1 Source κV V Source εV V Total data statistical uncertainty 0.11 Total data statistical uncertainty 0.14 SR data statistical uncertainty 0.10 SR data statistical uncertainty 0.14 CR data statistical uncertainty 0.019 CR data statistical uncertainty 0.011 MC statistical uncertainty 0.035 MC statistical uncertainty 0.036 Total systematic uncertainty 0.12 Total systematic uncertainty 0.056 Theoretical uncertainty 0.10 Theoretical uncertainty 0.050 Top-quark bkg. 0.072 Top-quark bkg. 0.039 W W bkg. 0.062 W W bkg. 0.036 ggF bkg. 0.033 ggF bkg. 0.013 Z/γ ∗bkg. 0.017 Z/γ ∗bkg. 0.012 VBF signal 0.019 VBF signal 0.010 Experimental uncertainty 0.050 Experimental uncertainty 0.024 Jet 0.026 Modelling of pile-up 0.022 b-tagging 0.014 Jet 0.018 Luminosity 0.011 Misidentiﬁed leptons 0.010 Misidentiﬁed leptons 0.007 b-tagging 0.010 Total 0.17 Total 0.16 (a) (b) Fig. 9 Likelihood scans over a κV V and b εV V , with the other parameter proﬁled. The ﬁts are performed using both shape and rate information. All relevant experimental and theoretical systematic uncertainties are considered in the ﬁts electroweak-boson polarisation in the VBF H →W W channel. Both the shape and rate information is exploited in the ﬁt. 9 Conclusion The signal strength parameter for the ggF + 2 jets Higgs boson production mode is found to be μggF+2jets = 0.5 ± 0.4(stat.)+0.7 −0.6(syst.). The mixing angle for CP-even and CP- odd contributions to the effective Higgs–gluon interaction is determined to be tan(α) = 0.0 ± 0.4(stat.) ± 0.3(syst.) using both shape and rate information. These results are obtained with novel analysis techniques and complement existing studies of the CP properties of the Higgs boson per- formed using tt H production. This article presents constraints on the CP structure of gluon– gluon fusion Higgs boson production and on the polarisations of the vector bosons in the HV V coupling. The results are obtained using H(→W W ∗→eνμν) j j ﬁnal states in data corresponding to 36.1 fb−1 of √s = 13 TeV proton–proton collisions recorded by the ATLAS detector at the LHC in 2015–2016. Total event yields as well as shapes of selected kinematical distributions in signal and control regions are exploited. The reported results for the VBF Higgs boson produc- tion mode include constraints on coupling-strength form fac- tors to longitudinally and transversely polarised W and Z 12 3 Eur. Phys. J. C (2022) 82 :622 Page 17 of 33 622 bosons. It is the ﬁrst such measurement of the Higgs cou- plings performed. In one-dimensional ML ﬁts (where the other parameter was set to its SM value) shape informa- tion is sufﬁcient to constrain the coupling to transversely polarised bosons, aT, while to constrain aL the informa- tion about the rates signiﬁcantly improves the sensitivity. Proﬁling the other coupling-strength scale factor results in: aL = 0.91+0.10 −0.18(stat.)+0.09 −0.17(syst.) and aT = 1.2 ± 0.4(stat.)+0.2 −0.3(syst.), while aL = 1.00+0.08 −0.10(stat.)+0.08 −0.13(syst.) and aT = 1.0+0.4 −0.5(stat.)+0.2 −0.4(syst.) are expected in the SM. Data Availability Statement This manuscript has associated data in a data repository. [Authors’ comment: The associated data is available at: https://www.hepdata.net/record/ins1932467.] Open Access This article is licensed under a Creative Commons Attri- bution 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, pro- vide a link to the Creative Commons licence, and indicate if changes were made. References 1. ATLAS Collaboration, Observation of a new particle in the search for the Standard Model Higgs boson with the ATLAS detector at the LHC. Phys. Lett. B 716, 1 (2012). https://doi.org/10.1016/j. physletb.2012.08.020 arXiv:1207.7214 [hep-ex] Acknowledgements We thank CERN for the very successful oper- ation of the LHC, as well as the support staff from our institutions without whom ATLAS could not be operated efﬁciently. We acknowl- edge the support of ANPCyT, Argentina; YerPhI, Armenia; ARC, Aus- tralia; BMWFW and FWF, Austria; ANAS, Azerbaijan; SSTC, Belarus; CNPq and FAPESP, Brazil; NSERC, NRC and CFI, Canada; CERN; ANID, Chile; CAS, MOST and NSFC, China; Minciencias, Colombia; MEYS CR, Czech Republic; DNRF and DNSRC, Denmark; IN2P3- CNRS and CEA-DRF/IRFU, France; SRNSFG, Georgia; BMBF, HGF and MPG, Germany; GSRI, Greece; RGC and Hong Kong SAR, China; ISF and Benoziyo Center, Israel; INFN, Italy; MEXT and JSPS, Japan; CNRST, Morocco; NWO, Netherlands; RCN, Norway; MEiN, Poland; FCT, Portugal; MNE/IFA, Romania; JINR; MES of Russia and NRC KI, Russian Federation; MESTD, Serbia; MSSR, Slovakia; ARRS and MIZŠ, Slovenia; DSI/NRF, South Africa; MICINN, Spain; SRC and Wallenberg Foundation, Sweden; SERI, SNSF and Cantons of Bern and Geneva, Switzerland; MOST, Taiwan; TAEK, Turkey; STFC, United Kingdom; DOE and NSF, USA. In addition, individual groups and members have received support from BCKDF, CANARIE, Compute Canada and CRC, Canada; COST, ERC, ERDF, Horizon 2020 and Marie Skłodowska-Curie Actions, European Union; Investissements d’Avenir Labex, Investissements d’Avenir Idex and ANR, France; DFG and AvH Foundation, Germany; Herakleitos, Thales and Aristeia pro- grammes co-ﬁnanced by EU-ESF and the Greek NSRF, Greece; BSF- NSF and GIF, Israel; Norwegian 2014-2021, Norway; NCN and NAWA, Poland; La Caixa Banking Foundation, CERCA Programme Generali- tat de Catalunya and PROMETEO and GenT Programmes Generalitat Valenciana, Spain; Göran Gustafssons Stiftelse, Sweden; The Royal Society and Leverhulme Trust, UK. The crucial computing support from all WLCG partners is acknowledged gratefully, in particular from CERN, the ATLAS Tier-1 facilities at TRIUMF (Canada), NDGF (Den- mark, Norway, Sweden), CC-IN2P3 (France), KIT/GridKA (Germany), INFN-CNAF (Italy), NL-T1 (Netherlands), PIC (Spain), ASGC (Tai- wan), RAL (UK) and BNL (USA), the Tier-2 facilities worldwide and large non-WLCG resource providers. Major contributors of computing resources are listed in Ref. [86]. 2. CMS Collaboration, Observation of a new boson at a mass of 125 GeV with the CMS experiment at the LHC. Phys. Lett. B 716, 30 (2012). https://doi.org/10.1016/j.physletb.2012.08.021 arXiv:1207.7235 [hep-ex] 3. 9 Conclusion The images or other third party material in this article are included in the article’s Creative Commons licence, unless indi- cated 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 permit- ted use, you will need to obtain permission directly from the copy- right holder. To view a copy of this licence, visit http://creativecomm ons.org/licenses/by/4.0/. 0.5 0.4 With an approximate mapping to pseudo-observables the following constraints are obtained in a ﬁt where the other parameter is proﬁled: κV V = 0.91+0.10 −0.18(stat.)+0.09 −0.17(syst.) and ϵV V = 0.13+0.28 −0.20 (stat.)+0.08 −0.10(syst.), while κV V = 1.00+0.08 −0.10(stat.)+0.08 −0.13(syst.) and ϵV V = 0.00+0.22 −0.24 (stat.)+0.11 −0.15 (syst.) are expected. In this parameterisation the sensitivity to the on-shell coupling κV V is affected by the event yields, while the off-shell coupling εV V is sensitive to both shape and rate information. All measurements are consistent with the expectations for the SM Higgs boson. g y Funded by SCOAP3. 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Connelly57 , F. Conventi69a,am , A. M. Cooper-Sarkar133 , F. Cormier173 , L. D. Corpe94 , M. Corradi72a,72b , E. E. Corrigan96 , F. Corriveau103,aa , M. J. Costa172 , F. Costanza5 , D. Costanzo148 , G. Cowan93 , J. W. Cowley32 , J. Crane100 , K. Cranmer124 , R. A. Creager135 , S. Crépé-Renaudin58 , F. Crescioli134 , M. Cristinziani24 , M. Cristoforetti75a,75b , V. Croft168 , G. Crosetti41a,41b , A. Cueto5 , T. Cuhadar Donszelmann169 , H. Cui15a,15d , A. R. Cukierman152 , W. R. Cunningham57 , S. Czekierda84 , P. Czodrowski36 , M. M. Czurylo61b , M. J. Da Cunha Sargedas De Sousa60b , J. V. Da Fonseca Pinto80b , 12 3 622 Page 22 of 33 Eur. Phys. J. C (2022) 82 :622 622 Page 22 of 33 C. Da Via100 , W. Dabrowski83a , F. Dachs36 , T. Dado47 , S. Dahbi33e , T. Dai105 , C. Dallapiccola102 , M. Dam40 , G. D’amen29 , V. D’Amico74a,74b , J. Damp99 , J. R. Dandoy135 , M. F. Daneri30 , M. Danninger151 , V. Dao36 , G. Darbo55b , O. Dartsi5, A. Dattagupta130 , S. D’Auria68a,68b , C. David166b , T. 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Freund109 , W. S. Freund80b , E. M. Freundlich47 , D. C. Frizzell127 , D. Froidevaux36 , J. A. Frost133 , M. ATLAS Collaboration⋆ Fujimoto125 , E. Fullana Torregrosa172 , T. Fusayasu115, J. Fuster172 , A. Gabrielli23a,23b , A. Gabrielli36 , P. Gadow114 , G. Gagliardi55a,55b , L. G. Gagnon109 , G. E. Gallardo133 , E. J. Gallas133 , B. J. Gallop142 , R. Gamboa Goni92 , K. K. Gan126 , S. Ganguly178 , J. Gao60a , Y. Gao50 , Y. S. Gao31,m , F. M. Garay Walls145a , C. García172 , J. E. García Navarro172 , J. A. García Pascual15a , M. Garcia-Sciveres18 , R. W. Gardner37 , S. Gargiulo52 , C. A. Garner165, V. Garonne132 , S. J. Gasiorowski147 , P. Gaspar80b , G. Gaudio70a , P. Gauzzi72a,72b , I. L. Gavrilenko110 , A. Gavrilyuk123 , C. Gay173 , G. Gaycken46 , E. N. Gazis10 , A. A. Geanta27b , C. M. Gee144 , C. N. P. Gee142 , J. Geisen96 , M. Geisen99 , C. Gemme55b , M. H. Genest58 , C. Geng105, 12 3 Page 23 of 33 622 Page 23 of 33 622 Eur. Phys. J. C (2022) 82 :622 I. Gnesi41b,c , M. 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Hartman152 , N. M. Hartmann113 , Y. Hasegawa149 , A. Hasib50 , S. Hassani143 , S. Haug20 , R. Hauser106 , M. Havranek140 , C. M. Hawkes21 , R. J. Hawkings36 , S. Hayashida116 , D. Hayden106 , C. Hayes105 , R. L. Hayes173 , C. P. Hays133 , J. M. Hays92 , H. S. Hayward90 , S. J. Haywood142 , F. He60a , Y. He163 , M. P. Heath50 , V. Hedberg96 , A. L. Heggelund132 , N. D. Hehir92 , C. Heidegger52 , K. K. Heidegger52 , W. D. Heidorn78 , J. Heilman34 , S. Heim46 , T. Heim18 , B. Heinemann46,aj , J. G. Heinlein135 , J. J. Heinrich130 , L. Heinrich36 , J. Hejbal139 , L. Helary46 , A. Held124 , S. Hellesund132 , C. M. Helling144 , S. Hellman45a,45b , C. Helsens36 , R. C. W. Henderson89, L. Henkelmann32 , A. M. Henriques Correia36, H. Herde152 , Y. Hernández Jiménez33e , H. Herr99, M. G. Herrmann113 , T. Herrmann48 , G. Herten52 , R. Hertenberger113 , L. Hervas36 , N. P. Hessey166a , H. Hibi82 , S. Higashino81 , E. Higón-Rodriguez172 , K. Hildebrand37, J. C. Hill32 , K. K. Hill29 , K. H. Hiller46, S. J. Hillier21 , M. Hils48 , I. Hinchliffe18 , F. Hinterkeuser24 , M. Hirose131 , S. Hirose167 , D. Hirschbuehl180 , B. Hiti91 , O. Hladik139, J. Hobbs154 , R. Hobincu27e , N. Hod178 , M. ATLAS Collaboration⋆ C. Hodgkinson148 , A. Hoecker36 , D. Hohn52 , D. Hohov64, T. Holm24 , T. R. Holmes37 , M. Holzbock114 , L. B. A. H. Hommels32 , T. M. Hong137 , J. C. Honig52 , A. Hönle114 , B. H. Hooberman171 , W. H. Hopkins6 , Y. Horii116 , P. Horn48 , L. A. Horyn37 , S. Hou157 , J. Howarth57 , J. Hoya88 , M. Hrabovsky129 , A. Hrynevich108 , T. Hryn’ova5 , P. J. Hsu63 , S.-C. Hsu147 , Q. Hu39 , S. Hu60c , Y. F. Hu15a,15d,an , D. P. Huang94 , X. Huang15c , Y. Huang60a , Y. Huang15a , Z. Hubacek140 , F. Hubaut101 , M. Huebner24 , F. Huegging24 , T. B. Huffman133 , M. Huhtinen36 , R. Hulsken58 , R. F. H. Hunter34 , N. Huseynov79,ab , J. Huston106 , J. Huth59 , R. Hyneman152 , S. Hyrych28a , G. Iacobucci54 , G. Iakovidis29 , I. Ibragimov150 , L. Iconomidou-Fayard64 , P. Iengo36 , R. 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Kuprash52 , H. Kurashige82 , L. L. Kurchaninov166a , Y. A. Kurochkin107 , A. Kurova111 , M. G. Kurth15a,15d, E. S. Kuwertz36 , M. Kuze163 , A. K. Kvam147 , J. Kvita129 , T. Kwan103 , C. Lacasta172 , F. Lacava72a,72b , D. P. J. Lack100 , H. Lacker19 , D. Lacour134 , E. Ladygin79 , R. Lafaye5 , B. Laforge134 , T. Lagouri145c , S. Lai53 , I. K. Lakomiec83a , J. E. Lambert127 , S. Lammers65, W. Lampl7 , C. Lampoudis161 , E. Lançon29 , U. Landgraf52 , M. P. J. Landon92 , V. S. Lang52 , J. C. Lange53 , R. J. Langenberg102 , A. J. Lankford169 , F. Lanni29 , K. Lantzsch24 , A. Lanza70a , A. Lapertosa55a,55b , J. F. Laporte143 , T. Lari68a , F. Lasagni Manghi23b , M. Lassnig36 , V. Latonova139 , T. S. Lau62a , A. Laudrain99 , A. Laurier34 , M. Lavorgna69a,69b , S. D. Lawlor93 , M. Lazzaroni68a,68b , B. Le100, A. Lebedev78 , M. LeBlanc7 , T. LeCompte6 , F. Ledroit-Guillon58 , A. C. A. Lee94, C. A. Lee29 , G. R. Lee17 , L. Lee59 , S. C. Lee157 , S. Lee78 , B. Lefebvre166a , H. P. Lefebvre93 , M. Lefebvre174 , C. Leggett18 , K. Lehmann151 , N. Lehmann20 , G. Lehmann Miotto36 , W. A. Leight46 , A. Leisos161,v , M. A. L. Leite80c , C. E. Leitgeb113 , R. Leitner141 , K. J. C. Leney42 , T. Lenz24 , S. Leone71a , C. Leonidopoulos50 , A. Leopold134 , C. Leroy109 , R. Les106 , C. G. Lester32 , M. Levchenko136 , J. ATLAS Collaboration⋆ Levêque5 , D. Levin105 , L. J. Levinson178 , D. J. Lewis21 , B. Li15b , B. Li105 , C-Q. Li60c,60d , F. Li60c, H. Li60a , H. Li60b , J. Li60c , K. Li147 , L. Li60c , M. Li15a,15d , Q. Y. Li60a , S. Li60c,60d,b , X. Li46 , Y. Li46 , Z. Li60b , Z. Li133 , Z. Li103 , Z. Li90, Z. Liang15a , M. Liberatore46 , B. Liberti73a , K. Lie62c , C. Y. Lin32 , K. Lin106 , R. A. Linck65 , R. E. Lindley7, J. H. Lindon21 , A. Linss46 , A. L. Lionti54 , E. Lipeles135 , A. Lipniacka17 , T. M. Liss171,ak , A. Lister173 , J. D. Little8 , B. Liu78 , B. X. Liu151 , J. B. Liu60a , J. K. K. Liu37 , K. Liu60c,60d , M. Liu60a , M. Y. Liu60a , P. Liu15a , X. Liu60a , Y. Liu46 , Y. Liu15a,15d , Y. L. Liu105 , Y. W. Liu60a , M. Livan70a,70b , A. Lleres58 , J. Llorente Merino151 , S. L. Lloyd92 , E. M. Lobodzinska46 , P. Loch7 , S. Loffredo73a,73b , T. Lohse19 , K. Lohwasser148 , M. Lokajicek139 , J. D. Long171 , R. E. Long89 , I. Longarini72a,72b , L. Longo36 , R. Longo171 , I. Lopez Paz100 , A. Lopez Solis148 , J. Lorenz113 , N. Lorenzo Martinez5 , A. M. Lory113 , A. Lösle52 , X. Lou45a,45b , X. Lou15a , A. Lounis64 , J. Love6 , P. A. Love89 , J. J. Lozano Bahilo172 , M. Lu60a , S. Lu135 , Y. J. Lu63 , H. J. Lubatti147 , C. Luci72a,72b , F. L. Lucio Alves15c , A. Lucotte58 , F. Luehring65 , I. Luise154 , L. Luminari72a, B. Lund-Jensen153 , N. A. Luongo130 , M. S. Lutz160 , D. Lynn29 , H. Lyons90, R. Lysak139 , E. Lytken96 , F. Lyu15a , V. Lyubushkin79 , T. Lyubushkina79 , H. Ma29 , L. L. Ma60b , Y. Ma94 , D. M. Mac Donell174 , G. Maccarrone51 , C. M. Macdonald148 , J. C. MacDonald148 , J. Machado Miguens135 , R. Madar38 , W. F. Mader48 , M. 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Martinelli74a,74b , M. Martinez14,w , P. Martinez Agullo172 , V. I. Martinez Outschoorn102 , S. Martin-Haugh142 , V. S. Martoiu27b , A. C. Martyniuk94 , A. Marzin36 , S. R. Maschek114 , L. Masetti99 , T. Mashimo162 , R. Mashinistov110 , J. Masik100 , A. L. Maslennikov121a,121b , L. Massa23b , P. Massarotti69a,69b , P. Mastrandrea71a,71b , A. Mastroberardino41a,41b , T. Masubuchi162 , D. Matakias29, T. Mathisen170 , A. Matic113 , N. Matsuzawa162, J. Maurer27b , B. Maˇcek91 , D. A. Maximov121a,121b , R. Mazini157 , I. Maznas161 , S. M. Mazza144 , C. Mc Ginn29 , J. P. Mc Gowan103 , S. P. Mc Kee105 , T. G. McCarthy114 , W. P. McCormack18 , E. F. McDonald104 , A. E. McDougall119 , J. A. Mcfayden18 , G. Mchedlidze158b , M. A. McKay42, K. D. McLean174 , S. J. McMahon142 , P. C. McNamara104 , C. J. McNicol176 , R. A. McPherson174,aa , J. E. Mdhluli33e , Z. A. Meadows102 , S. Meehan36 , T. Megy38 , S. Mehlhase113 , A. Mehta90 , B. Meirose43 , D. Melini159 , B. R. Mellado Garcia33e , F. 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Nagai133 , K. Nagano81 , J. L. Nagle29 , E. Nagy101 , A. M. Nairz36 , Y. Nakahama116 , K. Nakamura81 , H. Nanjo131 , F. Napolitano61a , R. F. Naranjo Garcia46 , R. Narayan42 , I. Naryshkin136 , M. Naseri34 , T. Naumann46 , G. Navarro22a , J. Navarro-Gonzalez172 , P. Y. Nechaeva110 , F. Nechansky46 , T. J. Neep21 , A. Negri70a,70b , M. Negrini23b , C. Nellist118 , C. Nelson103 , M. E. Nelson45a,45b , S. Nemecek139 , M. Nessi36,f , M. S. Neubauer171 , F. Neuhaus99 , M. Neumann180, R. Newhouse173 , P. R. ATLAS Collaboration⋆ Marchiori134 , M. Marcisovsky139 , L. Marcoccia73a,73b , C. Marcon96 , M. Marjanovic127 , Z. Marshall18 , 170 172 176 50 17 123 123 12 3 622 Page 26 of 33 Eur. Phys. J. C (2022) 82 :622 F. Parodi55a,55b , E. W. Parrish120 , J. A. Parsons39 , U. Parzefall52 , L. Pascual Dominguez134 , V. R. Pascuzzi18 , J. M. P. Pasner144 , F. Pasquali119 , E. Pasqualucci72a , S. Passaggio55b , F. Pastore93 , P. Pasuwan45a,45b , J. R. Pater100 , A. Pathak179,j , J. Patton90, T. 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A. Raine54 , S. Rajagopalan29 , K. Ran15a,15d , D. F. Rassloff61a , D. M. Rauch46 , S. Rave99 , B. Ravina57 , I. Ravinovich178 , M. Raymond36 , A. L. Read132 , N. P. Readioff148 , M. Reale67a,67b , D. M. Rebuzzi70a,70b , G. Redlinger29 , K. Reeves43 , D. Reikher160 , A. Reiss99, A. Rej150 , C. Rembser36 , A. Renardi46 , M. Renda27b , M. B. Rendel114, A. G. Rennie57 , S. Resconi68a , E. D. Resseguie18 , S. Rettie94 , B. Reynolds126, E. Reynolds21 , O. L. Rezanova121a,121b , P. Reznicek141 , E. Ricci75a,75b , R. Richter114 , S. Richter46 , E. Richter-Was83b , M. Ridel134 , P. Rieck114 , O. Rifki46 , M. Rijssenbeek154 , A. Rimoldi70a,70b , M. Rimoldi46 , L. Rinaldi23a,23b , T. T. Rinn171 , G. Ripellino153 , I. Riu14 , P. Rivadeneira46 , J. C. Rivera Vergara174 , F. Rizatdinova128 , E. Rizvi92 , C. Rizzi36 , S. H. Robertson103,aa , M. Robin46 , D. Robinson32 ,C. M. Robles Gajardo145d,M. Robles Manzano99 ,A. Robson57 ,A. Rocchi73a,73b ,C. Roda71a,71b , S. Rodriguez Bosca172 , A. 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Sahu180 , M. Saimpert36 , M. Saito162 , T. Saito162 , D. Salamani54, G. Salamanna74a,74b , A. Salnikov152 , J. Salt172 , A. Salvador Salas14 , D. Salvatore41a,41b , F. Salvatore155 , A. Salzburger36 , D. Sammel52 , D. Sampsonidis161 , D. Sampsonidou60c,60d , J. Sánchez172 , A. Sanchez Pineda36,66a,66c , H. Sandaker132 , C. O. Sander46 , I. G. Sanderswood89 , M. Sandhoff180 , C. Sandoval22b , D. P. C. Sankey142 , M. Sannino55a,55b , Y. Sano116 , A. Sansoni51 , C. Santoni38 , H. Santos138a,138b , S. N. Santpur18 , A. Santra178 , K. A. Saoucha148 , A. Sapronov79 , J. G. Saraiva138a,138d , J. Sardain134 , O. Sasaki81 , K. Sato167 , F. Sauerburger52 , E. Sauvan5 , P. Savard165,al , R. Sawada162 , C. Sawyer142 , L. Sawyer95 , I. Sayago Galvan172, C. Sbarra23b , A. Sbrizzi66a,66c , T. Scanlon94 , J. Schaarschmidt147 , P. Schacht114 , D. Schaefer37 , L. Schaefer135 , U. Schäfer99 , A. C. Schaffer64 , D. Schaile113 , R. D. Schamberger154 , E. Schanet113 , C. Scharf19 , N. Scharmberg100 , V. A. Schegelsky136 , D. Scheirich141 , F. Schenck19 , M. Schernau169 , C. Schiavi55a,55b , L. K. Schildgen24 , Z. M. Schillaci26 , E. J. Schioppa67a,67b , M. Schioppa41a,41b , K. E. Schleicher52 , S. Schlenker36 , K. R. Schmidt-Sommerfeld114 , K. Schmieden99 , C. Schmitt99 , S. Schmitt46 , L. Schoeffel143 , A. Schoening61b , P. G. Scholer52 , E. Schopf133 , M. Schott99 , J. F. P. Schouwenberg118 , J. Schovancova36 , S. Schramm54 , F. Schroeder180 , A. Schulte99 , H-C. Schultz-Coulon61a , M. Schumacher52 , B. A. Schumm144 , Ph. Schune143 , A. Schwartzman152 , 1 F. Parodi55a,55b , E. W. Parrish120 , J. A. Parsons39 , U. Parzefall52 , L. Pascual Dominguez134 , V. R. Pascuzzi18 , 12 3 Eur. Phys. J. C (2022) 82 :622 Page 27 of 33 622 Page 27 of 33 622 T. A. Schwarz105 , Ph. Schwemling143 , R. Schwienhorst106 , A. Sciandra144 , G. Sciolla26 , F. Scuri71a , F. Scutti104, L. M. Scyboz114 , C. D. Sebastiani90 , K. Sedlaczek47 , P. Seema19 , S. C. Seidel117 , A. Seiden144 , B. D. Seidlitz29 , T. Seiss37 , C. Seitz46 , J. M. ATLAS Collaboration⋆ Seixas80b , G. Sekhniaidze69a , S. J. Sekula42 , N. Semprini-Cesari23a,23b , S. Sen49 , C. Serfon29 , L. Serin64 , L. Serkin66a,66b , M. Sessa60a , H. Severini127 , S. Sevova152 , F. Sforza55a,55b , A. Sfyrla54 , E. Shabalina53 , J. D. Shahinian135 , N. W. Shaikh45a,45b , D. Shaked Renous178 , L. Y. Shan15a , M. Shapiro18 , A. Sharma36 , A. S. Sharma1 , P. B. Shatalov123 , K. Shaw155 , S. M. Shaw100 , M. Shehade178, Y. Shen127, P. Sherwood94 , L. Shi94 , C. O. Shimmin181 , Y. Shimogama177 , M. Shimojima115 , J. D. Shinner93 , I. P. J. Shipsey133 , S. Shirabe163 , M. Shiyakova79,y , J. Shlomi178 , M. J. Shochet37 , J. Shojaii104 , D. R. Shope153 , S. Shrestha126 , E. M. Shrif33e , M. J. Shroff174 , E. Shulga178 , P. Sicho139 , A. M. Sickles171 , E. Sideras Haddad33e , O. Sidiropoulou36 , A. Sidoti23b , F. Siegert48 , Dj. Sijacki16 , M. V. Silva Oliveira36 , S. B. Silverstein45a , S. Simion64, R. Simoniello99 , C. J. Simpson-allsop21, S. Simsek12b , P. Sinervo165 , V. Sinetckii112 , S. Singh151 , S. Sinha33e , M. Sioli23a,23b , I. Siral130 , S. Yu. Sivoklokov112 , J. Sjölin45a,45b , A. Skaf53 , E. Skorda96 , P. Skubic127 , M. Slawinska84 , K. Sliwa168 , V. Smakhtin178, B. H. Smart142 , J. Smiesko28b , N. Smirnov111 , S. Yu. Smirnov111 , Y. Smirnov111 , L. N. Smirnova112,s , O. Smirnova96 , E. A. Smith37 , H. A. Smith133 , M. Smizanska89 , K. Smolek140 , A. Smykiewicz84 , A. A. Snesarev110 , H. L. Snoek119 , I. M. Snyder130 , S. Snyder29 , R. Sobie174,aa , A. Soffer160 , A. Søgaard50 , F. Sohns53 , C. A. Solans Sanchez36 , E. Yu. Soldatov111 , U. Soldevila172 , A. A. Solodkov122 , A. Soloshenko79 , O. V. Solovyanov122 , V. Solovyev136 , P. Sommer148 , H. Son168 , A. Sonay14 , W. Y. Song166b , A. Sopczak140 , A. L. Sopio94, F. Sopkova28b , S. Sottocornola70a,70b , R. Soualah66a,66c , A. M. Soukharev121a,121b , D. South46 , S. Spagnolo67a,67b , M. Spalla114 , M. Spangenberg176 , F. Spanò93 , D. Sperlich52 , T. M. Spieker61a , G. Spigo36 , M. Spina155 , D. P. Spiteri57 , M. Spousta141 , A. Stabile68a,68b , R. Stamen61a , M. Stamenkovic119 , A. ATLAS Collaboration⋆ Yorita177 , K. Yoshihara78 , C. J. S. Young36 , C. Young152 , R. Yuan60b,i , X. Yue61a , M. Zaazoua35e , B. Zabinski84 , G. Zacharis10 , E. Zaffaroni54 , A. M. Zaitsev122,ag , T. Zakareishvili158b , N. Zakharchuk34 , S. Zambito36 , D. Zanzi52 , S. V. Zeißner47 , C. Zeitnitz180 , G. Zemaityte133 , J. C. Zeng171 , O. Zenin122 , T. Ženiš28a , S. Zenz92 , S. Zerradi35a , D. Zerwas64 , M. Zgubiˇc133 , B. Zhang15c , D. F. Zhang15b , G. Zhang15b , J. Zhang6 , K. Zhang15a , L. Zhang15c , L. Zhang60a , M. Zhang171 , R. Zhang179 , S. Zhang105, X. Zhang60c , X. Zhang60b , Y. Zhang15a,15d , Z. Zhang64 , P. Zhao49 , Y. Zhao144 , Z. Zhao60a , A. Zhemchugov79 , Z. Zheng105 , D. Zhong171 , B. Zhou105, C. Zhou179 , H. Zhou7 , M. Zhou154 , N. Zhou60c , Y. Zhou7, C. G. Zhu60b , C. Zhu15a,15d , H. L. Zhu60a , H. Zhu15a , J. Zhu105 , Y. Zhu60a , X. Zhuang15a , K. Zhukov110 , V. Zhulanov121a,121b , D. Zieminska65 , N. I. Zimine79 , S. Zimmermann52,* , Z. Zinonos114, M. Ziolkowski150 , L. Živkovi´c16 , A. Zoccoli23a,23b , K. Zoch53 , T. G. Zorbas148 , R. Zou37 , L. Zwalinski36 1 Department of Physics, University of Adelaide, Adelaide, Australia 2 Physics Department SUNY Albany Albany NY USA K. Zoch53 , T. G. Zorbas148 , R. Zou37 , L. Zwalinski36 K. Zoch53 , T. G. Zorbas148 , R. Zou37 , L. Zwalinski36 1 Department of Physics, University of Adelaide, Adelaide, Australia 1 Department of Physics, University of Adelaide, Adelaide, Australia 2 Physics Department, SUNY Albany, Albany, NY, USA 2 Physics Department, SUNY Albany, Albany, NY, USA 3 Department of Physics, University of Alberta, Edmonton, AB, Canada 4 (a)Department of Physics, Ankara University, Ankara, Turkey; (b)Istanbul Aydin University, Appl ( ) 4 (a)Department of Physics, Ankara University, Ankara, Turkey; (b)Istanbul Aydin University, Application and Research Center for Advanced Studies, Istanbul, Turkey; (c)Division of Physics, TOBB University of Economics and Technology, Ankara, Turkey 4 (a)Department of Physics, Ankara University, Ankara, Turkey; (b)Istanbul Aydin University, Application and Research Center for Advanced Studies, Istanbul, Turkey; (c)Division of Physics, TOBB University of Economics and Technology, Ankara Turkey 5 LAPP, Univ. Savoie Mont Blanc, CNRS/IN2P3, Annecy, France 5 LAPP, Univ. T. R. Van Daalen14 , P. Van Gemmeren6 , S. Van Stroud94 , I. Van Vulpen119 , M. Vanadia73a,73b , W. Vandelli36 , M. Vandenbroucke143 , E. R. Vandewall128 , D. Vannicola72a,72b , R. Vari72a , E. W. Varnes7 , C. Varni55a,55b , T. Varol157 , D. Varouchas64 , K. E. Varvell156 , M. E. Vasile27b , G. A. Vasquez174 , F. Vazeille38 , D. Vazquez Furelos14 , T. Vazquez Schroeder36 , J. Veatch53 , V. Vecchio100 , M. J. Veen119 , L. M. Veloce165 , F. Veloso138a,138c , S. Veneziano72a , A. Ventura67a,67b , A. Verbytskyi114 , M. Verducci71a,71b , C. Vergis24 , W. Verkerke119 , A. T. Vermeulen119 , J. C. Vermeulen119 , C. Vernieri152 , P. J. Verschuuren93 , M. L. Vesterbacka124 , M. C. Vetterli151,al , N. Viaux Maira145d , T. Vickey148 , O. E. Vickey Boeriu148 , G. H. A. Viehhauser133 , L. Vigani61b , M. Villa23a,23b , M. Villaplana Perez172 , E. M. Villhauer50, E. Vilucchi51 , M. G. Vincter34 , G. S. Virdee21 , A. Vishwakarma50 , C. Vittori23a,23b , I. Vivarelli155 , M. Vogel180 , P. Vokac140 , J. Von Ahnen46 , S. E. von Buddenbrock33e , E. Von Toerne24 , V. Vorobel141 , K. Vorobev111 , M. Vos172 , J. H. Vossebeld90 , M. Vozak100 , N. Vranjes16 , M. Vranjes Milosavljevic16 , V. Vrba140,, M. Vreeswijk119 , N. K. Vu101 , R. Vuillermet36 , I. Vukotic37 , S. Wada167 , C. Wagner102, P. Wagner24 , W. Wagner180 , S. Wahdan180 , H. Wahlberg88 , R. Wakasa167 , V. M. Walbrecht114 , J. Walder142 , R. Walker113 , S. D. Walker93, W. Walkowiak150 , V. Wallangen45a,45b, A. M. Wang59 , A. Z. Wang179 , C. Wang60a , C. Wang60c , H. Wang18 , J. Wang62a , P. Wang42 , R.-J. Wang99 , R. Wang60a , R. Wang120 , S. M. Wang157 , S. Wang60b, T. Wang60a , W. T. Wang60a , W. X. Wang60a , Y. Wang60a , Z. Wang105 , C. Wanotayaroj36 , A. Warburton103 , C. P. Ward32 , R. J. Ward21 , N. Warrack57 , A. T. Watson21 , M. F. Watson21 , G. Watts147 , B. M. Waugh94 , A. F. Webb11 , C. Weber29 , M. S. Weber20 , S. A. Weber34 , S. M. Weber61a , Y. Wei133 , A. R. Weidberg133 , J. Weingarten47 , M. Weirich99 , C. Weiser52 , P. S. Wells36 , T. Wenaus29 , B. Wendland47 , T. Wengler36 , S. Wenig36 , N. Wermes24 , M. Wessels61a , T. D. Weston20, K. Whalen130 , A. M. Wharton89 , A. S. White105 , A. White8 , M. J. White1 , D. Whiteson169 , B. W. Whitmore89 , W. Wiedenmann179 , C. Wiel48 , M. Wielers142 , N. Wieseotte99, C. Wiglesworth40 , L. A. M. Wiik-Fuchs52 , H. G. Wilkens36 , L. J. Wilkins93 , D. M. Williams39 , H. H. Williams135, S. Williams32 , S. Willocq102 , P. J. Windischhofer133 , I. Wingerter-Seez5 , E. Winkels155 , F. Winklmeier130 , B. T. Winter52 , M. Wittgen152, M. Wobisch95 , A. Wolf99 , R. Wölker133 , J. Wollrath52, M. W. Wolter84 , H. Wolters138a,138c , V. W. S. Wong173 , A. F. Wongel46 , N. L. Woods144 , S. D. Worm46 , B. K. Wosiek84 , K. W. Wo´zniak84 , K. Wraight57 , S. L. Wu179 , X. Wu54 , Y. Wu60a , J. Wuerzinger133 , T. R. Wyatt100 , B. M. Wynne50 , S. Xella40 , J. Xiang62c , X. Xiao105 , X. Xie60a , I. Xiotidis155, D. Xu15a , H. Xu60a, H. Xu60a , L. Xu29 , R. Xu135 , T. Xu60a , W. Xu105 , Y. Xu15b , Z. Xu60b , Z. Xu152 , B. Yabsley156 , S. Yacoob33a , D. P. Yallup94 , N. Yamaguchi87 , Y. Yamaguchi163 , M. Yamatani162, H. Yamauchi167 , T. Yamazaki18 , Y. Yamazaki82 , J. Yan60c, Z. Yan25 , H. J. Yang60c,60d , H. T. Yang18 , S. Yang60a , T. Yang62c , X. Yang60a , X. Yang15a , Y. Yang162 , Z. Yang105,60a , W-M. Yao18 , Y. C. Yap46 , H. Ye15c , J. Ye42 , S. Ye29 , I. Yeletskikh79 , M. R. Yexley89 , P. Yin39 , K. Yorita177 , K. Yoshihara78 , C. J. S. Young36 , C. Young152 , R. Yuan60b,i , X. Yue61a , M. Zaazoua35e , B. Zabinski84 , G. Zacharis10 , E. Zaffaroni54 , A. M. Zaitsev122,ag , T. Zakareishvili158b , N. Zakharchuk34 , S. Zambito36 , D. Zanzi52 , S. V. Zeißner47 , C. Zeitnitz180 , G. Zemaityte133 , J. C. Zeng171 , O. Zenin122 , T. Ženiš28a , S. Zenz92 , S. Zerradi35a , D. Zerwas64 , M. Zgubiˇc133 , B. Zhang15c , D. F. Zhang15b , G. Zhang15b , J. Zhang6 , K. Zhang15a , L. Zhang15c , L. Zhang60a , M. Zhang171 , R. Zhang179 , S. Zhang105, X. Zhang60c , X. Zhang60b , Y. Zhang15a,15d , Z. Zhang64 , P. Zhao49 , Y. Zhao144 , Z. Zhao60a , A. Zhemchugov79 , Z. Zheng105 , D. Zhong171 , B. Zhou105, C. Zhou179 , H. Zhou7 , M. Zhou154 , N. Zhou60c , Y. Zhou7, C. G. Zhu60b , C. Zhu15a,15d , H. L. Zhu60a , H. Zhu15a , J. Zhu105 , Y. Zhu60a , X. Zhuang15a , K. Zhukov110 , V. Zhulanov121a,121b , D. Zieminska65 , N. I. Zimine79 , S. Zimmermann52, , Z. Zinonos114, M. Ziolkowski150 , L. Živkovi´c16 , A. Zoccoli23a,23b , K. Zoch53 , T. G. Zorbas148 , R. Zou37 , L. Zwalinski36 ATLAS Collaboration⋆ Stampekis21 , E. Stanecka84 , B. Stanislaus133 , M. M. Stanitzki46 , M. Stankaityte133 , B. Stapf119 , E. A. Starchenko122 , G. H. Stark144 , J. Stark58 , P. Staroba139 , P. Starovoitov61a , S. Stärz103 , R. Staszewski84 , G. Stavropoulos44 , P. Steinberg29 , A. L. Steinhebel130 , B. Stelzer151,166a , H. J. Stelzer137 , O. Stelzer-Chilton166a , H. Stenzel56 , T. J. Stevenson155 , G A Stewart36 M C Stockton36 G Stoicea27b M Stolarski138a S Stonjek114 A Straessner48 123 123 12 622 Page 28 of 33 Eur. Phys. J. C (2022) 82 :622 T. R. Van Daalen14 , P. Van Gemmeren6 , S. Van Stroud94 , I. Van Vulpen119 , M. Vanadia73a,73b , W. Vandelli36 , M. Vandenbroucke143 , E. R. Vandewall128 , D. Vannicola72a,72b , R. Vari72a , E. W. Varnes7 , C. Varni55a,55b , T. Varol157 , D. Varouchas64 , K. E. Varvell156 , M. E. Vasile27b , G. A. Vasquez174 , F. Vazeille38 , D. Vazquez Furelos14 , T. Vazquez Schroeder36 , J. Veatch53 , V. Vecchio100 , M. J. Veen119 , L. M. Veloce165 , F. Veloso138a,138c , S. Veneziano72a , A. Ventura67a,67b , A. Verbytskyi114 , M. Verducci71a,71b , C. Vergis24 , W. Verkerke119 , A. T. Vermeulen119 , J. C. Vermeulen119 , C. Vernieri152 , P. J. Verschuuren93 , M. L. Vesterbacka124 , M. C. Vetterli151,al , N. Viaux Maira145d , T. Vickey148 , O. E. Vickey Boeriu148 , G. H. A. Viehhauser133 , L. Vigani61b , M. Villa23a,23b , M. Villaplana Perez172 , E. M. Villhauer50, E. Vilucchi51 , M. G. Vincter34 , G. S. Virdee21 , A. Vishwakarma50 , C. Vittori23a,23b , I. Vivarelli155 , M. Vogel180 , P. Vokac140 , J. Von Ahnen46 , S. E. von Buddenbrock33e , E. Von Toerne24 , V. Vorobel141 , K. Vorobev111 , M. Vos172 , J. H. Vossebeld90 , M. Vozak100 , N. Vranjes16 , M. Vranjes Milosavljevic16 , V. Vrba140,*, M. Vreeswijk119 , N. K. Vu101 , R. Vuillermet36 , I. Vukotic37 , S. Wada167 , C. Wagner102, P. Wagner24 , W. Wagner180 , S. Wahdan180 , H. Wahlberg88 , R. Wakasa167 , V. M. Walbrecht114 , J. Walder142 , R. Walker113 , S. D. Walker93, W. Walkowiak150 , V. Wallangen45a,45b, A. M. Wang59 , A. Z. Wang179 , C. Wang60a , C. Wang60c , H. Wang18 , J. Wang62a , P. Wang42 , R.-J. Wang99 , R. ATLAS Collaboration⋆ Wang60a , R. Wang120 , S. M. Wang157 , S. Wang60b, T. Wang60a , W. T. Wang60a , W. X. Wang60a , Y. Wang60a , Z. Wang105 , C. Wanotayaroj36 , A. Warburton103 , C. P. Ward32 , R. J. Ward21 , N. Warrack57 , A. T. Watson21 , M. F. Watson21 , G. Watts147 , B. M. Waugh94 , A. F. Webb11 , C. Weber29 , M. S. Weber20 , S. A. Weber34 , S. M. Weber61a , Y. Wei133 , A. R. Weidberg133 , J. Weingarten47 , M. Weirich99 , C. Weiser52 , P. S. Wells36 , T. Wenaus29 , B. Wendland47 , T. Wengler36 , S. Wenig36 , N. Wermes24 , M. Wessels61a , T. D. Weston20, K. Whalen130 , A. M. Wharton89 , A. S. White105 , A. White8 , M. J. White1 , D. Whiteson169 , B. W. Whitmore89 , W. Wiedenmann179 , C. Wiel48 , M. Wielers142 , N. Wieseotte99, C. Wiglesworth40 , L. A. M. Wiik-Fuchs52 , H. G. Wilkens36 , L. J. Wilkins93 , D. M. Williams39 , H. H. Williams135, S. Williams32 , S. Willocq102 , P. J. Windischhofer133 , I. Wingerter-Seez5 , E. Winkels155 , F. Winklmeier130 , B. T. Winter52 , M. Wittgen152, M. Wobisch95 , A. Wolf99 , R. Wölker133 , J. Wollrath52, M. W. Wolter84 , H. Wolters138a,138c , V. W. S. Wong173 , A. F. Wongel46 , N. L. Woods144 , S. D. Worm46 , B. K. Wosiek84 , K. W. Wo´zniak84 , K. Wraight57 , S. L. Wu179 , X. Wu54 , Y. Wu60a , J. Wuerzinger133 , T. R. Wyatt100 , B. M. Wynne50 , S. Xella40 , J. Xiang62c , X. Xiao105 , X. Xie60a , I. Xiotidis155, D. Xu15a , H. Xu60a, H. Xu60a , L. Xu29 , R. Xu135 , T. Xu60a , W. Xu105 , Y. Xu15b , Z. Xu60b , Z. Xu152 , B. Yabsley156 , S. Yacoob33a , D. P. Yallup94 , N. Yamaguchi87 , Y. Yamaguchi163 , M. Yamatani162, H. Yamauchi167 , T. Yamazaki18 , Y. Yamazaki82 , J. Yan60c, Z. Yan25 , H. J. Yang60c,60d , H. T. Yang18 , S. Yang60a , T. Yang62c , X. Yang60a , X. Yang15a , Y. Yang162 , Z. Yang105,60a , W-M. Yao18 , Y. C. Yap46 , H. Ye15c , J. Ye42 , S. Ye29 , I. Yeletskikh79 , M. R. Yexley89 , P. Yin39 , K. ATLAS Collaboration⋆ Righi, Università di Bologna, Bologna, Italy; (b)INFN Sezione di Bologna, Bologna, Italy 23 (a)Dipartimento di Fisica e Astronomia A. ATLAS Collaboration⋆ Savoie Mont Blanc, CNRS/IN2P3, Annecy, France 6 High Energy Physics Division, Argonne National Laboratory, Argonne, IL, USA 7 Department of Physics, University of Arizona, Tucson, AZ, USA 8 7 Department of Physics, University of Arizona, Tucson, AZ, USA 8 Department of Physics, University of Texas at Arlington, Arlington, TX, USA 8 Department of Physics, University of Texas at Arlington, Arlington, TX, USA 12 3 Eur. Phys. J. ATLAS Collaboration⋆ C (2022) 82 :622 Page 29 of 33 622 622 9 Physics Department, National and Kapodistrian University of Athens, Athens, Greece 10 Physics Department, National Technical University of Athens, Zografou, Greece 11 Department of Physics, University of Texas at Austin, Austin, TX, USA 12 (a)Bahcesehir University, Faculty of Engineering and Natural Sciences, Istanbul, Turkey; (b)Facul Natural Sciences, Istanbul Bilgi University, Istanbul, Turkey; (c)Department of Physics, Bogazici Turkey; (d)Department of Physics Engineering, Gaziantep University, Gaziantep, Turkey 13 Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan 14 Institut de Física d’Altes Energies (IFAE), Barcelona Institute of Science and Technology, Bar 14 Institut de Física d’Altes Energies (IFAE), Barcelona Institute of Science and Technology, Barcelona, Spain 15 (a)Institute of High Energy Physics, Chinese Academy of Sciences, Beijing, China; (b)Physics Department, Tsinghua University, Beijing, China; (c)Department of Physics, Nanjing University, Nanjing, China; (d)University of Chinese Academy of Science (UCAS) Beijing China 15 (a)Institute of High Energy Physics, Chinese Academy of Sciences, Beijing, China; (b)Physics Department, Tsinghua University, Beijing, China; (c)Department of Physics, Nanjing University, Nanjing, China; (d)University of Chinese Academy of Science (UCAS), Beijing, China 16 Institute of Physics, University of Belgrade, Belgrade, Serbia 16 Institute of Physics, University of Belgrade, Belgrade, Serbia 17 Department for Physics and Technology, University of Bergen, Bergen, Norway 17 Department for Physics and Technology, University of Bergen, Bergen, Norway 18 Physics Division, Lawrence Berkeley National Laboratory and University of California, B 18 Physics Division, Lawrence Berkeley National Laboratory and University of California, Berkeley, CA, USA 19 Institut für Physik, Humboldt Universität zu Berlin, Berlin, Germany 20 19 Institut für Physik, Humboldt Universität zu Berlin, Berlin, Germany 20 20 Albert Einstein Center for Fundamental Physics and Laboratory for High Energy Physics, University of Bern, Bern, Switzerland 20 Albert Einstein Center for Fundamental Physics and Laboratory for High Energy Physics, University of Bern, Bern, Switzerland 21 School of Physics and Astronomy, University of Birmingham, Birmingham, UK 21 School of Physics and Astronomy, University of Birmingham, Birmingham, UK 22 ( ) (b) 21 School of Physics and Astronomy, University of Birmingham, Birmingham, UK 22 ( ) 22 (a)Facultad de Ciencias y Centro de Investigaciónes, Universidad Antonio Nariño, Bogotá, Co 22 (a)Facultad de Ciencias y Centro de Investigaciónes, Universidad Antonio Nariño, Bogotá, Colombia; (b)Departamento de Física, Universidad Nacional de Colombia, Bogotá, Colombia 23 ( ) (b) 22 (a)Facultad de Ciencias y Centro de Investigaciónes, Universidad de Física, Universidad Nacional de Colombia, Bogotá, Colombia 22 (a)Facultad de Ciencias y Centro de Investigaciónes, Universidad A de Física, Universidad Nacional de Colombia, Bogotá, Colombia y g , , g , ; p de Física, Universidad Nacional de Colombia, Bogotá, Colombia 23 (a)Dipartimento di Fisica e Astronomia A Righi Università di Bologna Bologna Italy; (b)INFN Sezione di Bologna de Física, Universidad Nacional de Colombia, Bogotá, Colombia g 23 (a)Dipartimento di Fisica e Astronomia A. ATLAS Collaboration⋆ Righi, Università di Bologna, Bologna, Italy; (b)INFN Sezione di Bologna, Bologna, Italy 24 Physikalisches Institut, Universität Bonn, Bonn, Germany 24 Physikalisches Institut, Universität Bonn, Bonn, Germany 25 Department of Physics, Boston University, Boston, MA, USA 25 Department of Physics, Boston University, Boston, MA, USA 26 Department of Physics, Brandeis University, Waltham, MA, USA Department of Physics, Brandeis University, Waltham, MA, USA 27 (a)Transilvania University of Brasov, Brasov, Romania; (b)Horia Hulubei National Institute of Physics and Nuclear Engineering, Bucharest, Romania; (c)Department of Physics, Alexandru Ioan Cuza University of Iasi, Iasi, Romania; (d)National Institute for Research and Development of Isotopic and Molecular Technologies, Physics Department, Cluj-Napoca, Romania; (e)University Politehnica Bucharest, Bucharest, Romania; (f)West University in Timisoara, Timisoara, Romania a)Transilvania University of Brasov, Brasov, Romania; (b)Horia Hulubei National Institute of Physic ( ) 27 (a)Transilvania University of Brasov, Brasov, Romania; (b)Horia Hulubei National Institute of Phy Engineering, Bucharest, Romania; (c)Department of Physics, Alexandru Ioan Cuza University of 27 (a)Transilvania University of Brasov, Brasov, Romania; (b)Horia Hulubei National Institute of Physics and Nuclear Engineering, Bucharest, Romania; (c)Department of Physics, Alexandru Ioan Cuza University of Iasi, Iasi, Romania; (d)National Institute for Research and Development of Isotopic and Molecular Technologies, Physics Department Cluj Napoca Romania; (e)University Politehnica Bucharest Bucharest Romania; (f)West University in Engineering, Bucharest, Romania; (c)Department of Physics, Alexandru Ioan Cuza University of Iasi Romania; (d)National Institute for Research and Development of Isotopic and Molecular Technolo Romania; National Institute for Research and Development of Isotopic and Molecular Technologies, Physics Department, Cluj-Napoca, Romania; (e)University Politehnica Bucharest, Bucharest, Romania; (f)West University in Timisoara, Timisoara, Romania 28 (a)Faculty of Mathematics, Physics and Informatics, Comenius University, Bratislava, Slovak Republic; (b)Department of Subnuclear Physics, Institute of Experimental Physics of the Slovak Academy of Sciences, Kosice, Slovak Republic 28 (a)Faculty of Mathematics, Physics and Informatics, Comenius University, Bratislava, Slovak Republic; (b)Department of Subnuclear Physics, Institute of Experimental Physics of the Slovak Academy of Sciences, Kosice, Slovak Republic 28 (a)Faculty of Mathematics, Physics and Informatics, Comenius University, Bratislava, Slovak Republic; (b)Department of Subnuclear Physics, Institute of Experimental Physics of the Slovak Academy of Sciences, Kosice, Slovak Republic 29 Physics Department, Brookhaven National Laboratory, Upton, NY, USA 29 Physics Department, Brookhaven National Laboratory, Upton, NY, USA 30 29 Physics Department, Brookhaven National Laboratory, Upton, NY, USA 30 Departamento de Física (FCEN) and IFIBA, Universidad de Buenos Aires and CONICET, Buenos Aires, Argentina 31 California State University, CA, USA 31 California State University, CA, USA 32 32 Cavendish Laboratory, University of Cambridge, Cambridge, UK 32 Cavendish Laboratory, University of Cambridge, Cambridge, UK 32 Cavendish Laboratory, University of Ca 33 ( ) 33 (a)Department of Physics, University of Cape Town, Cape Town, South Africa; (b)iThemba Labs, Western Cape, South Africa; (c)Department of Mechanical Engineering Science, University of Johannesburg, Johannesburg, South Africa; (d)Department of Physics, University of South Africa, Pretoria, South Africa; (e)School of Physics, University of the Witwatersrand, Johannesburg, South Africa 33 (a)Department of Physics, University of Cape Town, Cape Town, South Africa; (b)iThemba Labs, Western Cape, South Africa; (c)Department of Mechanical Engineering Science, University of Johannesburg, Johannesburg, South Africa; (d)Department of Physics, University of South Africa, Pretoria, South Africa; (e)School of Physics, University of the Witwatersrand, Johannesburg, South Africa the Witwatersrand, Johannesburg, South Africa 34 Department of Physics, Carleton University, Ottawa, ON, Canada 34 Department of Physics, Carleton University, Ottawa, ON, Canada 35 (a)Faculté des Sciences Ain Chock, Réseau Universitaire de Physique des Hautes Energies, Unive Casablanca, Morocco; (b)Faculté des Sciences, Université Ibn-Tofail, Kénitra, Morocco; (c)Facult 35 (a)Faculté des Sciences Ain Chock, Réseau Universitaire de Physique des Hautes Energies, Université Hassan II, Casablanca, Morocco; (b)Faculté des Sciences, Université Ibn-Tofail, Kénitra, Morocco; (c)Faculté des Sciences Semlalia, Université Cadi Ayyad, LPHEA-Marrakech, Morocco; (d)LPMR, Faculté des Sciences, Université Mohamed Premier, Oujda, Morocco; (e)Faculté des sciences, Université Mohammed V, Rabat, Morocco Sciences Ain Chock, Réseau Universitaire de Physique des Hautes Energies, Université Hassan II, Morocco; (b)Faculté des Sciences, Université Ibn-Tofail, Kénitra, Morocco; (c)Faculté des Sciences , y q g , , Casablanca, Morocco; (b)Faculté des Sciences, Université Ibn-Tofail, Kénitra, Morocco; (c)Faculté des Sciences Semlalia, Université Cadi Ayyad, LPHEA-Marrakech, Morocco; (d)LPMR, Faculté des Sciences, Université Mohamed Premier, Oujda, Morocco; (e)Faculté des sciences, Université Mohammed V, Rabat, Morocco 36 Casablanca, Morocco; Faculté des Sciences, Université Ibn Tofail, Kénitra, Morocco; Faculté des Sciences Semlalia, Université Cadi Ayyad, LPHEA-Marrakech, Morocco; (d)LPMR, Faculté des Sciences, Université Mohamed Premier, Oujda, Morocco; (e)Faculté des sciences, Université Mohammed V, Rabat, Morocco 36 CERN G S i l d Semlalia, Université Cadi Ayyad, LPHEA-Marrakech, Morocco; (d)LPMR, Faculté des Sciences, Un ( ) Premier, Oujda, Morocco; (e)Faculté des sciences, Université Mohammed V, Rabat, Morocco 36 CERN, Geneva, Switzerland 37 Enrico Fermi Institute, University of Chicago, Chicago, IL, USA 37 Enrico Fermi Institute, University of Chicago, Chicago, IL, USA 38 LPC, Université Clermont Auvergne, CNRS/IN2P3, Clermont-Ferrand, France 38 LPC, Université Clermont Auvergne, CNRS/IN2P3, Clermont-Ferrand, France 39 Nevis Laboratory, Columbia University, Irvington, NY, USA 39 Nevis Laboratory, Columbia University, Irvington, NY, USA 40 Niels Bohr Institute, University of Copenhagen, Copenhagen, Denmark 41 (a)Dipartimento di Fisica, Università della Calabria, Rende, Italy; (b)INFN Gruppo Collegato di Cosenza, Laboratori N i li di F i F i I l 40 Niels Bohr Institute, University of Copenhagen, Copenhagen, Denmark b Niels Bohr Institute, University of Copenhagen, Copenhagen, Denmark 41 (a)Dipartimento di Fisica, Università della Calabria, Rende, Italy; (b)INFN Gruppo Collegato di Cosenza, Laboratori Nazionali di Frascati, Frascati, Italy , y p g , p g , 41 (a)Dipartimento di Fisica, Università della Calabria, Rende, Italy; (b)INFN Gruppo Collegato di Cosenza, Laboratori Nazionali di Frascati, Frascati, Italy 42 Physics Department, Southern Methodist University, Dallas, TX, USA 42 Physics Department, Southern Methodist University, Dallas, TX, USA 12 3 622 Page 30 of 33 Eur. ATLAS Collaboration⋆ Fermi, Università di Pisa, Pisa, I 71 (a)INFN Sezione di Pisa, Pisa, Italy; (b)Dipartimento di Fisica E. ATLAS Collaboration⋆ Physikalisches Institut, Justus-Liebig-Universität Giessen, Giessen, Germany 58 LPSC, Université Grenoble Alpes, CNRS/IN2P3, Grenoble INP, Grenoble, France 58 LPSC, Université Grenoble Alpes, CNRS/IN2P3, Grenoble INP, Grenoble, France 59 Laboratory for Particle Physics and Cosmology, Harvard University, Cambridge, MA, USA 60 ( ) 60 (a)Department of Modern Physics and State Key Laboratory of Particle Detection and Electronics, University of Science and Technology of China, Hefei, China; (b)Institute of Frontier and Interdisciplinary Science and Key Laboratory of Particle Physics and Particle Irradiation (MOE), Shandong University, Qingdao, China; (c)School of Physics and Astronomy, Shanghai Jiao Tong University, Key Laboratory for Particle Astrophysics and Cosmology (MOE), SKLPPC, Particle Physics and Particle Irradiation (MOE), Shandong University, Qingdao, China; (c)School of Astronomy, Shanghai Jiao Tong University, Key Laboratory for Particle Astrophysics and Cosmolog Shanghai, China; (d)Tsung-Dao Lee Institute, Shanghai, China hanghai, China; (d)Tsung-Dao Lee Institute, Shanghai, China Shanghai, China; (d)Tsung-Dao Lee Institute, Shanghai, China 61 (a)Kirchhoff-Institut für Physik, Ruprecht-Karls-Universität Heidelberg, Heidelberg, Germany; (b)Physikalisches Institut, g , ; g , g , 61 (a)Kirchhoff-Institut für Physik, Ruprecht-Karls-Universität Heidelberg, Heidelbe g g g a)Kirchhoff-Institut für Physik, Ruprecht-Karls-Universität Heidelberg, Heidelberg, Germany; (b)Ph 61 (a)Kirchhoff-Institut für Physik, Ruprecht-Karls-Universität He Ruprecht-Karls-Universität Heidelberg, Heidelberg, Germany Ruprecht-Karls-Universität Heidelberg, Heidelberg, Germany 62 (a)Department of Physics Chinese University of Hong Kong Sh Ruprecht-Karls-Universität Heidelberg, Heidelberg, Germany 62 (a) f h i hi i i f h Ruprecht-Karls-Universität Heidelberg, Heidelberg, Germany Ruprecht-Karls-Universität Heidelberg, Heidelberg, Germany 62 (a)Department of Physics, Chinese University of Hong Kong, Shatin N.T., Hong Kong, China; Ruprecht-Karls-Universität Heidelberg, Heidelberg, Germany 62 (a)Department of Physics, Chinese University of Hong Kong, Shatin N.T., Hong Kong, China; (b)Department of Physics, p g g y 62 (a)Department of Physics, Chinese University of Hong Kong, Shatin N.T., Hong Kon p g g y a)Department of Physics, Chinese University of Hong Kong, Shatin N.T., Hong Kong, China; (b)Dep Physics, Chinese University of Hong Kong, Shatin N.T., Hong Kong, China; (b)Department of Phys University of Hong Kong, Hong Kong, China; (c)Department of Physics and Institute for Advanced S University of Hong Kong, Hong Kong, China; (c)Department of Physics and Institute for Adva University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China 63 University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China 63 Department of Physics, National Tsing Hua University, Hsinchu, Taiwan 63 Department of Physics, National Tsing Hua University, Hsinchu, Taiwan 64 IJCLab, Université Paris-Saclay, CNRS/IN2P3, 91405, Orsay, France 64 IJCLab, Université Paris-Saclay, CNRS/IN2P3, 91405, Orsay, France 65 Department of Physics, Indiana University, Bloomington, IN, USA 65 Department of Physics, Indiana University, Bloomington, IN, USA 66 (a)INFN Gruppo Collegato di Udine, Sezione di Trieste, Udine, Italy; (b)ICTP, Trieste, Italy; (c)Dipartimento Politecnico di Ingegneria e Architettura, Università di Udine, Udine, Italy 66 (a)INFN Gruppo Collegato di Udine, Sezione di Trieste, Udine, Italy; (b)ICTP, Trieste, Italy; (c)Dipartimento Politecnico di Ingegneria e Architettura, Università di Udine, Udine, Italy g g y 67 (a)INFN Sezione di Lecce, Lecce, Italy; (b)Dipartimento di Mat 67 (a)INFN Sezione di Lecce, Lecce, Italy; (b)Dipartimento di Matematica e Fisica, Università del Salento, Lecce, Italy 68 (a)INFN Sezione di Milano Milan Italy; (b)Dipartimento di Fisica Università di Milano Milan Italy 67 (a)INFN Sezione di Lecce, Lecce, Italy; (b)Dipartimento di Matematica e Fisica, Università del Salento, Lecce, Italy 68 (a)INFN S i di Mil Mil It l (b)Di ti t di Fi i U i ità di Mil Mil It l 69 (a)INFN Sezione di Napoli, Naples, Italy; (b)Dipartimento di Fisica, Università di Napoli, Naples, 69 (a)INFN Sezione di Napoli, Naples, Italy; (b)Dipartimento di Fisica, Università di Napoli, Naples, Ital 70 (a)INFN Sezione di Pavia, Pavia, Italy; (b)Dipartimento di Fisica, Università di Pavia, Pavia, Italy 71 (a)INFN Sezione di Pisa, Pisa, Italy; (b)Dipartimento di Fisica E. ATLAS Collaboration⋆ Phys. J. C (2022) 82 :622 622 Page 30 of 33 43 Physics Department, University of Texas at Dallas, Richardson, TX, USA 44 National Centre for Scientiﬁc Research “Demokritos”, Agia Paraskevi, Greece National Centre for Scientiﬁc Research Demokritos , Agia Paraskevi, Greece 45 (a)Department of Physics, Stockholm University, Sweden; (b)Oskar Klein Centre, Stockholm, Sweden epartment of Physics, Stockholm University, Sweden; (b)Oskar Klein Centre, Stockholm, Sweden 45 (a)Department of Physics, Stockholm University, Sweden; (b)Oskar Klein Centre, Stockholm, tsches Elektronen-Synchrotron DESY, Hamburg and Zeuthen, Germany 46 Deutsches Elektronen-Synchrotron DESY, Hamburg and Zeuthen, Germany Fakultät Physik , Technische Universität Dortmund, Dortmund, Germany 47 Fakultät Physik , Technische Universität Dortmund, Dortmund, Germany y , , , y 48 Institut für Kern und Teilchenphysik Technische Universität Dresden Dresden Germany nstitut für Kern- und Teilchenphysik, Technische Universität Dresden, Dresden, Germany 48 Institut für Kern- und Teilchenphysik, Technische Universität Dresden, Dresden, Germany 49 Department of Physics, Duke University, Durham, NC, USA 50 SUPA-School of Physics and Astronomy, University of Edinburgh, Edinburgh, UK 51 INFN e Laboratori Nazionali di Frascati, Frascati, Italy 52 Physikalisches Institut, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany 53 II. Physikalisches Institut, Georg-August-Universität Göttingen, Göttingen, Germany 54 Département de Physique Nucléaire et Corpusculaire, Université de Genève, Geneva, Switzerland 54 Département de Physique Nucléaire et Corpusculaire, Université de Genève, Geneva, Switzerland 55 (a)Dipartimento di Fisica, Università di Genova, Genoa, Italy; (b)INFN Sezione di Genova, Ge 56 II. Physikalisches Institut, Justus-Liebig-Universität Giessen, Giessen, Germany hysikalisches Institut, Justus-Liebig-Universität Gi 56 II. ATLAS Collaboration⋆ Fermi, Università di Pisa, Pisa, Italy 72 ( ) (b) 72 (a)INFN Sezione di Roma, Rome, Italy; (b)Dipartimento di Fisica, Sapienza Università di Roma, R 73 (a)INFN Sezione di Roma Tor Vergata, Rome, Italy; (b)Dipartimento di Fisica, Università di Roma Tor Vergata, Rome, Italy 74 (a) i di l (b) i i di i i i i i à l 73 (a)INFN Sezione di Roma Tor Vergata, Rome, Italy; (b)Dipartimento di Fisica, Università di Rom Italy 73 (a)INFN Sezione di Roma Tor Vergata, Rome, Italy; (b)Dipartimento di Fisica, Università di Roma Tor Vergata, Rome, Italy y 74 (a)INFN Sezione di Roma Tre, Rome, Italy; (b)Dipartimento di Matematica e Fisica, Università Roma Tre, Rome, Italy 75 (a) (b) N Sezione di Roma Tre, Rome, Italy; (b)Dipartimento di Matematica e Fisica, Università Roma Tre, R 74 (a)INFN Sezione di Roma Tre, Rome, Italy; (b)Dipartimento di Matematica e Fisica, Unive 75 (a)INFN-TIFPA, Povo, Italy; (b)Università degli Studi di Trento, Trento, Italy 75 (a)INFN-TIFPA, Povo, Italy; (b)Università degli Studi di Trento, Trento, Italy 76 Institut für Astro- und Teilchenphysik, Leopold-Franzens-Universität, Innsbruck, Austria 76 Institut für Astro- und Teilchenphysik, Leopold-Franzens-Universität, Innsbruck, Austr 77 University of Iowa, Iowa City, IA, USA 78 Department of Physics and Astronomy, Iowa State University, Ames, IA, USA 78 Department of Physics and Astronomy, Iowa State University, Ames, IA, USA 79 Joint Institute for Nuclear Research, Dubna, Russia 80 (a)Departamento de Engenharia Elétrica, Universidade Federal de Juiz de Fora (UFJF), Juiz de Fora, Brazil; (b)Universidade Federal do Rio De Janeiro COPPE/EE/IF, Rio de Janeiro, Brazil; (c)Instituto de Físic Universidade de São Paulo, São Paulo, Brazil epartamento de Engenharia Elétrica, Universidade Federal de Juiz de Fora (UFJF), Juiz de Fora, 80 (a)Departamento de Engenharia Elétrica, Universidade Federal de Juiz de Fora (UFJF), Juiz de Brazil; (b)Universidade Federal do Rio De Janeiro COPPE/EE/IF, Rio de Janeiro, Brazil; (c)Instituto de Física Universidade de São Paulo São Paulo Brazil Brazil; (b)Universidade Federal do Rio De Janeiro COPPE/EE/IF, Rio de Janeiro, Brazil; (c)Instituto de Física Universidade de São Paulo, São Paulo, Brazil 81 KEK, High Energy Accelerator Research Organization, Tsukuba, Japan 81 KEK, High Energy Accelerator Research Orga 81 KEK, High Energy Accelerator Research Organization, Tsukuba, Japan ate School of Science, Kobe University, Kobe, Jap 83 (a)AGH University of Science and Technology, Faculty of Physics and Applied Computer Science, Kraków, 83 (a)AGH University of Science and Technology, Faculty of Physics and Applied Computer Scie University of Science and Technology, Faculty of Poland; (b)Marian Smoluchowski Institute of Physics, Jagiellonian University, Kraków, Poland (b)Marian Smoluchowski Institute of Physics, Jagiellonian University, Kraków, Poland 3 Page 31 of 33 622 Eur. Ljubljana, Slovenia Lomonosov Moscow State University, Moscow, Russia 113 Fakultät für Physik, Ludwig-Maximilians-Universität München, Munich, Germany 114 Max-Planck-Institut für Physik (Werner-Heisenberg-Institut), Munich, Germany 114 Max-Planck-Institut für Physik (Werner-Heisenberg-Institut), Munich, Germany 115 Nagasaki Institute of Applied Science, Nagasaki, Japan 116 Graduate School of Science and Kobayashi-Maskawa Institute, Nagoya University, Nagoya, Japan 116 Graduate School of Science and Kobayashi-Maskawa Instit 116 Graduate School of Science and Kobayashi-Maskawa Institute, Nagoya University, Nagoya, Japan G aduate Sc oo o Sc e ce a d obayas as awa st tute, Nagoya U ve s ty, Nagoya, J 117 Department of Physics and Astronomy, University of New Mexico, Albuquerque, NM, USA 118 Institute for Mathematics, Astrophysics and Particle Physics, Radboud University/Nikhef, Nijmegen, The Netherlands 119 Nikhef National Institute for Subatomic Physics and University of Amsterdam, Amsterdam, The Netherlands 120 Department of Physics, Northern Illinois University, DeKalb, IL, USA 118 Institute for Mathematics, Astrophysics and Particle Physics, Radboud University/Nikhef, Nijmegen, The Netherlands 119 Nikhef National Institute for Subatomic Physics and University of Amsterdam, Amsterdam, The Netherlands 120 Department of Physics, Northern Illinois University, DeKalb, IL, USA 120 Department of Physics, Northern Illinois University, DeKalb, IL, US 121 (a)Budker Institute of Nuclear Physics and NSU, SB RAS, Novosibirsk, Russia; (b)Novosibirsk State University, Novosibirsk, Russia 122 Novosibirsk, Russia 122 Institute for High Energy Physics of the National Research Centre Kurchatov Institute, Protvin Institute for High Energy Physics of the National Research Centre Kurchatov Institute, Protvino, Russia 123 Institute for Theoretical and Experimental Physics named by A.I. Alikhanov of National Research Centre “Kurchatov Institute”, Moscow, Russia 123 Institute for Theoretical and Experimental Physics named by A.I. Alikhanov of National Research Centre “Kurchatov Institute”, Moscow, Russia 24 Department of Physics, New York University, New York, NY, USA 124 Department of Physics, New York University, New York, NY, USA 5 125 Ochanomizu University, Otsuka, Bunkyo-ku, Tokyo, Japan 125 Ochanomizu University, Otsuka, Bunkyo-ku, Tokyo, Japan 126 126 Ohio State University, Columbus, OH, USA 127 Homer L. Dodge Department of Physics and Astronomy, University of Oklahoma, Norman, OK, USA 128 127 Homer L. Ljubljana, Slovenia 92 School of Physics and Astronomy, Queen Mary University of London, London, UK 92 School of Physics and Astronomy, Queen Mary University of London, London, UK 93 Department of Physics, Royal Holloway University of London, Egham, UK 94 Department of Physics and Astronomy, University College London, London, UK 95 Louisiana Tech University, Ruston, LA, USA 96 Fysiska institutionen, Lunds universitet, Lund, Sweden 97 Centre de Calcul de l’Institut National de Physique Nucléaire et de Physique des Particules (IN2P3), Villeurbanne, France 97 Centre de Calcul de l’Institut National de Physique Nucléaire et de Physique des Particules (IN2P3), Villeurbanne, France Departamento de Física Teorica C-15 and CIAFF, Universidad Autónoma de Madrid, Madrid, Spain 98 Departamento de Física Teorica C-15 and CIAFF, Universidad Autónoma de Madrid, Madrid, 99 99 Institut für Physik, Universität Mainz, Mainz, Germany 99 Institut für Physik, Universität Mainz, Mainz, Germany 00 School of Physics and Astronomy, University of Manchester, Manchester, UK 100 School of Physics and Astronomy, University of Manchester, Manchester, UK 01 CPPM, Aix-Marseille Université, CNRS/IN2P3, Marseille, France 101 CPPM, Aix-Marseille Université, CNRS/IN2P3, Marseille, France 102 Department of Physics, University of Massachusetts, Amherst, MA, USA 02 Department of Physics, University of Massachusetts, Amherst, MA, USA 103 Department of Physics, McGill University, Montreal, QC, Canada 03 Department of Physics, McGill University, Montreal, QC, Canada 104 School of Physics, University of Melbourne, Melbourne, VIC, Australia 105 Department of Physics, University of Michigan, Ann Arbor, MI, USA p y , y g , , , 106 Department of Physics and Astronomy Michigan State University East Lansing MI USA 106 Department of Physics and Astronomy, Michigan State University, East Lansing, MI, USA 107 B.I. Stepanov Institute of Physics, National Academy of Sciences of Belarus, Minsk, Belarus 108 Research Institute for Nuclear Problems of Byelorussian State University, Minsk, Belarus 109 Group of Particle Physics, University of Montreal, Montreal, QC, Canada 110 P.N. Lebedev Physical Institute of the Russian Academy of Sciences, Moscow, Russia 111 National Research Nuclear University MEPhI, Moscow, Russia 112 D.V. Skobeltsyn Institute of Nuclear Physics, M.V. Lomonosov Moscow State University, Moscow, Russi 112 D.V. Skobeltsyn Institute of Nuclear Physics, M.V. Lomonosov Moscow Stat 112 D.V. Skobeltsyn Institute of Nuclear Physics, M.V. ATLAS Collaboration⋆ Phys. J. C (2022) 82 :622 84 Institute of Nuclear Physics Polish Academy of Sciences, Kraków, Poland 85 Faculty of Science, Kyoto University, Kyoto, Japan 86 Kyoto University of Education, Kyoto, Japan 87 Research Center for Advanced Particle Physics and Department of Physics, Kyushu University, Fukuoka , Japan 88 Instituto de Física La Plata, Universidad Nacional de La Plata and CONICET, La Plata, Argentina 89 earch Center for Advanced Particle Physics and Department of Physics, Kyushu University, Fukuok 87 Research Center for Advanced Particle Physics and Department of Physics, Kyushu 87 Research Center for Advanced Particle Physic 87 Research Center for Advanced Particle Physics and Department of Physics, Kyushu University, Fukuoka , Japan 88 Instituto de Física La Plata, Universidad Nacional de La Plata and CONICET, La Plata, Argentina Research Center for Advanced Particle Physics and Department of Physics, Kyushu University, Fukuoka , Japan 88 Instituto de Física La Plata, Universidad Nacional de La Plata and CONICET, La Plata, Argentina tuto de Física La Plata, Universidad Nacional de La Plata and CONICET, La Plata, Argentina 88 Instituto de Física La Plata, Universidad Nacional de La Plata and CONICET, La Pl 88 Instituto de Física La Plata, Universidad Nacional de La Plata and CONICET, La Plata, Argentina 89 89 Physics Department, Lancaster University, Lancaster, UK 90 Oliver Lodge Laboratory, University of Liverpool, Liverpool, UK 91 Department of Experimental Particle Physics, Jožef Stefan Institute and Department of Physics, University of Ljubljana, Ljubljana, Slovenia 91 Department of Experimental Particle Physics, Jožef Stefan Institute and Department of Physics, University of Ljubljana, Ljubljana, Slovenia 91 Department of Experimental Particle Physics, Jožef Stefan Institute and Department of Physi Ljubljana, Slovenia Ljubljana, Slovenia Peters 136 Konstantinov Nuclear Physics Institute of National Research Centre “Kurchatov Institute”, PN 137 Department of Physics and Astronomy, University of Pittsburgh, Pittsburgh, PA, USA Department of Physics and Astronomy, University of Pittsburgh, Pittsburgh, PA, USA 138 (a)Laboratório de Instrumentação e Física Experimental de Partículas-LIP, Lisbon, Portugal; (b)Departamento de Física, Faculdade de Ciências, Universidade de Lisboa, Lisbon, Portugal; (c)Departamento de Física, Universidade de Coimbra, Coimbra, Portugal; (d)Centro de Física Nuclear da Universidade de Lisboa, Lisbon, Portugal; (e)Departamento de Física, Universidade do Minho, Braga, Portugal; (f)Departamento de Física Teórica y del Cosmos, Universidad de Granada, Granada, Spain; (g)Dep Física and CEFITEC of Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Caparica Portugal; (h)Instituto Superior Técnico Universidade de Lisboa Lisbon Portugal 138 (a)Laboratório de Instrumentação e Física Experimental de Partículas-LIP, Lisbon, Portugal; (b)Departamento de Física, Faculdade de Ciências, Universidade de Lisboa, Lisbon, Portugal; (c)Departamento de Física, Universidade de Coimbra, Coimbra, Portugal; (d)Centro de Física Nuclear da Universidade de Lisboa, Lisbon, Portugal; (e)Departamento de Física, Universidade do Minho, Braga, Portugal; (f)Departamento de Física Teórica y del Cosmos, Universidad de Granada, Granada, Spain; (g)Dep Física and CEFITEC of Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Faculdade de Ciências, Universidade de Lisboa, Lisbon, Portugal; ( )Departamento de Física, Universidade de Coimbra, Coimbra, Portugal; (d)Centro de Física Nuclear da Universidade de Lisboa, Lisbon, Portugal; (e)Departamento de Física, Universidade do Minho, Braga, Portugal; (f)Departamento de Física Teórica y del Cosmos, Universidad de Granada, Granada, Spain; (g)Dep Física and CEFITEC of Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, C i P l(h)I i S i Té i U i id d d Li b Li b P l Faculdade de Ciências, Universidade de Lisboa, Lisbon, Portugal; ( )Departamento de Física, Universidade de Coimbra, Coimbra, Portugal; (d)Centro de Física Nuclear da Universidade de Lisboa, Lisbon, Portugal; (e)Departamento de Física, Universidade do Minho, Braga, Portugal; (f)Departamento de Física Teórica y del Cosmos, Universidad de Granada, Granada, Spain; (g)Dep Física and CEFITEC of Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, C i l(h) i S i é i i id d d i b i b l Caparica, Portugal; (h)Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal 139 Institute of Physics of the Czech Academy of Sciences, Prague, Czech Republic 140 Czech Technical University in Prague, Prague, Czech Republic University, Faculty of Mathematics and Physics, P 141 Charles University, Faculty of Mathematics and Physics, Prague, Czech Republic 141 Charles University, Faculty of Mathematics and Physics, Prague, Czech Republic Physics Department, Rutherford Appleton Laborat 143 IRFU, CEA, Université Paris-Saclay, Gif-sur-Yvette, France 143 IRFU, CEA, Université Paris-Saclay, Gif-sur-Yvette, France EA, Université Paris-Saclay, Gif-sur-Yvette, Fran Santa Cruz Institute for Particle Physics, University of California Santa Cruz, Santa Cruz, CA, USA 144 Santa Cruz Institute for Particle Physics, University of California Santa Cruz, Santa Cruz, CA, USA 145 (a)Departamento de Física, Pontiﬁcia Universidad Católica de Chile, Santiago, Chile; (b)Universidad Andres B Department of Physics, Santiago, Chile; (c)Instituto de Alta Investigación, Universidad de Tarapacá, Arica, Chile; (d)Departamento de Física, Universidad Técnica Federico Santa María, Valparaiso, Chile 146 144 Santa Cruz Institute for Particle Physics, University of California Santa Cruz, Santa Cruz, CA, USA 145 (a)Departamento de Física, Pontiﬁcia Universidad Católica de Chile, Santiago, Chile; (b)Universidad Andres Bello, Department of Physics, Santiago, Chile; (c)Instituto de Alta Investigación, Universidad de Tarapacá, Arica, Chile; (d)Departamento de Física, Universidad Técnica Federico Santa María, Valparaiso, Chile 146 i id d d l d S d l i ( S ) S d l i il 44 Santa Cruz Institute for Particle Physics, University of California Santa Cruz, Santa Cruz, CA, US 45 (a)Departamento de Física, Pontiﬁcia Universidad Católica de Chile, Santiago, Chile; (b)Universid Department of Physics Santiago Chile; (c)Instituto de Alta Investigación Universidad de Tarapac Santa Cruz Institute for Particle Physics, University of California Santa Cruz, Santa Cruz, CA, USA a)Departamento de Física, Pontiﬁcia Universidad Católica de Chile, Santiago, Chile; (b)Universidad Department of Physics, Santiago, Chile; (c)Instituto de Alta Investigación, Universidad de Tarapacá, Department of Physics, Santiago, Chile; ( )Instituto de Alta Investigación, Universidad de Tarapac Chile; (d)Departamento de Física, Universidad Técnica Federico Santa María, Valparaiso, Chile 146 Chile; (d)Departamento de Física, Universidad Técnica Federico Santa María, Valparaiso, Chile 146 Universidade Federal de São João del Rei (UFSJ), São João del Rei, Brazil 146 Universidade Federal de São João del Rei (UFSJ), São João del Rei, Brazi 147 Department of Physics, University of Washington, Seattle, WA, USA 147 Department of Physics, University of Washington, Seattle, WA, USA 148 Department of Physics and Astronomy, University of Shefﬁeld, Shefﬁeld, UK 148 Department of Physics and Astronomy, University of Shefﬁeld, Shefﬁeld, UK 149 Department of Physics, Shinshu University, Nagano, Japan 149 Department of Physics, Shinshu University, Nagano, Japan 150 Department Physik, Universität Siegen, Siegen, Germany 150 Department Physik, Universität Siegen, Siegen, Germany 151 Department of Physics, Simon Fraser University, Burnaby, BC, Canada 151 Department of Physics, Simon Fraser University, Burnaby, BC, Canada 152 SLAC National Accelerator Laboratory, Stanford, CA, USA 152 SLAC National Accelerator Laboratory, Stanford, CA, USA 53 Department of Physics, Royal Institute of Technology, Stockholm, Sweden 153 Department of Physics, Royal Institute of Technology, Stockholm, Sweden 54 Department of Physics and Astronomy, Stony Brook University, Stony Brook, NY, USA 154 Department of Physics and Astronomy, Stony Brook University, Stony Brook, NY, US 55 Department of Physics and Astronomy, University of Sussex, Brighton, UK 155 Department of Physics and Astronomy, University of Sussex, Brighton, UK 156 School of Physics, University of Sydney, Sydney, Australia 156 School of Physics, University of Sydney, Sydney, Australia nstitute of Physics, Academia Sinica, Taipei, Taiwa y p 158 (a)E. 34 LPNHE, Sorbonne Université, Université de Paris, CNRS/IN2P3, Paris, France Ljubljana, Slovenia Andronikashvili Institute of Physics, Iv. Javakhishvili Tbilisi State University, Tbilisi, Georgia; (b)High Energy Physics Institute Tbilisi State University Tbilisi Georgia 158 (a)E. Andronikashvili Institute of Physics, Iv. Javakhishvili Tbili Physics Institute, Tbilisi State University, Tbilisi, Georgia (a)E. Andronikashvili Institute of Physics, Iv. Java 158 (a)E. Andronikashvili Institute of Physics, Iv. Javakhishvili Tbilisi State University, Tbilisi, Georgia; (b)High Energy Physics Institute Tbilisi State University Tbilisi Georgia 158 (a)E. Andronikashvili Institute of Physics, Iv. Ljubljana, Slovenia Dodge Department of Physics and Astronomy, University of Oklahoma, Norman, OK, USA 128 Department of Physics, Oklahoma State University, Stillwater, OK, USA 129 Joint Laboratory of Optics, Palacký University, Olomouc, Czech Republic 129 Joint Laboratory of Optics, Palacký University, Olomouc, Czech Republic or Fundamental Science, University of Oregon, Eu 131 Graduate School of Science, Osaka University, Osaka, Ja 132 Department of Physics, University of Oslo, Oslo, Norway 12 123 Eur. Phys. J. C (2022) 82 :622 622 Page 32 of 33 622 Page 32 of 33 133 Department of Physics, Oxford University, Oxford, UK 134 LPNHE, Sorbonne Université, Université de Paris, CNRS/IN2P3, Paris, France 135 Department of Physics, University of Pennsylvania, Philadelphia, PA, USA 135 Department of Physics, University of Pennsylvania, Philadelphia, PA, USA ntinov Nuclear Physics Institute of National Research Centre “Kurchatov Institute”, PNPI, St. 35 Department of Physics, University of Pennsylvania, Philadelphia, PA, USA Ljubljana, Slovenia Javakhishvili Tbilisi State University, Tbilisi, Georgia; (b)High Energy y y g 159 Department of Physics, Technion, Israel Institute of Technology, Haifa, Israel 159 Department of Physics, Technion, Israel Institute of Technology, Haifa, Israel 159 Department of Physics, Technion, Israel Institute of Technology, Haifa, Israel 60 Raymond and Beverly Sackler School of Physics and Astronomy, Tel Aviv University, Tel Av 6 160 Raymond and Beverly Sackler School of Physics and Astronomy, Tel Aviv Unive 161 Department of Physics, Aristotle University of Thessaloniki, Thessaloniki, Greece 162 161 Department of Physics, Aristotle University of Thessaloniki, Thessaloniki, Greece 162 62 International Center for Elementary Particle Physics and Department of Physics, University of To 162 International Center for Elementary Particle Physics and Department of Physics, Uni 63 Department of Physics, Tokyo Institute of Technology, Tokyo, Japan 163 Department of Physics, Tokyo Institute of Technology, Tokyo, Japan 164 Tomsk State University, Tomsk, Russia 165 Department of Physics, University of Toronto, Toronto, ON, Canada 65 Department of Physics, University of Toronto, Toronto, ON, Canada 66 (a)TRIUMF, Vancouver, BC, Canada; (b)Department of Physics and Astronomy, York University, 6 166 (a)TRIUMF, Vancouver, BC, Canada; (b)Department of Physics and A F, Vancouver, BC, Canada; (b)Department of Physics and Astronomy, York University, Toronto, ON f Ph i d T C f h Hi f h U i F l f P d A li d S i 166 (a)TRIUMF, Vancouver, BC, Canada; (b)Department of Physics and Astronomy, York University, Toronto, ON, Canada 167 Division of Physics and Tomonaga Center for the History of the Universe, Faculty of Pure and Applied Sciences, University of Tsukuba, Tsukuba, Japan 166 (a)TRIUMF, Vancouver, BC, Canada; (b)Department of Physics and Astronomy, York University, Toronto, ON, Canada 167 Division of Physics and Tomonaga Center for the History of the Universe, Faculty of Pure and Applied Sciences, University of Tsukuba, Tsukuba, Japan 167 Division of Physics and Tomonaga Center for the History of the Universe, Faculty of Pure and Applied Sciences, University of Tsukuba, Tsukuba, Japan 67 Division of Physics and Tomonaga Center for the History of the Universe, Faculty of Pure and Ap 167 Division of Physics and Tomonaga Center for the History of the Universe, Faculty of Pure and University of Tsukuba, Tsukuba, Japan 168 Department of Physics and Astronomy, Tufts University, Medford, MA, USA 68 Department of Physics and Astronomy, Tufts University, Medford, MA, USA 169 Department of Physics and Astronomy, University of California Irvine, Irvine, CA, US 169 Department of Physics and Astronomy, University of California Irvine, Irvine, CA, US 170 Department of Physics and Astronomy, University of Uppsala, Uppsala, Sweden 171 Department of Physics, University of Illinois, Urbana, IL, USA 172 Instituto de Física Corpuscular (IFIC), Centro Mixto Universidad de Valencia-CSIC, Valen 173 Department of Physics, University of British Columbia, Vancouver, BC, Cana 173 Department of Physics, University of British Columbia, Vancouver, BC, Canada 175 Fakultät für Physik und Astronomie, Julius-Maximilians-Universität Würzburg, Würzbu 175 Fakultät für Physik und Astronomie, Julius-Maximilians-Universität Würzburg, Würzburg, Germany Fakultät für Physik und Astronomie, Julius-Maxim 176 Department of Physics, University of Warwick, Coventry, UK 123 Page 33 of 33 622 Eur. Ljubljana, Slovenia Phys. J. C (2022) 82 :622 622 177 Waseda University, Tokyo, Japan 78 Department of Particle Physics and Astrophysics, Weizmann Institute of Science, Rehovot, Israel 179 Department of Physics, University of Wisconsin, Madison, WI, USA 180 F k ltät fü M th tik d N t i h ft F h Ph ik B i h U i ität W t l W t l 179 Department of Physics, University of Wisconsin, Madison, WI, USA 180 179 Department of Physics, University of Wisconsin, Madison, WI, USA 180 Fakultät für Mathematik und Naturwissenschaften, Fachgruppe Physik, Bergische Universität Wuppertal, Wuppertal, Germany 181 Department of Physics, Yale University, New Haven, CT, USA a Also at Borough of Manhattan Community College, City University of New York, New York, Borough of Manhattan Community College, City University of New York, New York, NY, USA b Also at Center for High Energy Physics, Peking University, China c Also at Centro Studi e Ricerche Enrico Fermi, Rome, Italy d Also at CERN, Geneva, Switzerland e Also at CPPM, Aix-Marseille Université, CNRS/IN2P3, Marseille, France e Also at CPPM, Aix-Marseille Université, CNRS/IN2P3, Marseille, France e Also at CPPM, Aix-Marseille Université Also at Département de Physique Nucléaire et Corpusculaire, Université de Genève, Geneve, Switze épartement de Physique Nucléaire et Corpusculaire f Also at Département de Physique Nucléaire et Corpusculaire, Université de G g Also at Departament de Fisica de la Universitat Autonoma de Barcelona, Barcelona, Spain h Also at Department of Financial and Management Engineering, University of the Aegean, Chios, i Also at Department of Physics and Astronomy, Michigan State University, East Lansing, MI, US j Also at Department of Physics and Astronomy, University of Louisville, Louisville, KY, USA k Also at Department of Physics, Ben Gurion University of the Negev, Beer Sheva, Israel l Also at Department of Physics, California State University, East Bay, USA m Also at Department of Physics, California State University, Fresno, USA n Also at Department of Physics, California State University, Sacramento, USA p y g g p Also at Department of Physics, St. Petersburg State Polytechnical University, St. Petersburg, Rus p Also at Department of Physics, St. Petersburg State Polytechnical University, St. Petersburg, Russia q Also at Department of Physics, University of Fribourg, Fribourg, Switzerland r Also at Dipartimento di Matematica, Informatica e Fisica, Università di Udine, Udine, Italy s Also at Faculty of Physics, M.V. Ljubljana, Slovenia Lomonosov Moscow State University, Moscow, Russia t Also at Giresun University, Faculty of Engineering, Giresun, Turkey u Also at Graduate School of Science, Osaka University, Osaka, Japan v Also at Hellenic Open University, Patras, Greece w Also at Institucio Catalana de Recerca i Estudis Avancats, ICREA, Barcelona, Spain w Also at Institucio Catalana de Recerca i Estudis Avancats, ICREA, Barcelona, Spain x Also at Institut für Experimentalphysik, Universität Hamburg, Hamburg, Germany x Also at Institut für Experimentalphysik, Universität Hamburg, Hamburg, Germany y Also at Institute for Nuclear Research and Nuclear Energy (INRNE) of the Bulgarian Academy o Bulgaria y Also at Institute for Nuclear Research and Nuclear Energy (INRNE) of the Bulgarian Academ Bulgaria z Also at Institute for Particle and Nuclear Physics, Wigner Research Centre for Physics, Budapest, Hungary z Also at Institute for Particle and Nuclear Physics, Wigner Research Centre for Physics, Budapest, Hungary z Also at Institute for Particle and Nuclear Physics, Wigner Research Centre for Physics, aa Also at Institute of Particle Physics (IPP), Montreal, Canada aa Also at Institute of Particle Physics (IPP), Montreal, Canada ab Also at Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan ab Also at Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan ac Also at Institute of Theoretical Physics, Ilia State University, Tbilisi, Georgia ac Also at Institute of Theoretical Physics, Ilia State University, Tbilisi, Georgia ad Also at Instituto de Fisica Teorica, IFT-UAM/CSIC, Madrid, Spain ad Also at Instituto de Fisica Teorica, IFT-UAM/CSIC, Madrid, Spain ae Also at Dept. of Physics, Istanbul University, Istanbul, Turkey af Also at Joint Institute for Nuclear Research, Dubna, Russia t Joint Institute for Nuclear Research, Dubna, Russ ag Also at Moscow Institute of Physics and Technology State University, Dolgoprudny, Russia ag Also at Moscow Institute of Physics and Technology State University, Dolgoprudny, Russia ah Also at National Research Nuclear University MEPhI, Moscow, Russia ah Also at National Research Nuclear University MEPhI, Moscow, Russia ai Also at Physics Department, An-Najah National University, Nablus, Palestine ai Also at Physics Department, An-Najah National University, Nablus, Palestine aj Also at Physikalisches Institut, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany aj Also at Physikalisches Institut, Albert-Ludwigs-Universität Freiburg, Freiburg, G ak Also at The City College of New York, New York, NY, USA ak Also at The City College of New York, New York, NY, USA al Also at TRIUMF, Vancouver, BC, Canada al Also at TRIUMF, Vancouver, BC, Canada al Also at TRIUMF, Vancouver, BC, Canada am Also at Università di Napoli Parthenope, Naples, Italy am Also at Università di Napoli Parthenope, N an Also at University of Chinese Academy of Sciences (UCAS), Beijing, China ∗Deceased an Also at University of Chinese Academy of Sciences (UCAS), Beijing, China ∗Deceased 12 123
https://openalex.org/W2131701602	https://europepmc.org/articles/pmc3392188?pdf=render	English	null	Coastal Staphylinidae (Coleoptera): A worldwide checklist, biogeography and natural history	ZooKeys	2,011	cc-by	47,279	ZooKeys 107: 1–98 (2011) doi: 10.3897/zookeys.107.1651 www.zookeys.org ZooKeys 107: 1–98 (2011) doi: 10.3897/zookeys.107.1651 www.zookeys.org aphylinidae (C checklist Coastal Staphylinidae (Coleoptera): A worldwide checklist, biogeography and natural history J. H. Frank1, Kee-Jeong Ahn2 1 Entomology and Nematology Department, University of Florida, Gainesville, FL 32611-0630, USA 2 De- partment of Biology, Chungnam National University, Daejeon 305-764, Republic of Korea Corresponding author: Kee-Jeong Ahn (kjahn@cnu.ac.kr) emic editor: Jan Klimaszewski \| Received 8 September 2010 \| Accepted 14 February 2011 \| Published 16 June 201 Citation: Frank JH, Ahn K-J (2011) Coastal Staphylinidae (Coleoptera): A worldwide checklist, biogeography and natural history. ZooKeys 107: 1–98. doi: 10.3897/zookeys.107.1651 Copyright J.H. Frank, K.-J. Ahn. 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. Keywords seashore Staphylinidae, marine Staphylinidae, littoral Staphylinidae, intertidal Staphylinidae, habitat, be- havior J. H. Frank & Kee-Jeong Ahn / ZooKeys 107: 1–98 (2011) 2 J. H. Frank & Kee-Jeong Ahn / ZooKeys 107: 1–98 (2011) The Pacific Ocean basin was the origin and contributed to the dispersal of the majority of specialist coastal Staphylinidae at the level of genus. However, at the level of species, species belonging to non- coastal-specialist genera are about as likely to occur on the shores of other oceans as on the shores of the Pacific. This difference is a reflection of the antiquity of coastal genera and species. ihfl A complete bibliography, and habitat and habitus photographs of some representative coastal Staphylinidae species are provided. Abstract We provide a list of the 392 described species of Staphylinidae confined to coastal habitats worldwide. The list is in taxonomic sequence by subfamily, tribe, and genus and includes 91 genera. We provide the page reference of the original description of every species and genus listed and of many synonyms. We note the existence of recent reviews, phylogenies and keys of each of the tribes and genera included. Coastal Staphylinidae contain eight subfamilies: Microsilphinae, Omaliinae, Pselaphinae, Aleocharinae, Oxyteli- nae, Scydmaeninae, Paederinae, and Staphylininae. By ‘coastal habitats’ we mean habitats existing on the sea coast and subject to inundation or at least splashing by the very highest tides. This includes rocky, boulder, coral, sandy, and muddy seashores, and at least portions of salt-marshes, estuaries, and mangrove swamps. We exclude the sand dune habitat and higher parts of sea-cliffs. The list notes distribution of all the species, first according to the ocean or sea on whose shores it has been recorded, and second by country (and for the larger countries by province or state). Although this distribution is undoubtedly incomplete, it provides a basis for future development of a dedicated database. The ‘Habitats, Habits, and Classificatory Notes’ section is designed to provide ecologists with further taxonomic and ecological information. It includes references to descriptions of the immature stages, be- havior of adults and immatures, their food, natural enemies, and habitat. We would have preferred to sep- arate these entities, but current knowledge of ecology is developed in few instances beyond natural history. 2 Introduction We struggled to find an appropriate title for this work, but eventually rejected the ex- pressions intertidal, marine, littoral, and seashore, all of which have been used by other authors. By “coastal” we mean species that dwell on sea coasts and are restricted to such habitats. However, we restrict the definition to habitats to those that are normally or occasionally inundated by tides, excluding cliff and dune habitats, as well as inland salt-laden habitats. Thus, those species included dwell in the intertidal zone and at the drift line, and in mangrove swamps, salt marshes, and estuaries where they may be inundated by the tides. We do not employ the terms halophile and halobiont because they refer to organisms that dwell in salt-laden habitats, which are not restricted to coastal areas; indeed saline lakes and ponds occur hundreds of kilometers from coasts, and we do not wish to consider these. A book chapter on intertidal Staphylinidae (Moore and Legner 1976) was un- doubtedly Ian Moore’s major contribution to this, his favorite, subject. Moore had already published numerous papers on intertidal Staphylinidae of the Pacific coast of North America. In this pioneering treatment he endeavored to summarize the world literature on intertidal staphylinids. To do so it was necessary to separate the literature on intertidal species from that on non-marine species with which it was intermixed. The task was daunting because of the number of species in the family (over 54,000 are now recognized), a huge polyglot literature, and the lack of habitat information in many of the early taxonomic publications. He included keys to identification of adults to the level of genus. In the subsequent 35 years, some genera that Moore dealt with have been re- vised (see particularly studies by Ahn, Ashe, Assing, Gusarov, Haghebaert, Herman, Jeon, Klimaszewski, Maruyama, and Zerche), additional species have been described, synonymies have been reported, there has been much change in the higher classifica- tion of Staphylinidae, and there have been some studies of the behavior of intertidal Staphylinidae. These changes make an updated contribution worth undertaking. This contribution is not simply an updating of Moore and Legner (1976), but it addition- ally lists all the staphylinid species (not just genera) that are believed to be restricted to Coastal Staphylinidae (Coleoptera) 3 coastal habitats. Introduction This list is augmented by page-references to their original descriptions in the literature, to references to generic revisions, and to publications on behavior of the species in question. We believe this will enable interested readers to access the origi- nal literature more readily. In this contribution, however, we do not include keys to identification of adults. For these the reader is urged to consult the cited literature. This contribution is intended for the reader who is willing and able to tackle the taxonomic literature, even if the ultimate objective is ecological or ethological.h j g g This contribution lists some 392 species, in 91 genera, of Staphylinidae that are be- lieved to be confined to coastal habitats. Some genera are confined to coastal habitats. Others include species that are confined to coastal habitats (and primarily only those with such restricted habitat are included in this treatment). One large genus, Bledius Leach, is exceptional in that its members live on banks of either freshwater or saline water bodies. Among the latter group it is in many instances unclear whether they are restricted to marine saline habitats. Moore and Legner (1976) included not only genera that restricted to intertidal habitats, but also genera whose members had frequently or occasionally been found there. Our contribution is more selective in that it tries to admit only those species for which there is evidence of restriction to such habitats. This attempt to concen- trate on true coastal species is emphasized by Smetana (2009) in his critical review of a paper published by Majka et al. (2008). Smetana’s (2009) viewpoint is to exclude those species that are occasional or even frequent visitors to the coastal habitat, and to concentrate on those that are confined to the habitat. There is some difficulty in treat- ing members of the large genus Bledius because some species occupy not only coastal habitats, but also inland saline habitats; we attempt to exclude species that are not restricted to coastal habitats. The current epitome of a study of regional coastal staphylinids is that by Ham- mond (2000). It discusses systematics and distribution of the British species, but it includes species that are not restricted to this habitat. Introduction The converse is perhaps Hase- gawa and Kanie (1992) which provided a list of Staphylinidae collected at one seashore locality in a 2-year time period, making no distinction between those species restricted to seashores and those incidentally found there, and makes no mention of their wider distribution. Lengerken (1929) published an extensive compilation of the distribution of coastal species of the North, Baltic, Irish, Mediterranean, Black, and Caspian seas. Audisio and Taglianti (2010) presented a compilation of Coleoptera found on Italian coasts, but did not indicate which ones among the included species are restricted to such habitats. The adjective halophilous seems first to have been used in English in the late 19th century to mean plants that are salt-loving, or growing in salt marshes (OED 1971). Since then, a set of terms evolved to describe adaptations of organisms to saline envi- ronments: 1. Halobionts (obligate inhabitants of saline habitats), 2. Halophiles (facultative inhabitants of saline habitats), J. H. Frank & Kee-Jeong Ahn / ZooKeys 107: 1–98 (2011) 4 4 3. Haloxenes (halotolerant species),i 3. Haloxenes (halotolerant species), 4. Incidentals (species not specifically associated with saline habitats but regularly found there. p 4. Incidentals (species not specifically associated with saline habitats but regularly found there. Such terms (in German) were used by Lengerken (1929) and other authors in English and French. We do not use these terms because they do not describe exactly what we want to include (and exclude), which is species that are restricted to sea coasts. We sup- pose that all of the species we list are halobionts, but we exclude halobionts living on the shores of inland saline lakes. Such terms (in German) were used by Lengerken (1929) and other authors in English and French. We do not use these terms because they do not describe exactly what we want to include (and exclude), which is species that are restricted to sea coasts. We sup- pose that all of the species we list are halobionts, but we exclude halobionts living on the shores of inland saline lakes. The checklist This checklist is the first to attempt to enumerate all coastal staphylinids, and their dis- tribution. Arrangement is taxonomic including subfamily, tribe, and genus; subtribes are included where defined. References to original generic and specific description are given. Generic and species synonyms are listed, each with original bibliographic reference. Listing of names of species within genera, genera within tribes, and tribes within subfamilies are mostly alphabetical, but names of subfamilies are arranged in taxonomic sequence. The arrangement followed for the higher categories is that of Lawrence and Newton (2000), Newton and Thayer (2007), and Grebennikov and Newton (2009) (Fig. 1). g To the far right of the taxonomic entries for genera (and in one instance for a tribe) reference to recent taxonomic revisions and phylogenies and keys to identification of adults [e.g., rev. Ahn 1996a; phy. Ahn and Ashe 1996b; key Moore and Legner 1976] is given. Because this work deals with coastal species, primary geographical entries are given according to the oceans and seas on which species are found. Secondary entries are the names of the countries they inhabit, and tertiary entries (if any) are the (mainly politi- cal) subunits of larger countries, or islands belonging to the former. Compression of this information into a checklist required the use of abbreviations, which are as follows: A. Codes used for oceans have 3 letters: ACO (Arctic Ocean), INO (Indian Ocean), NAO (North Atlantic Ocean), NPO (North Pacific Ocean), SAO (South Atlantic Ocean), and SPO (South Pacific Ocean). Names of seas and gulfs are spelled out: Anda- man Sea, Arabian Sea, Arafura Sea, Bali Sea, Baltic Sea, Bering Sea, Bismarck Sea, Black Sea, Caribbean Sea, Celebes Sea, East China Sea, East Sea [sometimes called Sea of Ja- pan, but that name is disputed (Wikipedia 2010)], Gulf of California (sometimes called Sea of Cortez), Gulf of Mexico, Irish Sea, Java Sea, Mediterranean Sea (here including Adriatic, Aegean, Ionian, Ligurian and Tyrrhenian Seas), North Sea, Sea of Okhotsk, Philippine Sea, Red Sea, South China Sea, Sulu Sea, Tasman Sea, and Timor Sea. pp Names of seas are not used throughout. We have used the name of the ocean in the broad sense (of which the sea is part) in instances where the name of the sea is not ap- Coastal Staphylinidae (Coleoptera) 5 5 p y p Figure 1. Phylogeny of the Staphylinidae. The checklist Bold indicates eight subfamilies containing coastal species Modified from Newton and Thayer (2007) and Grebennikov and Newton (2009). Micropeplinae Empelinae Glypholomatinae Omaliinae Proteininae Microsilphinae Neophoninae Dasycerinae Protopselaphinae Pselaphinae Aleocharinae Trichophyinae Habrocerinae Phloeocharinae Olisthaerinae Tachyporinae Osoriinae Piestinae Oxytelinae Scaphidiinae Trigonurinae Apateticinae Steninae Euaesthetinae Scydmaeninae Paederinae Staphylininae Pseudopsinae Leptotyphlinae Solieriinae Megalopsidiinae Oxyporinae Oxyporinae Steninae Euaesthetinae Scydmaeninae Paederinae Staphylininae Pseudopsinae Leptotyphlinae Figure 1. Phylogeny of the Staphylinidae. Bold indicates eight subfamilies containing coastal species. Modified from Newton and Thayer (2007) and Grebennikov and Newton (2009). parent from the literature. For example, some species known from New Zealand may be known from the west coast (the Tasman Sea), but if that was not apparent from the literature, we ascribed them to SPO (the South Pacific Ocean). parent from the literature. For example, some species known from New Zealand may be known from the west coast (the Tasman Sea), but if that was not apparent from the literature, we ascribed them to SPO (the South Pacific Ocean). B. Country codes have 2 letters and are the International Standards Organization (ISO) abbreviations. They are given in parentheses. Those used are: AG=Antigua and J. H. Frank & Kee-Jeong Ahn / ZooKeys 107: 1–98 (2011) 6 6 Barbuda, AL=Albania, AR=Argentina, AU=Australia, BB=Barbados, BE=Belgium, BG=Bulgaria, BM=Bermuda, BR=Brazil, BS=Bahamas, CA=Canada, CL=Chile, CN=China, CO=Columbia, CU=Cuba, CY=Cyprus, DE=Germany, DJ=Djibouti, DK=Denmark, DM=Dominica, DO=Dominican Republic, DZ=Algeria, EC=Ecuador, EE=Estonia, EG=Egypt, ER=Eritrea, ES=Spain, ET=Ethiopia, FI=Finland, FJ=Fiji, FP=French Polynesia, FR=France, GB=Great Britain, GD=Grenada, GE=Georgia, GH=Ghana, GL=Greenland, GP=Guadeloupe, GR=Greece, HR=Croatia, HT=Haiti, ID=Indonesia, IE=Ireland, IL=Israel, IN=India, IQ=Iraq, IS=Iceland, IT=Italy, JM=Jamaica, JP=Japan, KE=Kenya, KN=St. Kitts Nevis, KP=North Korea, KR=South Korea, KY=Cayman Island, LB=Lebanon, LC=St. Lucia, LK=Sri Lanka, LY=Libya, MA=Morocco, MG=Madagascar, MM=Myanmar, MR=Mauritania, MS=Montserrat, MT=Malta, MU=Mauritius, MX=Mexico, MY=Malaysia, NA=Namibia, NC=New Caledonia, NG=Nigeria, NL=Netherlands, NO=Norway, NZ=New Zealand, PE=Peru, PG=Papua New Guinea, PH=Philippines, PL=Poland, PR=Puerto Rico, PT=Portugal, RE=Reunion, RO=Romania, RU=Russian Federation, SA=Saudi Arabia, SC=Seychelles, SD=Sudan, SG=Singapore, SN=Senegal, SE=Sweden, SO=Somalia, TH=Thailand, TN=Tunisia, TR=Turkey, TT=Trinidad and Tobago, TW=Taiwan, TZ=Tanzania, UA=Ukraine, UK=United Kingdom, US=USA, UY=Uruguay, VE=Venezuela, VI=US Virgin Islands, VN=Vietnam, WS=Samoa (formerly Western Samoa, not American Samoa), YE=Yemen, YU=former Yugoslavia, ZA=South Africa. C. Where places within countries are mentioned, they are given after a colon (:) follow- ing the abbreviation of the country name, and either are spelled out or are abbreviated. The checklist For the USA and Canada, the abbreviations are the 2-letter postal codes (BC=British Columbia, NB=New Brunswick, NL=Newfoundland and Labrador, NS=Nova Sco- tia, NT=Northwest Territories, PE=Prince Edward Island, QC=Quebec, YT=Yukon Territory); for Mexico they have 2 letters (BN=Baja California, BS=Baja Califor- nia Sur, CA=Campeche, CH=Chiapas, GU=Guerrero, JA=Jalisco, MI=Michoacán, NA=Nayarit, OA=Oaxaca, QR=Quintana Roo, SI=Sinaloa, SO=Sonora, TB=Tabasco, TM=Tamaulipas, and VC=Veracruz); for Japan, designations are for major islands and is- land groups: (HK=Hokkaido, HN=Honshu, KY=Kyushu, RY=Ryukyu, SH=Shikoku). For Great Britain (GB) they are England, N. Ireland, Scotland, and Wales. g Compilation of the checklist is a first step in mapping of the distribution of all species. It draws upon information given in the original species descriptions as well as later sources that are believed reliable but are not cited in the bibliography. An ideal compilation would be an online database with a map for each species compiled from published collection records and linked directly to a bibliographic entry. Habits and habitats A checklist by itself reveals nothing about how the insects live. The ultimate in autecol- ogy is the numerical assessment of the dynamics of populations. An intermediate step Coastal Staphylinidae (Coleoptera) 7 is the study of habitat and behavior, by which can be learned the kinds of limitations to population size. This kind of information is sketchy for most coastal staphylinids, and is hard to present in tabular form. Therefore, this section presents textual information for genera and species whose habitat and behavior are known. Although this information is fundamental to population dynamics, it is also useful for the purposes of zoogeography. Works on seashores and their fauna at large have traditionally been published by marine biologists. If we wanted to learn about the identification of the fauna on Euro- pean shores, we might consult Barrett and Yonge (1958) or its replacement (Hayward et al. 1996), but we would be disappointed in coverage of Staphylinidae. If we wanted to read about sandy beaches and their fauna, we might consult McLachlan and Brown (2006), or Little et al. (2009) for rocky shores, or Hogarth (2007) for mangroves and seagrasses. Although we might learn much about those environments, we would again be disappointed in coverage of Staphylinidae. Not until we consult Cheng (1976) for marine insects do we gain an appreciation that many staphylinid species dwell on seashores (the included chapter by Moore and Legner), or Morris et al. (1980) for intertidal invertebrates of California (the included chapter by Evans), or Sherwood et al. (2000) for British coastal invertebrates (the included chapter by Hammond). The fault, if there is one, is the slowness in development of comprehensive treatment by entomologists of coastal staphylinids, and that the groundwork (taxonomic and behavioral studies) may not be in publications that marine biologists usually consult. y p g y Habitats of coastal staphylinids are drifted seaweed, the intertidal zone, sandy beaches, pebble beaches, rocky shores, muddy beaches and flats, salt marshes, and man- grove swamps (Figs 2–5), but these are not necessarily mutually exclusive. Staphylini- dae are very often associated with plants and algae. These may be growing in estuaries (especially marshgrasses, such as Spartina), on rocky shores (green, brown, and red algae), or on sandy shores (diatoms). Or they may be plants or seagrasses (Thalassia, Zostera, etc.) torn loose and deposited on any of those shores. Habits and habitats Few staphylinid stud- ies have identified associated plants specifically or even generically. Often, the drifted seaweeds (i.e., seaweeds deposited onshore by the tide) are called ‘wrack’. Backlund (1945) used the term to include not only the brown alga Fucus, but many other al- gae, and the sea-grass Zostera (a vascular plant) deposited onshore. Barrett and Yonge (1958), in contrast, referred to large brown algae including the genus Laminaria as ‘kelp’, confining the term ‘wrack’ to moderately sized brown algae including Pelvetia, Fucus, and Ascophyllum. They apply these terms to growing seaweed as well as to sea- weed deposited onshore. Moore and Legner (1973) defined wrack as drifted kelp. We employ the term ‘drifted seaweed’ to all algae deposited on shorelines, and we distin- guish sea-grasses and marsh-grasses. Drifted seaweed may be ephemeral and sparse and greatly subject to drying, to deep and decomposing and more or less permanent depending upon location and tides. Backlund (1945) published an ecological study of drifted seaweed in more or less permanent beds on seashores in Sweden and Finland, including its insect inhabitants. Staphylinids may occupy the sand under sparse accretions of seaweed. In thick seaweed beds, staphylinids inhabit the seaweed together with other invertebrates. Organisms J. H. Frank & Kee-Jeong Ahn / ZooKeys 107: 1–98 (2011) 8 Figure 2. Habitats of coastal Staphylinidae. A Seagrasses on a sandy beach in Florida, USA B Seaweeds on a sandy beach in Jejudo Island, Korea C Kelp on a rocky shore in Greymouth, New Zealand D Rocky shore in San Diego, USA E Seaweeds on rocks of a sandy beach in California, USA F Rock crevice on a rocky shore in Plymouth, England G Rock covered with barnacles in Baeksu, Korea H Close-up of barnacles. Figure 2. Habitats of coastal Staphylinidae. A Seagrasses on a sandy beach in Florida, USA B Seaweeds on a sandy beach in Jejudo Island, Korea C Kelp on a rocky shore in Greymouth, New Zealand D Rocky shore in San Diego, USA E Seaweeds on rocks of a sandy beach in California, USA F Rock crevice on a rocky shore in Plymouth, England G Rock covered with barnacles in Baeksu, Korea H Close-up of barnacles. Coastal Staphylinidae (Coleoptera) 9 p y p Figure 3. Habitats of coastal Staphylinidae. Habits and habitats A Estuary of Carmel River in California, USA B Salt marsh in Florida, USA C Mangrove forests in Cat Ba National Park, Vietnam D Under stones of mangrove in Cat Ba National Park, Vietnam E Mud flat in Gungpyongri, Korea F Pebbles and rocks on beach in Kaikoura, New Zealand G Seagrasses on a sandy beach in Haiphong, Vietnam H Dead fishes on a sandy beach in Florida, USA. Figure 3. Habitats of coastal Staphylinidae. A Estuary of Carmel River in California, USA B Salt marsh in Florida, USA C Mangrove forests in Cat Ba National Park, Vietnam D Under stones of mangrove in Cat Ba National Park, Vietnam E Mud flat in Gungpyongri, Korea F Pebbles and rocks on beach in Kaikoura, New Zealand G Seagrasses on a sandy beach in Haiphong, Vietnam H Dead fishes on a sandy beach in Florida, USA. J. H. Frank & Kee-Jeong Ahn / ZooKeys 107: 1–98 (2011) 10 Figure 4. Coastal Staphylinidae. A Aleochara zerchei on a sandy beach in Donghae, Korea B Aleochara puetzi on a sandy beach in Donghae, Korea C Bryothinusa koreana under a stone on rocky headland in Dangjin, Korea D Atheta tokiokai on a sandy beach in Jejudo Island, Korea E Paramblopusa borealis under a stone on pebble beach in Alaska, USA F Diaulota aokii with barnacles on a rocky shore in Baeksu, Korea G Diaulota aokii with fresh seaweeds on a rocky shore in Jejudo Island, Korea H Larva of Diaulota aokii with barnacles on a rocky shore in Baeksu, Korea. Figure 4. Coastal Staphylinidae. A Aleochara zerchei on a sandy beach in Donghae, Korea B Aleochara puetzi on a sandy beach in Donghae, Korea C Bryothinusa koreana under a stone on rocky headland in Dangjin, Korea D Atheta tokiokai on a sandy beach in Jejudo Island, Korea E Paramblopusa borealis under a stone on pebble beach in Alaska, USA F Diaulota aokii with barnacles on a rocky shore in Baeksu, Korea G Diaulota aokii with fresh seaweeds on a rocky shore in Jejudo Island, Korea H Larva of Diaulota aokii with barnacles on a rocky shore in Baeksu, Korea. Coastal Staphylinidae (Coleoptera) Coastal Staphylinidae (Coleoptera) 11 Coastal Staphylinidae (Coleoptera) 11 Figure 5. Coastal Staphylinidae. Habits and habitats A Phucobius simulator on a sandy beach in Guryongpo, Korea B Liusus hilleri on a sandy beach in Donghae, Korea C Overwintering staphylinine species (Cafius histrio, Liusus hilleri and Philonthus nudus) under a wooden board on a sandy beach in Jindo Island, Korea D Larva of Cafius sp. under fresh seaweeds on a sandy beach in Jejudo Island, Korea E Cafius bistriatus on a sandy beach in North Carolina, USA F Cafius seminites under decaying seaweeds on a sandy beach in Califor- nia, USA G Cafius rufescens on a sandy beach in Jindo Island, Korea H Philonthus nudus under decaying seaweeds on a sandy beach in Jindo Island, Korea. Figure 5. Coastal Staphylinidae. A Phucobius simulator on a sandy beach in Guryongpo, Korea B Liusus hilleri on a sandy beach in Donghae, Korea C Overwintering staphylinine species (Cafius histrio, Liusus hilleri and Philonthus nudus) under a wooden board on a sandy beach in Jindo Island, Korea D Larva of Cafius sp. under fresh seaweeds on a sandy beach in Jejudo Island, Korea E Cafius bistriatus on a sandy beach in North Carolina, USA F Cafius seminites under decaying seaweeds on a sandy beach in Califor- nia, USA G Cafius rufescens on a sandy beach in Jindo Island, Korea H Philonthus nudus under decaying seaweeds on a sandy beach in Jindo Island, Korea. J. H. Frank & Kee-Jeong Ahn / ZooKeys 107: 1–98 (2011) 12 that eat and decompose seaweed are more abundant, including amphipods (Crustacea) and seaweed flies (Diptera: Coelopidae), both fed upon by many predacious Staphyli- nidae, including Cafius spp. Amphipods (commonly called ‘beach-hoppers’ or ‘beach- fleas’) are innocuous with respect to humans, but coelopids, (i.e., Coelopa spp.) are more problematic. “The flies normally pass their whole lives on the weed [and] are abundant in the wrack all the year around [and] their numbers are not appreciated un- til something makes them come out into the open [and] they may fly in a band, a little above the ground [and] occasionally they stray far inland [and] can be a great nuisance when large numbers… congregate in shops, garages, and particularly dry-cleaners’ (Ol- droyd 1965: 173–174).” By feeding on these flies, Cafius spp. and other staphylinids can be considered as beneficial species. Habits and habitats Poleward movement of seaweed deposits and their coelopid inhabitants has been noted and attributed to global warming, and there is a suspicion that Laminaria is in decline and that Fucus is expected to decline in the future in the British Isles (Edward et al. 2007). Rocky shores offer refuges for specialist staphylinids (those not found in other habitats) in crevices or empty barnacle shells that trap pockets of air. Such shorelines often support the growth of algae, and these algae exhibit zonation according to spe- cies (Jones 1968). Staphylinids likewise distribute themselves according to such zones where they find refuge among algal holdfasts (Jones 1968; Topp and Ring 1988b). Rocky shores not only provide a substrate for barnacles and living algae, with which some staphylinids are associated, but they also frequently include tidal pool habitats. Tidal pools are inhabited by a few staphylinids, e.g., Rothium. A few species such as Micralymma marinum (which is believed to prey on Collembola) attain higher eleva- tions above sea level on rocky cliffs (Thayer 1985).i fh Coral reefs, even five km from the shoreline, are habitat for a species of Brachypro- nomaea (Sawada 1956). Its food there remained unknown until the abundant Collem- bola present at one such site suggested a probable food source (Ahn et al. 2003). Off the coast of the Aru Islands in the Indonesian Archipelago, among coral polyps, Fauvel (1878a) found unusual staphylinids and described the genera Corallis and Polypea. Their food and way of life have not yet been determined. h Shores may be graded from solid rock to boulders, cobbles, pebbles, gravels, and sand, and even finer particles typical of mud flats, salt marshes, and mangrove swamps. All of those substrates have their complement of coastal Staphylinidae. Table 1 is our attempt to summarize information about habitats across all genera; it is incomplete because the available information is incomplete. We summarize exist- ing information not just about habitats but also about behavior and physiology in the section on Habits, Habitats, and Classificatory Notes under the name of each genus. Zoogeography Based on taxonomy and distribution, we provide some provisional ideas about the dispersion of the taxa. Coastal Staphylinidae (Coleoptera) 13 Table 1. Genera of the Staphylinidae containing coastal species with their known numbers and habitats. Subfamily Tribe Genus No. species Tidal zone Habitat Microhabitat Microsilphinae Microsilpha 1 not known sand spit not known Omaliinae Aphaenostemmini Giulianium 3 HM SB UD, UP Omaliini Crymus 2 not known not known US Macralymma 1 not known SB US Micralymma 2 ML RH RC Omaliomimus 10 ML not known US Omalium 4 not known not known US, UG, UD Pselaphinae Batrisini Arthromelus 1 HM MA UD Batriscenites 2 HM MA UD Batrisocenus 1 HM MA UD Brachyglutini Brachygluta 6 not known SM UG, UP Briara 1 PH MA UP, UD Briaraxis 1 not known not known UD, UP, US Mangalobythus 3 HM MA cavities in log Nisaxis 2 not known SM UD, UG Pedisinops 1 not known coral reef not known Physoplectus 4 not known not known UP Prosthecarthron 1 not known MF, SM UP Aleocharinae Aleocharini Aleochara 16 PH, HM SB, RH US, UD Athetini Acticola 1 not known not known US Adota 6 PH SM, SB US, UD Atheta 6 PH SM, ES, SB US, UD Brundinia 2 not known SM, ES UD J. H. Frank & Kee-Jeong Ahn / ZooKeys 107: 1–98 (2011) 14 Tribe Genus No. Zoogeography species Tidal zone Habitat Microhabitat Thinobius 3 PH, HM BS, SB, SM, MF US ydmaeninae Cephenniini Cephennodes 1 PH BS UP ederinae Paederini Chetocephalus 1 PH SB US Medon 4 PH SB US, UD Ophioomma 1 PH SB UD Sunius 2 PH SB, MA US aphylininae Staphylinini Bisnius 1 PH SB US Cafius 44 PH SB US, UG, UD Gabronthus 1 not known not known not known Hadropinus 1 PH SB US Hadrotes 2 PH SB US Heterothops 1 PH SB US, UD Liusus 2 PH SB US, UG, UD Orthidus 1 PH SB US,UP Philonthus 1 PH SB, SM US, UG, UD Phucobius 8 PH SB US, UG, UD Quediocafus 3 PH SB UG Remus 4 PH SB US, UG, UD Thinocafius 1 PH SB US Thinopinus 1 PH SB US : proximal to high tide zone; HM: high to mid tide zone; ML: mid to low tide zone; VL: very low tide zone. BS: boulder shores (band of gravels/pebbles/ bles); ES: estuary; MA: mangrove; MF: mud/sand flats; RH: rocky headland; SB: sandy beach; SM: salt marsh. EB: Inside of empty barnacles/shells; RA: rock h algae; RC: rock crevices; UB: under beach sand; UD: under debris; UG: under seagrasses; UP: under stones (gravels/pebbles/cobbles); US: under seaweeds. Subfamily Tribe Genus No. species Tidal zone Habitat Microhabitat Thinobius 3 PH, HM BS, SB, SM, MF US Scydmaeninae Cephenniini Cephennodes 1 PH BS UP Paederinae Paederini Chetocephalus 1 PH SB US Medon 4 PH SB US, UD Ophioomma 1 PH SB UD Sunius 2 PH SB, MA US Staphylininae Staphylinini Bisnius 1 PH SB US Cafius 44 PH SB US, UG, UD Gabronthus 1 not known not known not known Hadropinus 1 PH SB US Hadrotes 2 PH SB US Heterothops 1 PH SB US, UD Liusus 2 PH SB US, UG, UD Orthidus 1 PH SB US,UP Philonthus 1 PH SB, SM US, UG, UD Phucobius 8 PH SB US, UG, UD Quediocafus 3 PH SB UG Remus 4 PH SB US, UG, UD Thinocafius 1 PH SB US Thinopinus 1 PH SB US PH: proximal to high tide zone; HM: high to mid tide zone; ML: mid to low tide zone; VL: very low tide zone. BS: boulder shores (band of gravels/pebbles/ cobbles); ES: estuary; MA: mangrove; MF: mud/sand flats; RH: rocky headland; SB: sandy beach; SM: salt marsh. Zoogeography species Tidal zone Habitat Microhabitat Halobrecta 7 not known SM, ES US, UD Hydrosmecta 1 PH SB US Iotarphia 1 not known not known not known Osakatheta 1 HM ES, MF UP Pontomalota 2 PH, HM SB US, UD Psammopora 1 not known not known not known Psammostiba 5 PH, HM SB US Tarphiota 3 PH, HM SB US, UD Thinusa 2 PH, HM SB US, UD Diglottini Diglotta 8 PH SB, SM, ES, BS UP, UB Falagriini Bryobiota 2 PH, HM SB US, UD Myrmecopora 14 PH, HM SB US, UD Homalotini Cameronium 5 HM RH RC, US Heterota 10 PH SB US, UD Linoglossa 1 not known MA not known Paractocharis 3 PH SB US Pseudopasilia 1 not known BS UP, US Thinobiosus 1 not known SB US Liparocephalini Amblopusa 5 HM BS UP, UB, US Baeostethus 1 PH SB UP Diaulota 8 ML, VL RH EB, RC, RA Halorhadinus 3 HM BS UP, US Ianmoorea 1 PH SB UB Liparocephalus 4 ML, VL RH RC, RA Subfamily Coastal Staphylinidae (Coleoptera) 15 Tribe Genus No. species Tidal zone Habitat Microhabitat Paramblopusa 2 HM SB UP Myllaenini Brachypronomaea 4 ML RH coral reef Bryothinusa 30 HM, ML SB, BS, MF, MA UP, UB, RC, EB Corallis 1 VL not known under coral polyp Lautaea 1 not known MA, MF not known Myllaena 1 PH BS UP Polypea 1 VL not known under coral polyp Rothium 6 HM, ML RH RA Oxypodini Chilodera 1 not known not known US Dasydera 1 not known not known US Gyronotus 1 not known not known not known Oreuryalea 1 PH SB US, UD Phytosini Actocharis 2 PH SB UB Arena 2 PH SB, MF UB Euphytosus 1 not known not known not known Phytosus 8 not known SB UB, US, UD Incertae sedis Salinamexus 3 PH SB US, UD, UP e Oxytelini Anotylus 1 PH SB US, UD Blediotrogus 4 PH SB US Pareiobledius 3 PH SB US Sartallus 1 PH SB US, UD Thinobiini Bledius 57 PH, HM MF, SM, ES UB, US, UD Carpelimus 1 PH MF, SM, ES UB, US, UD Teropalpus 9 PH SB US, UD Subfamily J. H. Frank & Kee-Jeong Ahn / ZooKeys 107: 1–98 (2011) 16 bfamily Tribe Genus No. OMALIINAE OMALIINAE APHAENOSTEMMINI Giulianium Moore 1976: 56 [rev Ahn and Ashe 1999] G. alaskanum Ahn and Ashe 1999: 162 - NPO (US: AK; JP: HK) G. campbelli Moore 1976: 57 - NPO (US: CA) G. newtoni Ahn and Ashe 1999: 163 - NPO (US: CA) OMALIINAE APHAENOSTEMMINI Giulianium Moore 1976: 56 [rev Ahn and Ashe 1999] G. alaskanum Ahn and Ashe 1999: 162 - NPO (US: AK; JP: HK) G. campbelli Moore 1976: 57 - NPO (US: CA) G. newtoni Ahn and Ashe 1999: 163 - NPO (US: CA) Micralymma Westwood 1838: 129 M. marinum (Strøm) 1783: 65 - NAO (CA: NB, NL, NS, QC; US: MA, ME, NH; FR; GB: England, N. Ireland, Scotland; GL; IE; IS; NO), North and Baltic and Irish Seas (BE; DE; GB: England, Scotland, N. Ireland, Wales; NL; SE; RU: Karelia) = brevipenne (Gyllenhal) 1810: 234 = johnstonis Westwood 1838: 130 = stimpsonii LeConte 1863: 57 = brevipenne (Gyllenhal) 1810: 234 = johnstonis Westwood 1838: 130 = stimpsonii LeConte 1863: 57 p M. laticolle Motschulsky 1860: 549 ACO (RU: Siberia) [probably does not belong to Micralymma] l J l 1940 117 M. laticolle Motschulsky 1860: 549 ACO (RU: Siberia) [probably does not belong to Micralymma] OMALIINI Crymus Fauvel 1904b: 92 = Arpediopsis Cameron 1917a: 124 = Arpediomimus Cameron 1917f: 277 C. antarticus Fauvel 1904b: 93 - SAO (South Georgia; Falkland Islands) = falklandicus (Cameron) 1917a: 125 C. kronii (Kiesenwetter) 1877: 161 SPO (NZ: Antipodes Island, Auckland Island, Campbell Island, South Island) = longiceps (Broun) 1914: 89 Macralymma Cameron 1945c: 179 M. punctiventre Cameron 1945c: 179 - SPO (NZ) l d A Checklist of coastal Staphylinidae and their distribution MICROSILPHINAE Microsilpha Broun 1886: 889 M. litorea Broun 1886: 890 - SPO (NZ) Crymus Fauvel 1904b: 92 p C. antarticus Fauvel 1904b: 93 - SAO (South Georgia; Falkland Islands) = falklandicus (Cameron) 1917a: 125 C. kronii (Kiesenwetter) 1877: 161 SPO (NZ: Antipodes Island, Auckland Island, Campbell Island, South Island) = longiceps (Broun) 1914: 89 Macralymma Cameron 1945c: 179 M. punctiventre Cameron 1945c: 179 - SPO (NZ) Macralymma Cameron 1945c: 179 M. punctiventre Cameron 1945c: 179 - SPO (NZ) Micralymma Westwood 1838: 129 Zoogeography EB: Inside of empty barnacles/shells; RA: rock with algae; RC: rock crevices; UB: under beach sand; UD: under debris; UG: under seagrasses; UP: under stones (gravels/pebbles/cobbles); US: under seaweeds. PH: proximal to high tide zone; HM: high to mid tide zone; ML: mid to low tide zone; VL: very low tide zone. BS: boulder shores (band of gravels/pebbles/ cobbles); ES: estuary; MA: mangrove; MF: mud/sand flats; RH: rocky headland; SB: sandy beach; SM: salt marsh. EB: Inside of empty barnacles/shells; RA: rock with algae; RC: rock crevices; UB: under beach sand; UD: under debris; UG: under seagrasses; UP: under stones (gravels/pebbles/cobbles); US: under seaweeds. 17 17 Coastal Staphylinidae (Coleoptera) Arthromelus Jeannel 1949: 149 Arthromelus Jeannel 1949: 149 A. quadratus Tanokuchi 1989: 88 - South China Sea (SG) Batriscenites Jeannel 1952: 96 B. celer Tanokuchi 1989: 91 - South China Sea (SG) B. humicola Tanokuchi 1989: 95 - South China Sea (SG) Batrisocenus Raffray 1903: 48 B. foveiterminalis Tanokuchi 1989: 97 - South China Sea (SG) A. quadratus Tanokuchi 1989: 88 - South China Sea (SG) B. celer Tanokuchi 1989: 91 - South China Sea (SG) B. humicola Tanokuchi 1989: 95 - South China Sea (SG)f B. foveiterminalis Tanokuchi 1989: 97 - South China Sea (SG) Omaliomimus Jeannel 1940: 117 Omaliomimus Jeannel 1940: 117 O. actobius (Broun) 1893: 1035 - SPO (NZ) O. actobius (Broun) 1893: 1035 - SPO (NZ) O. albipennis (Kiesenwetter) 1877: 162 - SPO (AU: Macquarie Island; NZ: Campbell Island, Auckland Island) = variipennis (Lea) 1920: 30 = flavipennis Cameron 1948: 723 O. carinigerus (Broun) 1893: 1036 - SPO (NZ) O. chalmeri (Broun) 1893: 1037 - SPO (NZ) O. conicus (Fauvel) 1878b: 484 - SPO (NZ) O. albipennis (Kiesenwetter) 1877: 162 - SPO (AU: Macquarie Island; NZ: O. albipennis (Kiesenwetter) 1877: 162 - SPO (AU: Macquarie Island; NZ: Campbell Island, Auckland Island) = variipennis (Lea) 1920: 30 = flavipennis Cameron 1948: 723 O. albipennis (Kiesenwetter) 1877: 162 Campbell Island, Auckland Island) = variipennis (Lea) 1920: 30 = flavipennis Cameron 1948: 723 O. carinigerus (Broun) 1893: 1036 - SPO (NZ) O. chalmeri (Broun) 1893: 1037 - SPO (NZ) O. conicus (Fauvel) 1878b: 484 - SPO (NZ) J. H. Frank & Kee-Jeong Ahn / ZooKeys 107: 1–98 (2011) 18 O. laetipennis (Broun) 1910: 19 - SPO (NZ) O. litoreus (Broun) 1886: 942 - SPO (NZ) O. robustus (Broun) 1911: 96 - SPO (NZ: Chatham Islands, Pitt Island) O. setipes (Broun) 1909b: 230 - SPO (NZ) O. venator (Broun) 1909a: 98 SPO (AU: Macquarie Island; NZ: mainland Antipodes Island, Auckland Island, Campbell Island, Snares Island) p O. litoreus (Broun) 1886: 942 - SPO (NZ) O. robustus (Broun) 1911: 96 - SPO (NZ: Chatham Islands, Pitt Island) O. setipes (Broun) 1909b: 230 - SPO (NZ) O. setipes (Broun) 1909b: 230 - SPO (NZ) p O. venator (Broun) 1909a: 98 SPO (AU: Macquarie Island; NZ: mainland, p O. venator (Broun) 1909a: 98 SPO (AU: Macquarie Island; NZ: mainland, Antipodes Island, Auckland Island, Campbell Island, Snares Island) Omalium Gravenhorst 1802: 111 O. algarum Casey 1885: 316 - NPO (CA: BC; US: CA, OR) O. algarum Casey 1885: 316 - NPO (CA: BC; US: CA, OR) g y O. laeviusculum Gyllenhal 1827: 464 NAO (FR; GB: England, Scotland; IE; O. laeviusculum Gyllenhal 1827: 464 NAO (FR; GB: England, Scotland; IE; IS; NO), ACO (RU), North Sea (BE; DE; DK; GB: England, Scotland; NL), Irish Sea (GB: England, Wales; IE), Baltic Sea (FI; SE)h O. laeviusculum Gyllenhal 1827: 464 NAO (FR; GB: England, Scotland; IE; IS; NO), ACO (RU), North Sea (BE; DE; DK; GB: England, Scotland; NL), Irish Sea (GB: England, Wales; IE), Baltic Sea (FI; SE) O. riparium Thomson 1857: 224 - NAO (ES; FR; GB: England, Scotland; MA; PT; DK: Faroes), Irish Sea (GB: England, Wales; IE), North Sea (BE; DE; DK; FR; GB: England, Scotland; NL), Baltic Sea (DE; DK; EE; FI; PL; SE), Mediterranean Sea (DZ; ES; FR; IT; MA; YU) O. riparium Thomson 1857: 224 - NAO (ES; FR; GB: England, Scotland; MA; PT; DK: Faroes), Irish Sea (GB: England, Wales; IE), North Sea (BE; DE; DK; FR; GB: England, Scotland; NL), Baltic Sea (DE; DK; EE; FI; PL; SE), Mediterranean Sea (DZ; ES; FR; IT; MA; YU) O. rugulipenne Rye 1864: 58 - NAO (GB: England, Scotland), Irish Sea (GB: England; IE), North Sea (BE; DE; FR; GB: England, Scotland; NL) O. rugulipenne Rye 1864: 58 - NAO (GB: England, Scotland), Irish Sea (GB: England; IE), North Sea (BE; DE; FR; GB: England, Scotland; NL) PSELAPHINAE BATRISITAE BATRISINI Arthromelus Jeannel 1949: 149 A. quadratus Tanokuchi 1989: 88 - South China Sea (SG) Batriscenites Jeannel 1952: 96 B. celer Tanokuchi 1989: 91 - South China Sea (SG) B. humicola Tanokuchi 1989: 95 - South China Sea (SG) Batrisocenus Raffray 1903: 48 B. foveiterminalis Tanokuchi 1989: 97 - South China Sea (SG Brachygluta Thomson 1859: 54 Brachygluta Thomson 1859: 54 Brachygluta Thomson 1859: 54 h B. abdominalis (Aubé) 1833: 27 - NAO (eastern US; CA: NB, NS) B. cavicornis (Brendel) 1865a: 30 - NAO (eastern US: NY, MD, DC, VA) B. curvicera (Motschulsky) 1854: 4 - NAO (eastern US: NY)l B. floridana (Brendel) 1865b: 257 - NAO (eastern US: NY, MD, VA, NC, SC, FL) B. luniger (LeConte) 1849: 87 - NAO (eastern US: MA, NY, NJ, MD, VA) B. ulkei (Brendel) 1866: 193 - NAO (eastern US: MD, DC, DE, VA, SC, GA, FL) ( d ) 93 N O ( US , C, , V , SC, G , FL) p Rica) Mangalobythus Tanokuchi 1989: 104 Mangalobythus Tanokuchi 1989: 104 M. acutifolius Tanokuchi 1989: 109 - South China Sea (TH) M. furcifer Tanokuchi 1989: 106 - South China Sea (SG) M. murphyi Tanokuchi 1989: 111 - South China Sea (SG) Nisaxis Casey 1886: 183 N. maritima Casey 1887: 468 - Gulf of Mexico (US: LA, MS, TX) N. tomentosa (Aubé) 1833: 33 - NAO (eastern US: CT, NY, NJ, DE, MD, N. tomentosa (Aubé) 1833: 33 - NAO (eastern US: CT, NY, NJ, DE, MD, DC, NC), Gulf of Mexico (FL, Al, MS, TX), Caribbean Sea = minuta (Brendel) 1865a: 30 = cincinnata Casey 1887: 466 DC, NC), Gulf of Mexico (FL, Al, MS, TX), Caribbean Sea = minuta (Brendel) 1865a: 30 y Pedisinops Newton and Chandler 1989: 43 = Pedinopsis Raffray 1890: 102 = Halohermatus Sawada 1991: 148 P. regulus (Sawada) 1991: 150 - NPO (JP: RY) Physoplectus Reitter 1882: 210 = Halorabyxis Jeannel 1954: 338 = Thalassomerus Sawada 1992: 55 P. irritans Chandler 2001: 349 - SPO (AU: Queensland) P. miyakei (Sawada) 1992: 58 - NPO (JP: RY) P. reikoae (Sawada) 1992: 56 - NPO (JP: HN) P. vinsoni (Jeannel) 1954: 341 - INO (MU) Prosthecarthron Raffray 1915: 2 P. sauteri Raffray 1915: 3 - NPO (KP; JP: HN, KY, SH, RY), SPO (TW; VN) = palpalis (Löbl) 1974: 97; 1977: 236 Pedisinops Newton and Chandler 1989: 43f P. regulus (Sawada) 1991: 150 - NPO (JP: RY) h P. irritans Chandler 2001: 349 - SPO (AU: Queensland) Q P. miyakei (Sawada) 1992: 58 - NPO (JP: RY) y J P. reikoae (Sawada) 1992: 56 - NPO (JP: HN) P. vinsoni (Jeannel) 1954: 341 - INO (MU) f P. sauteri Raffray 1915: 3 - NPO (KP; JP: HN, KY, SH, RY), SPO (TW; VN f = palpalis (Löbl) 1974: 97; 1977: 236 Briara Reitter 1882: 207 Briara Reitter 1882: 207 Briara Reitter 1882: 207 = Gonatocerus Schaufuss 1880: 30 [preoccupied] = Berlara Reitter 1882: 206 Coastal Staphylinidae (Coleoptera) 19 B. bella (Tanokuchi) 1989: 101 - South China Sea (SG) Briaraxis Brendel 1894: 158 Briaraxis Brendel 1894: 158 B. depressa Brendel 1894: 159 - Caribbean Sea (US: FL; TT: Tobago, Costa Rica) B. depressa Brendel 1894: 159 - Caribbean Sea (US: FL; TT: Tobago, Costa Rica) ALEOCHARINI ( riochara) nubis ( ss g) 995: 3 ast Sea (RU: Sa a , a c at a) A. (Triochara) trisulcata Weise 1877: 88 - NPO, East Sea (CN: Hong Kong; JP: HN; KR) A. (Triochara) trisulcata Weise 1877: 88 - NPO, East Sea (CN: Hong Kong; JP: HN; KR) A. (Triochara) zerchei (Assing) 1995: 231 - East Sea (KR; RU: Primorie, Sakha- lin) A. (Triochara) zerchei (Assing) 1995: 231 - East Sea (KR; RU: Primorie, Sakha- lin) ALEOCHARINI eochara Gravenhorst 1802: 67 [rev Nearctic Klimaszewski 1984, southern Africa Kli ki d J 1994 P l i A i 1995] Aleochara Gravenhorst 1802: 67 [rev Nearctic Klimaszewski 1984, southern Africa Klimaszewski and Jansen 1994, Palearctic Assing 1995] Klimaszewski and Jansen 1994, Palearctic Assing 1995] Klimaszewski and Jansen 1994, Palearctic Assing 1995] g A. (Coprochara) salsipotens Bernhauer 1912c: 209 - SAO (NA; ZA), INO (ZA) A. (Coprochara) salsipotens Bernhauer 1912c: 209 - SAO (NA; ZA), INO (ZA) A. (Coprochara) squalithorax Sharp 1888: 282 - East Sea (JP: HN; KR) A. (Coprochara) squalithorax Sharp 1888: 282 - East Sea (JP: HN; KR) A. (Coprochara) sulcicollis Mannerheim 1843: 225 NPO (CA: BC; US: AK, CA, OR, WA; MX: BN, BS), SPO (CL) A. (Emplenota) albopila (Mulsant and Rey) 1852: 171 - Mediterranean Sea (FR; GR; IT; YU), Black Sea (BG), NAO (ES: Canary Islands) A. (Emplenota) curtidens Klimaszewski 1984: 101 - NPO (CA: BC; US: CA) A. (Emplenota) fucicola Sharp 1874: 9 - NPO, South China Sea, East Sea (CN: Hong Kong; JP: HN; KR) = variolosa Weise 1877: 89 J. H. Frank & Kee-Jeong Ahn / ZooKeys 107: 1–98 (2011) 20 A. (Emplenota) litoralis (Mäklin) 1853: 182 - NAO (CA: NB, NL, NS, QC; US: FL, MA, NJ, NY, RI), NPO (CA: BC; US: AK, CA; MX: BN, BS, SO) A. (Emplenota) obscurella Gravenhorst 1806: 159 NAO (ES; FR; GB: England, Scotland; IE; NO), Irish Sea (GB: England, Wales; IE), North Sea (BE; DE; DK; GB: England, Scotland; NL; NO), Baltic Sea (DE; DK; PL; SE), Irish Sea (GB; IE) = algarum Fauvel 1862b: 92 A. (Emplenota) pacifica (Casey) 1894: 290 NPO (CA: BC; US: CA, WA; MX: BN) A. (Emplenota) phycophila Allen 1937: 219 - NAO (GB) A. (Emplenota) puetzi (Assing) 1995: 225 - East Sea (KR; RU: Sakhalin, Kam- chatka) A. (Polystomota) grisea Kraatz 1856: 96 - NAO (FR; GB: England, Scotland; IE; MA; NO; PT), Irish Sea (GB: Wales), North Sea (BE; DE; DK; GB: England, Scotland; NL; NO), Baltic Sea (DE; DK; FI; PL; SE), Mediter- ranean Sea (DZ; ES; FR; IT; YU) A. (Polystomota) punctatella Motschulsky 1858b: 240 - NAO (England, Scot- land; IE), Irish Sea (GB: England, Wales), North Sea (BE; GB: England, Scotland; NL), NAO (FR) A. (Triochara) nubis (Assing) 1995: 232 - East Sea (RU: Sakhalin, Kamchatka) . ATHETINI Acticola Cameron 1944b: 618 Acticola Cameron 1944b: 618 A. falkandica Cameron 1944b: 619 - SAO (Falkland Islands) Adota Casey 1910: 67 [rev Gusarov 2003a,b] = Panalota Casey 1910: 71 = Halostiba Yosii and Sawada 1976: 86 A. colpophila Gusarov 2003b: 16 - Gulf of California (MX: SO) A. gnypetoides (Casey) 1910: 69 - NPO (US: AK, CA) A. madida (Bernhauer) 1907: 400 - NPO (JP: HN, KY) A. magnipennis (Bernhauer) 1943: 184 - NPO, East Sea (JP: HN, KY; KR) A. maritima (Mannerheim) 1843: 224 - NPO (CA: BC; US: AK, CA), NAO (GB: England), North Sea (GB: England, Scotland) = massettensis Casey 1910: 68 = subintima Casey 1910: 68 = setosetarsis Casey 1910: 71 = insons Casey 1911: 125 Coastal Staphylinidae (Coleoptera) 21 21 = scolopacina Casey 1911: 124 = scortea Casey 1911: 124 = scortea Casey 1911: 124 y = immigrans (Easton) 1971: 25 - North Sea (GB), SPO (NZ) = immigrans (Easton) 1971: 25 - North Sea (GB), SPO (NZ) = immigrans (Easton) 1971: 25 - North Sea (GB), SPO (NZ) A. ushio (Sawada) 1971a: 304 - NPO, East Sea (JP: HN, KY)h g . ushio (Sawada) 1971a: 304 - NPO, East Sea (JP: HN, KY) g A. ushio (Sawada) 1971a: 304 - NPO, East Sea (JP: HN, KY) Atheta Thomson 1858: 36 Atheta Thomson 1858: 36fi meridionalis (Mulsant and Rey) 1853: 38 - NAO (FR; GB: England), Irish Sea England, Wales), North Sea (BE; GB: England), Mediteranean Sea (FR; IT)h Halobrecta Thomson 1858: 35 [rev Gusarov 2004, mainly Nearctic] obrecta Thomson 1858: 35 [rev Gusarov 2004, mainly Nearctic] H. algae (Hardy) 1851: 78 - Baltic Sea (DK; EE; FI; RU: Karelia; SE), North Sea (BE; GB: England, Scotland), NAO (FR; NO), SPO (AU) = puncticeps (Thomson) 1852: 134 = anthracina Fairmaire 1852: 687 [synonymy based on statement by Fair- maire 1856: 424 but not otherwise verified] H. algae (Hardy) 1851: 78 - Baltic Sea (DK; EE; FI; RU: Karelia; SE), North Sea (BE; GB: England, Scotland), NAO (FR; NO), SPO (AU) = puncticeps (Thomson) 1852: 134 = anthracina Fairmaire 1852: 687 [synonymy based on statement by Fair- maire 1856: 424 but not otherwise verified] h = anthracina Fairmaire 1852: 687 [synonymy based on statement by Fair- maire 1856: 424 but not otherwise verified] H. algophila (Fenyes) 1909: 419 - NPO (US: CA), SPO (NZ; CL: Palena), SAO (Tristan da Cunha: Inaccessible Island), NAO (GB), Mediterranean Sea (FR: Corsica) = barbarae (Casey) 1910: 18 = importuna (Casey) 1911: 111 Atheta Thomson 1858: 36fi A. (Actophylla) varendorffiana Bernhauer and Scheerpeltz [in Scheerpeltz1934: 1637] - North Sea (DE) d ffi B h 4 [ d] A. (Actophylla) varendorffiana Bernhauer and Scheerpeltz [in Scheerpeltz1934: 1637] - North Sea (DE) = varendorffi Bernhauer 1908a: 334 [preoccupied] A. (Actophylla) varendorffiana Bernhauer and Scheerpeltz [in Scheerpeltz1934: 1637] - North Sea (DE) = varendorffi Bernhauer 1908a: 334 [preoccupied] = varendorffi Bernhauer 1908a: 334 [preoccupied] ffi p p A. (Badura) ririkoae Sawada 1989b: 285 - NPO, East Sea (JP: HN; KR) ffi A. (Badura) ririkoae Sawada 1989b: 285 - NPO, East Sea (JP: HN; KR) A. (Badura) tokiokai (Sawada) 1971a: 306 - NPO, East Sea (JP: HN, KY; KR) A. (Badura) tokiokai (Sawada) 1971a: 306 - NPO, East Sea (JP: HN, KY; KR A. (Datomicra) acadiensis Klimaszewski and Majka 2007: 49 NAO (CA: NB, NS, PE, QC) A. (Datomicra) acadiensis Klimaszewski and Majka 2007: 49 NAO (CA: NB, NS, PE, QC) A. (Sipalatheta) algarum Pace 1999b: 680 - South China Sea (CN: Hong Kong) A. (Sipalatheta) algarum Pace 1999b: 680 - South China Sea (CN: Hong Kong) A. novaescotiae Klimaszewski and Majka [in Klimaszewski et al. 2006: 68] A. novaescotiae Klimaszewski and Majka [in Klimaszewski et al. 2006: 68] A. novaescotiae Klimaszewski and Majka [in Klimaszewski et al. 2006: 68] NAO (CA: NB, NS, NL, Sable Island; St Pierre and Miquelon: Miquelon) undinia Tottenham 1949: 78 NAO (CA: NB, NS, NL, Sable Island; St Pierre and Miquelon: Miquelon) undinia Tottenham 1949: 78 B. marina (Mulsant and Rey) 1853: 39 - NAO (FR; GB: England), North Sea (DE; GB: England, Scotland; NL), Baltic Sea (DE; SE), Irish Sea (GB: England, Wales; IE), Mediterranean Sea (FR; IT) = imbecilla (G. Waterhouse) 1858: 6074 (and 1859: 16) = thinobia Thomson 1861: 73 B. marina (Mulsant and Rey) 1853: 39 - NAO (FR; GB: England), North Sea (DE; GB: England, Scotland; NL), Baltic Sea (DE; SE), Irish Sea (GB: England, Wales; IE), Mediterranean Sea (FR; IT) = imbecilla (G. Waterhouse) 1858: 6074 (and 1859: 16) = thinobia Thomson 1861: 73 = thinobia Thomson 1861: 73 B. meridionalis (Mulsant and Rey) 1853: 38 - NAO (FR; GB: England), Irish Sea England, Wales), North Sea (BE; GB: England), Mediteranean Sea (FR; IT)h B. = pubes (Mulsant and Rey) 1873a: 660 = pubes (Mulsant and Rey) 1873a: 660 = puncticeps sensu Mulsant and Rey 1875: 12 = pocahontas (Casey) 1910: 19 = pocahontas (Casey) 1910: 19 p = vaticina (Casey) 1910: 19 = vaticina (Casey) 1910: 19 = incertula (Casey) 1910: 84 H. halensis Mulsant and Rey 1873b: 173 - Mediterranean Sea (FR) H. princeps (Sharp) 1869: 142 - NAO (GB: England) Hydrosmecta Thomson 1858: 33 yh H. subalgarum Pace 1999b: 672 - South China Sea (CN: Hong Kong) Iotarphia Cameron 1943: 352 h H. subalgarum Pace 1999b: 672 - South China Sea (CN: Hong Kong) Iotarphia Cameron 1943: 352 I. australis Cameron 1943: 352 - Tasman Sea (AU: New So Osakatheta Maruyama, Klimaszewski and Gusarov 2008: 40 I. australis Cameron 1943: 352 Tasman Sea (AU: New S Osakatheta Maruyama, Klimaszewski and Gusarov 2008: 40 akatheta Maruyama, Klimaszewski and Gusarov 2008: 40 O. yasukoae Maruyama, Klimaszewski and Gusarov 2008: 41 - N Pontomalota Casey 1885: 296 [rev Ahn and Ashe 1992] P. opaca (LeConte) 1863: 28 - NPO (CA: BC; US: CA, OR, WA; MX: BN) = californica Casey 1885: 298 = nigriceps Casey 1885: 299 = luctuosa Casey 1911: 164 = bakeri Bernhauer 1912b: 170 P. opaca (LeConte) 1863: 28 - NPO (CA: BC; US: CA, OR, WA; MX: BN) = californica Casey 1885: 298 = nigriceps Casey 1885: 299 = luctuosa Casey 1911: 164 = bakeri Bernhauer 1912b: 170 P. opaca (LeConte) 1863: 28 - NPO (CA: BC; US: CA, OR, WA; MX: BN) l f = nigriceps Casey 1885: 299 = luctuosa Casey 1911: 164 = bakeri Bernhauer 1912b: 170 = importuna (Casey) 1911: 111 H. cingulata (Cameron) 1920: 266 - South China Sea (SG) H. cingulata (Cameron) 1920: 266 - South China Sea (SG) = consors (Cameron) 1920: 266 = consors (Cameron) 1920: 266 H. discipula Pace 1999a: 171 - SPO (AU; CL: Valparaíso) p p H. flavipes Thomson 1861: 50 - NAO (NO), Baltic Sea (DK; EE; FI; SE), Mediterranean Sea (IT), North Sea (BE; DE; GB: England, Scotland), NAO (CA: NB; US: NY,VA), SPO (AU; CL: Llanquihue) = maritima (G. Waterhouse) 1863: 137 = halobrectha (Sharp) 1869: 139 p p H. flavipes Thomson 1861: 50 - NAO (NO), Baltic Sea (DK; EE; FI; SE), Mediterranean Sea (IT), North Sea (BE; DE; GB: England, Scotland), NAO (CA: NB; US: NY,VA), SPO (AU; CL: Llanquihue) = maritima (G. Waterhouse) 1863: 137 = halobrectha (Sharp) 1869: 139 22 J. H. Frank & Kee-Jeong Ahn / ZooKeys 107: 1–98 (2011) = pubes (Mulsant and Rey) 1873a: 660 = puncticeps sensu Mulsant and Rey 1875: 12 = pocahontas (Casey) 1910: 19 = vaticina (Casey) 1910: 19 = incertula (Casey) 1910: 84 halensis Mulsant and Rey 1873b: 173 - Mediterranean Sea (FR) P. terminalia Ahn and Ashe 1992: 356 - NPO (US: CA) geniculata (Mäklin) 1852: 308 - NPO (CA: BC; US: AK, CA, OR) = iota Casey 1910: 76 = insolita Casey 1910: 76 = litorina Casey 1910: 75 = seditiosa Casey 1910: 76 23 Coastal Staphylinidae (Coleoptera) Thinusa Casey 1894: 371 [rev Ahn 1997b] Thinusa Casey 1894: 371 [rev Ahn 1997b] T. fletcheri Casey 1906: 353 - NPO (CA: BC; US: AK, CA, OR, WA) = divergens Casey 1911: 213 = nigra Casey 1911: 214 = robustula Casey 1911: 215 T. maritima (Casey) 1885: 312 - NPO (CA: BC; US: CA, OR, WA; MX: BN) = obscura Casey 1906: 354 Thinusa Casey 1894: 371 [rev Ahn 1997b] T. fletcheri Casey 1906: 353 - NPO (CA: BC; US: AK, CA, OR, WA) = divergens Casey 1911: 213 = nigra Casey 1911: 214 = robustula Casey 1911: 215 T. maritima (Casey) 1885: 312 - NPO (CA: BC; US: CA, OR, WA; MX: B = obscura Casey 1906: 354 = obscura Casey 1906: 354 P. terminalia Ahn and Ashe 1992: 356 - NPO (US: CA) P. terminalia Ahn and Ashe 1992: 356 - NPO (US: CA) P. terminalia Ahn and Ashe 1992: 356 - N Psammopora Pace 2003: 154 P. delittlei Pace 2003: 157 - Tasman Sea (AU: Tasmania) Psammostiba Yosii and Sawada 1976: 82 [rev Gusarov 2003b] P. comparabilis (Mäklin) 1853: 181 - NPO (CA: BC, US: AK, CA) P. comparabilis (Mäklin) 1853: 181 - NPO (CA: BC, US: AK, CA) P. hilleri (Weise) 1877: 90 - NPO, East Sea (JP: HN, KY) = multipunctata (Sawada) 1971a: 301 P. jessoensis (Brundin) 1943: 22 - NPO (JP: HK, HN; RU: Maritime Territory) P. jessoensis (Brundin) 1943: 22 - NPO (JP: HK, HN; RU: Maritime Territory) P. kamtschatica (Brundin) 1943: 21 - NPO (JP: HK; RU: Kamchatka, Kuril Islands) P. jessoensis (Brundin) 1943: 22 - NPO (JP: HK, HN; RU: Maritime Territory) P. kamtschatica (Brundin) 1943: 21 - NPO (JP: HK; RU: Kamchatka, Kuril Islands) P. kamtschatica (Brundin) 1943: 21 - NPO (JP: HK; RU: Kamchatka, Kuril Islands) P. kenaii Gusarov 2003b: 28 - NPO (CA: BC; US: AK, CA) P. kenaii Gusarov 2003b: 28 - NPO (CA: BC; US: AK, CA) Tarphiota Casey 1894: 332 [rev Ahn 1996b, Ahn 1999, Klimaszewski et al. 2006] T. densa (Moore) 1978a: 115 - NPO (MX: BS, SO) Tarphiota Casey 1894: 332 [rev Ahn 1996b, Ahn 1999, Klimaszew T. densa (Moore) 1978a: 115 - NPO (MX: BS, SO) = hirsutula Casey 1910: 75 T. densa (Moore) 1978a: 115 - NPO (MX: BS, SO) = hirsutula Casey 1910: 75 y T. fucicola (Mäklin) 1852: 306 - NPO (CA: BC), Gulf of California (MX: BC, SO) = debilicollis Casey 1910: 75 p llidip C 1894 333 NPO (US CA) T. fucicola (Mäklin) 1852: 306 - NPO (CA: BC), Gulf of California (MX: BC, SO) = debilicollis Casey 1910: 75 = pallidipes Casey 1894: 333 - NPO (US: CA) = pallidipes Casey 1894: 333 - NPO (US: CA) p p T. geniculata (Mäklin) 1852: 308 - NPO (CA: BC; US: AK, CA, OR) = iota Casey 1910: 76 = insolita Casey 1910: 76 = litorina Casey 1910: 75 = seditiosa Casey 1910: 76 T. DIGLOTTINI [key Pace 1986] Diglotta Champion 1887: 228 (repeated 1899: 265) [rev Haghebaert 1991] = Diglossa Haliday 1837: 252 [preoccupied] D. brasiliensis Caron and Ribeiro-Costa 2008: 53 - SAO (BR: Paraná) Diglotta Champion 1887: 228 (repeated 1899: 265) [rev Haghebaert 1991] = Diglossa Haliday 1837: 252 [preoccupied] D. brasiliensis Caron and Ribeiro-Costa 2008: 53 - SAO (BR: Paraná) Diglotta Champion 1887: 228 (repeated 1899: 265) [rev Haghebaert 1991] = Diglossa Haliday 1837: 252 [preoccupied] g D. brasiliensis Caron and Ribeiro-Costa 2008: 53 - SAO (BR: Paraná) D. legneri Moore and Orth 1979a: 339 - NPO (US: CA) g D. littoralis (Horn) 1871: 331 - NAO (US: NJ) D. maritima Lea 1927: 277 - SPO (FJ: Levuka) D. mersa (Haliday) 1837: 252 North Sea (BE; DE; DK; FR; GB: England; NL), Irish Sea (GB: N. Ireland, Wales), Mediterranean Sea (DZ; IT), NAO (CA: NB; GB: England, Scotland; IE) = D. submarina Fairmaire 1856: 468 D. pacifica Fenyes 1921: 17 - NPO (US: CA, OR; MX: BN) D. secqi Pace 1992: 180 - Red Sea (DJ) D. sinuaticollis (Mulsant and Rey) 1870: 176 NAO (GB: England), Irish Sea (GB: England, Wales; IE) D. sinuaticollis (Mulsant and Rey) 1870: 176 NAO (GB: England), Irish Sea (GB: England, Wales; IE) = D. crassa (Mulsant and Rey) 1870: 180 (GB: England, Wales; IE) = D. crassa (Mulsant and Rey) 1870: 180 FALAGRIINI [rev Hoebeke 1985, phy Ahn and Ashe 1995] FALAGRIINI [rev Hoebeke 1985, phy Ahn and Ashe 1995] ALAGRIINI [rev Hoebeke 1985, phy Ahn and Ashe 1995] Bryobiota Casey 1894: 367 [rev Ahn and Ashe 1995] Bryobiota Casey 1894: 367 [rev Ahn and Ashe 1995] B. bicolor (Casey) 1885: 311 - NPO (CA: BC; US: WA, OR, CA; MX: BN) = californica (Scheerpeltz) 1965: 49 y y 9 3 7 [ 995] B. bicolor (Casey) 1885: 311 - NPO (CA: BC; US: WA, OR, CA; MX: BN) lif i (S h l ) 1965 49 B. bicolor (Casey) 1885: 311 - NPO (CA: BC; US: WA, OR, CA; MX: BN) B. bicolor (Casey) 1885: 311 - NPO (CA: BC; US: WA, OR, CA; MX: BN) = californica (Scheerpeltz) 1965: 49 f p B. giulianii (Moore) 1978a: 113 - NPO (US: CA, WA) Myrmecopora Saulcy 1864: 429 [rev Assing 1997a,b, Palaearctic] M. (Lamproxenusa) algarum (Sharp) 1874: 12 - NPO, East Sea (JP: HN, Toka- ra Island) = miyamotoi (Sawada) 1955: 85 M. (Lamproxenusa) algarum (Sharp) 1874: 12 - NPO, East Sea (JP: HN, Toka- ra Island) M. (Lamproxenusa) chinensis Cameron 1944c: 158 South China Sea (CN: Hong Kong) M. (Lamproxenusa) chinensis Cameron 1944c: 158 South China Sea (CN: Hong Kong) M. (Lamproxenusa) reticulata Assing 1997b: 344 - NPO (RU: Far East; KP) M. (Lamproxenusa) rufescens (Sharp) 1874: 11 - NPO, East Sea (JP: HN, KY p f p M. (Paraxenusa) laesa (Erichson) 1839b: 73 Mediterranean Sea (IT; PT; FR; DZ; TN; HR; ES: Balearic Islands), NAO (ES: Canary Islands) = tenuicornis (Küster) 1854: no. 3 M. (Paraxenusa) laesa (Erichson) 1839b: 73 Mediterranean Sea (IT; PT; FR; DZ; TN; HR; ES: Balearic Islands), NAO (ES: Canary Islands) = tenuicornis (Küster) 1854: no. 3 = carica Fagel 1970: 155 M. (Xenusa) uvida (Erichson) 1840: 916 NAO (GB: England), Mediterranean Sea (AL; BG; CY; ES; GR; TN; IT; HR; YU), North Sea (BE; DE; GB: England; NL), NAO (FR), Black Sea (UA; GE) = meridiogallica Scheerpeltz 1972: 101 = tenuicornis (Küster) 1854: no. 3 M. (Xenusa) anatolica (Fagel) 1969: 117 - Mediterranean Sea (CY; TR) M. (Xenusa) bernhaueri Koch 1936: 210 - Red Sea (EG; IL) 24 J. H. Frank & Kee-Jeong Ahn / ZooKeys 107: 1–98 (2011) M. (Xenusa) boehmi Bernhauer 1910: 259 Mediterranean Sea (FR: Corsica; IT; GR; TN; MA; CY) = sydowi Bernhauer 1927b: 97 = mediterranea Fagel 1970: 152 M. (Xenusa) brevipes Butler 1909: 29 NAO (FR; GB: England), Irish Sea (GB: Wales; IE), North Sea (GB: England) [but see Hammond 2000: 257] = oweni Assing 1997a: 114 [fide Hammond 2000: 257] M. (Xenusa) maritima (Wollaston) 1860: 51 NAO (ES: Canary Islands; PT: Madeira) M. (Xenusa) minima Bernhauer 1901a: 537 Black Sea (BG; RO) Mediterra- nean Sea (GR; MA) = buresi Rambousek 1910: 19 [in Czech] and 21 [in French] = pamphylica (Fagel) 1969: 120 M. (Xenusa) simillima (Wollaston) 1864: 534 NAO (ES: Canary Islands; GB: England, Scotland; IE; NO), Baltic Sea (DK; DE), North Sea (GB: Eng- land, Scotland), Mediterranean Sea (FR; ES; PT; TN; DZ; EG), NAO (PT: Azores) = lohmanderi Bernhauer 1927a: 167 M. (Xenusa) sulcata (Kiesenwetter) 1850: 218 Mediterranean Sea (AL; GR; FR: Corsica; IT: Sardinia, Sicily; HR), North Sea (GB), Black Sea (RO; BG; UA) = carica Fagel 1970: 155 Cameronium Koch 1936: 202 Cameronium Koch 1936: 202 C. flavipenne Cameron 1944a: 318 - INO (SO; TZ: Zanzibar) C. gomyi Pace 1985: 622 - INO (Comoros) C. lamuense Pace 1994: 155 - INO (KE: Lamu) C. obockianus (Fauvel) 1905: 146 - Red Sea [DJ; ET; YE: Barim (= Perim) Island] C. sonorensis Moore 1964a: 175 - Gulf of California (MX: SO) Heterota Mulsant and Rey 1874: 194 [rev Park et al. 2008] H. arenaria Cameron 1920: 251 - South China Sea (SG) H. brevicollis (Bernhauer) 1929: 187 - Red Sea [YE: Barim (= Perim) Island] H. gomyi Jarrige 1973: 257 - INO (MG) H. obscura Cameron 1938: 174 - INO (RE) H. pamphylica Fagel 1969: 123 - Mediterranean Sea (TR) H. pictipennis (Fauvel) 1905: 142 - Red Sea (DJ; ET), INO (SO) C. flavipenne Cameron 1944a: 318 - INO (SO; TZ: Zanzibar) C. lamuense Pace 1994: 155 - INO (KE: Lamu) C. obockianus (Fauvel) 1905: 146 - Red Sea [DJ; ET; YE: Barim (= Perim) Island] C. obockianus (Fauvel) 1905: 146 - Red Sea [DJ; ET; YE: Barim (= Perim) Island] C. sonorensis Moore 1964a: 175 - Gulf of California (MX: SO) Heterota Mulsant and Rey 1874: 194 [rev Park et al. 2008] H. arenaria Cameron 1920: 251 - South China Sea (SG) H. arenaria Cameron 1920: 251 - South China Sea (SG) H. brevicollis (Bernhauer) 1929: 187 - Red Sea [YE: Barim (= Perim) Island] H. gomyi Jarrige 1973: 257 - INO (MG) H b C 1938 1 4 INO (RE) H. brevicollis (Bernhauer) 1929: 187 - Red Sea [YE: Barim (= Perim) Island] H. obscura Cameron 1938: 174 - INO (RE) H. pamphylica Fagel 1969: 123 - Mediterranean Sea (TR) H. pictipennis (Fauvel) 1905: 142 - Red Sea (DJ; ET), INO (SO) Coastal Staphylinidae (Coleoptera) 25 H. plumbea (G. Waterhouse) 1858: 6074 (and 1859: 15) - Mediterranean Sea (Europe), NAO (ES: Canary Islands; GB: England; US: FL), Irish Sea (GB: Wales), Caribbean Sea (JM; MX: QR) = fairmairii (Brisout) 1859: ccxviii = godelinaisei (Fauvel) 1862b: 92 = trogophloeoides (Wollaston) 1864: 536 = impressa (Mulsant and Rey) 1875: 459 H. plumbea (G. Cameronium Koch 1936: 202 Waterhouse) 1858: 6074 (and 1859: 15) - Mediterranean Sea (Europe), NAO (ES: Canary Islands; GB: England; US: FL), Irish Sea (GB: Wales), Caribbean Sea (JM; MX: QR) = fairmairii (Brisout) 1859: ccxviii = godelinaisei (Fauvel) 1862b: 92 = trogophloeoides (Wollaston) 1864: 536 = impressa (Mulsant and Rey) 1875: 459 p y H. rougemonti Pace 1993: 137 - Bali Sea (ID: Bali) H. sunjaei Park and Ahn [in Park et al.] 2008: 111 - NPO (KR) H. vinsoni Cameron 1947a: 118 - INO (MG; MU; RE, Comoros) Linoglossa Kraatz 1859a: 10 L. murphyi Sawada 1991: 142 - South China Sea (SG) Paractocharis Cameron 1917c: 154 P. deharvengi Pace 1990: 81 - Luzon Sea (PH: Mindoro) P. fucicola Cameron 1917c: 155 - South China Sea (SG) P. orousseti Pace 1990: 79 - Luzon Sea (PH: Mindoro) Pseudopasilia Ganglbauer 1895: 211 p g P. testacea (Brisout) 1863: 16 NAO (FR; GB: England), North Sea (BE), Med- iterranean Sea (FR: mainland, Corsica; HR; IT; TN) P. testacea (Brisout) 1863: 16 NAO (FR; GB: England), North Sea (BE), Med it rr n n S (FR m inl nd C r i HR IT TN) P. testacea (Brisout) 1863: 16 NAO (FR; GB: England), Nor iterranean Sea (FR: mainland, Corsica; HR; IT; TN) P. testacea (Brisout) 1863: 16 NAO (FR; GB: England), North Sea (BE iterranean Sea (FR: mainland, Corsica; HR; IT; TN) g iterranean Sea (FR: mainland, Corsica; HR; IT; TN) Thinobiosus Moore and Legner 1977: 468 h T. salinus Moore and Legner 1977: 469 - Gulf of California (MX: SO) LIPAROCEPHALINI [phy Ahn and Ashe 1996b, Ahn et al. 2010] p Amblopusa Casey 1894: 355 [rev Ahn and Ashe 1996a, Zerche 1998] A. alaskana Ahn and Ashe 1996a: 143 - NPO (US: AK) A. brevipes Casey 1894: 356 - NPO (CA: BC; US: AK, CA) = pallida Casey 1911: 212 p A. hokkaidona Ahn and Ashe 1996a: 142 - NPO (JP: HK) A. magna Zerche 1998: 106 - NPO (JP: HK; RU: Kuril Islands)i A. pacifica (Sawada) 1991: 147 - NPO (JP: HK) i Baeostethus Broun 1909a: 96 [rev Leschen et al. 2002] i Baeostethus Broun 1909a: 96 [rev Leschen et al. 2002] Baeostethus Broun 1909a: 96 [rev Leschen et al. 2002] B. chiltoni Broun 1909a: 97 - SPO (NZ: Campbell, Auckland, Antipodes Is- land) B. chiltoni Broun 1909a: 97 - SPO (NZ: Campbell, Auckland, Antipodes Is- land) B. chiltoni Broun 1909a: 97 - SPO (NZ: Campbell, Auckland, Antipodes Is- land) Diaulota Casey 1894: 354 [rev Ahn 1996a, Zerche 1998] = Genoplectes Sawada 1955: 81 D. alaskana Ahn 1996a: 278 - NPO (US: AK) D. aokii Sawada 1971b: 104 - NPO (JP: HK, HN; KR; US: AK) D. densissima Casey 1894: 354 - NPO (CA: BC; US: CA, OR, WA) = D insolita Casey 1894: 355 Diaulota Casey 1894: 354 [rev Ahn 1996a, Zerche 1998] = Genoplectes Sawada 1955: 81 D. alaskana Ahn 1996a: 278 - NPO (US: AK) D. aokii Sawada 1971b: 104 - NPO (JP: HK, HN; KR; US: AK) D. densissima Casey 1894: 354 - NPO (CA: BC; US: CA, OR, WA) = D. insolita Casey 1894: 355 D. fulviventris Moore 1956a: 120 - NPO (US: CA; MX: BN) D. harteri Moore 1956a: 123 - NPO (US: CA; MX: BN) = D. megacephala Moore 1956a: 124 D. alaskana Ahn 1996a: 278 - NPO (US: AK) D. aokii Sawada 1971b: 104 - NPO (JP: HK, HN; KR; US: AK) D. densissima Casey 1894: 354 - NPO (CA: BC; US: CA, OR, WA) D. densissima Casey 1894: 354 - NPO (CA: BC; US: CA, OR, WA) = D. insolita Casey 1894: 355 D. pacifica Sawada 1971b: 101 - NPO (JP: HN; KR) borealis (Casey) 1906: 354 - NPO (CA: BC; US: AK, OR, WA; JP: HK) P. eoa Ahn and Maruyama 2000: 359 - NPO (RU: Kuril Islands) P. borealis (Casey) 1906: 354 - NPO (CA: BC; US: AK, OR, WA; JP: HK) P. eoa Ahn and Maruyama 2000: 359 - NPO (RU: Kuril Islands) D. pacifica Sawada 1971b: 101 - NPO (JP: HN; KR) D. pacifica Sawada 1971b: 101 - NPO (JP: HN; KR) p fi D. uenoi (Sawada) 1955: 82 - NPO (JP: HN, RY; KR) D. vandykei Moore 1956a: 125 - NPO (US: CA) Halorhadinus Sawada 1971b: 92 [rev Ahn 2001] D. vandykei Moore 1956a: 125 - NPO (US: CA) Halorhadinus Sawada 1971b: 92 [rev Ahn 2001] H. aequalis Sawada 1971b: 92 - NPO, East Sea (JP: HN; KR) q H. inaequalis Sawada 1971b: 95 - NPO, East Sea (JP: HN; KR) H. sawadai Maruyama and Hayashi 2009: 72 - East Sea (JP: HN) H. sawadai Maruyama and Hayashi 2009: 72 - East Sea (JP: HN) Ianmoorea Ahn 2006: 36 Moorea Ahn 2004: 255 Ianmoorea Ahn 2006: 36 = Moorea Ahn 2004: 255 I. zealandica (Ahn) 2004: 258 - SPO (NZ: North Island, South Island) Liparocephalus Mäklin 1853: 191 [rev Ahn 1997a, Maruyama and Ahn 2000b] L. brevipennis (Mäklin) 1853: 192 - NPO (US: AK) L. cordicollis LeConte 1880: 177 - NPO (CA: BC; US: AK, CA, OR, WA) L. litoralis Kirschenblatt 1938: 532 - NPO (RU: Kuril Islands; JP: HK) L. tokunagai Sakaguti 1944: 20 - NPO (JP: SH, KY) = Moorea Ahn 2004: 255 I. zealandica (Ahn) 2004: 258 - SPO (NZ: North Island, South Island) Liparocephalus Mäklin 1853: 191 [rev Ahn 1997a, Maruyama and Ahn 2000b] L. brevipennis (Mäklin) 1853: 192 - NPO (US: AK) L. cordicollis LeConte 1880: 177 - NPO (CA: BC; US: AK, CA, OR, WA) L l l K h bl 5 NPO (RU K l I l d JP HK) I. zealandica (Ahn) 2004: 258 - SPO (NZ: North Island, South Island) Liparocephalus Mäklin 1853: 191 [rev Ahn 1997a, Maruyama and Ahn 2000b I. zealandica (Ahn) 2004: 258 - SPO (NZ: North Island, South Island) Liparocephalus Mäklin 1853: 191 [rev Ahn 1997a, Maruyama and Ahn 2000b p p y L. brevipennis (Mäklin) 1853: 192 - NPO (US: AK) p L. cordicollis LeConte 1880: 177 - NPO (CA: BC; US: AK, CA, OR, WA) L. litoralis Kirschenblatt 1938: 532 - NPO (RU: Kuril Islands; JP: HK) L. tokunagai Sakaguti 1944: 20 - NPO (JP: SH, KY) Paramblopusa Ahn and Ashe 1996a: 148 [rev Ahn and Ashe 1996a, Maruyama and Ahn 2000a] P. borealis (Casey) 1906: 354 - NPO (CA: BC; US: AK, OR, WA; JP: HK) P. eoa Ahn and Maruyama 2000: 359 - NPO (RU: Kuril Islands) and Ahn 2000a] P. MYLLAENINI [phy Ahn and Ashe 2004] MYLLAENINI [phy Ahn and Ashe 2004] Brachypronomaea Sawada 1956: 197 [rev Ahn et al. 2003]h h p g B. esakii Sawada 1956: 197 - NPO (JP: RY) B. marchemarchadi (Jarrige) 1959: 64 - South China Sea (VN) B. nosybiana (Jarrige) 1959: 65 - INO (MG) B. sawadai Jarrige 1964: 178 - SPO (NC) Bryothinusa Casey 1904: 312 [rev Pace 1986, Haghebaert 1995, Ashe 2005] Bryothinusa Casey 1904: 312 [rev Pace 1986, Haghebaert 1995, Ashe 2005] = Halesthenus Sawada 1955: 83 Bryothinusa Casey 1904: 312 [rev Pace 1986, Haghebaert 1995, Ashe 2005] = Halesthenus Sawada 1955: 83 B. algarum Sawada 1971b: 90 - NPO (JP: HN, KY) B. cameroni (Fauvel) 1904a: 74 - Red Sea [ER; YE: Kameran (= Cameran) Island, Barim (= Perim) Island] = B. microphthalma (Bernhauer) 1929: 187 p B. catalinae Casey 1904: 313 - NPO (US: CA) B. catalinae Casey 1904: 313 - NPO (US: CA) B. celebensis (Fauvel) 1878a: 301 - Celebes Sea (ID: Sulawesi) B. chani Moore and Legner 1971: 107 - South China Sea (CN: Hong Kong) B. chani Moore and Legner 1971: 107 - South China Sea (CN: Hong Kong) B. chengae Ahn 1998: 335 - SPO (Caroline Island: Palau) g B. fluenta Moore and Legner 1975: 111 - South China Sea (CN: Hong Kong B. fluenta Moore and Legner 1975: 111 - South China Sea (CN: Hong Kong) fl g B. gangjinensis Ahn and Jeon 2004: 29 - NPO (KR) B. grootaerti Haghebaert 1995: 29 - Bismarck Sea (PG: Laing Island) B. hauseri Ashe 2005: 582 - South China Sea (MY: Malaya) = D. insolita Casey 1894: 355 D. fulviventris Moore 1956a: 120 - NPO (US: CA; MX: BN) f D. harteri Moore 1956a: 123 - NPO (US: CA; MX: BN) D. harteri Moore 1956a: 123 - NPO (US: = D. megacephala Moore 1956a: 124 = D. megacephala Moore 1956a: 124 26 J. H. Frank & Kee-Jeong Ahn / ZooKeys 107: 1–98 (2011) B. hauseri Ashe 2005: 582 - South China Sea (MY: Malaya) B. hongkongensis Moore, Legner and Chan 1973: 77 South China Sea (CN: Hong Kong) B. hongkongensis Moore, Legner and Chan 1973: 77 South China Sea (CN: Hong Kong) Coastal Staphylinidae (Coleoptera) 27 B. koreana Ahn and Jeon 2004: 31 - NPO (KR) B. madecassa Pace 2008: 568 - INO (MG) B. minuta (Sawada) 1955: 83 - NPO (JP: HN, RY; KR) B. nakanei (Sawada) 1955: 85 - NPO (JP: RY; KR) SO: Hartan Peninsula] B. testacea (Cameron) 1904: 157 - Red Sea [YE: Barim (= Perim) Island] B. testaceipennis (Cameron) 1919: 245 - South China Sea (SG) B. tsutsuii (Sawada) 1955: 84 - NPO (JP: HN, RY) B. tsutsuii (Sawada) 1955: 84 - NPO (JP: HN, RY) = B. serpentis (Sawada) 1955: 84 B. tsutsuii (Sawada) 1955: 84 NPO (JP: HN, RY) = B. serpentis (Sawada) 1955: 84 B. nakanei (Sawada) 1955: 85 - NPO (JP: RY; KR) B. neoguineensis Pace 2000a: 115 - Bismarck Sea (PG: Laing Island) B. orousseti Pace 1990: 66 - Luzon Sea (PH: Mindoro) B. papuensis Haghegbaert 1995: 31 - Bismarck Sea (PG: Cape Vogel Peninsula) B. papuensis Haghegbaert 1995: 31 - Bismarck Sea (PG: C p p g g p g B. parvula Haghebaert 1995: 27 - Bismarck Sea (PG: Laing Island) B. perexilis Pace 1994: 132 - INO (SO: Sar Uanle) B. peyerimhoffi (Fauvel) 1904a: 73 Red Sea, Gulf of Akkaba, Mediterranean Sea (IL) B. rothi Moore and Legner 1975: 110 - Gulf of California (MX: SO) B. sakishimana Sawada 1991: 144 - NPO (JP: RY) B. samoensis Pace 1984: 67 - SPO (WS: Upolu Island) B. sawadai Moore, Legner and Chan 1973: 75 - South China Sea (CN: Hong Kong) B. sinensis Moore, Legner and Chan 1973: 76 - South China Sea (CN: Hong Kong) B. subtilissima (Cameron) 1904: 157 Red Sea [YE: Barim (= Perim) Island; SO: Hartan Peninsula] Corallis Fauvel 1878a: 212 Corallis Fauvel 1878a: 212 C. polyporum Fauvel 1878a: 213 Arafura Sea [ID: Kepalauan Aru (Aru and Wokam), Kepalauan Kai (= Kei or Ke Island)] C. polyporum Fauvel 1878a: 213 Arafura Sea [ID: Kepalauan Aru (Aru and Wokam), Kepalauan Kai (= Kei or Ke Island)] Lautaea Sawada 1989a: 83 L. murphyi Sawada 1989a: 85 - South China Sea (SG) Lautaea Sawada 1989a: 83 L. murphyi Sawada 1989a: 85 - South China Sea (SG) L. murphyi Sawada 1989a: 85 - South China Sea (SG) Myllaena Erichson 1837: 382 M. insipiens Casey 1911: 237 - NAO, Gulf of Mexico (US: AL, FL, LA, NJ, PA) OXYPODINI Chilodera Cameron 1944b: 619 C. falklandica Cameron 1944b: 620 - SAO (Falkland Islands) C. falklandica Cameron 1944b: 620 - SAO (Falkland Islands) Dasydera Cameron 1948: 731 = Calonotus Cameron 1945c: 171 [preoccupied] = Mecrona Blackwelder 1952: 232 D. algophila (Broun) 1886: 941 - SPO (NZ: Mokohinau Island) Gyronotus Cameron 1948: 731 = Eurynotus Cameron 1945c: 170 [preoccupied] = Marecon Blackwelder 1952: 230 G. rufipennis (Broun) 1880: 92 - SPO (NZ: North Island) Oreuryalea Assing and Maruyama 2002: 210 O. watanabei Assing and Maruyama 2002: 217 - NPO, East Sea (RU: Primo- rie, Sakhalin; JP: HK) f ( ) Dasydera Cameron 1948: 731 = Calonotus Cameron 1945c: 171 [preoccupied] = Mecrona Blackwelder 1952: 232 D. algophila (Broun) 1886: 941 - SPO (NZ: Mokohinau Island) Gyronotus Cameron 1948: 731 = Eurynotus Cameron 1945c: 170 [preoccupied] = Marecon Blackwelder 1952: 230 G. rufipennis (Broun) 1880: 92 - SPO (NZ: North Island) Oreuryalea Assing and Maruyama 2002: 210 O. watanabei Assing and Maruyama 2002: 217 - NPO, East Sea (RU: rie, Sakhalin; JP: HK) G. rufipennis (Broun) 1880: 92 - SPO (NZ: North Island) O. watanabei Assing and Maruyama 2002: 217 - NPO, East Sea (RU: Primo- rie, Sakhalin; JP: HK) O. watanabei Assing and Maruyama 2002: 217 - NPO, East Sea (RU: Primo rie, Sakhalin; JP: HK) M. insipiens Casey 1911: 237 - NAO, Gulf of Mexico (US: AL, FL, LA, NJ PA) Polypea Fauvel 1878a: 301 P coralli Fauvel 1878a: 302 - Arafura Sea (ID: Kepalauan Aru) Polypea Fauvel 1878a: 301 P. coralli Fauvel 1878a: 302 - Arafura Sea (ID: Kepalauan Aru) Rothium Moore and Legner 1977: 460 [rev Ahn and Ashe 1996c] R. ashlocki Ahn and Ashe 1996c: 247 - SPO (EC: Galapagos) R. evansi Ahn and Ashe 1996c: 248 - SPO (EC: Esmeraldas, Guayas; PE: Piura) Rothium Moore and Legner 1977: 460 [rev Ahn and Ashe 1996c] R. ashlocki Ahn and Ashe 1996c: 247 - SPO (EC: Galapagos) R. evansi Ahn and Ashe 1996c: 248 - SPO (EC: Esmeraldas, Guayas; PE: Piura) Rothium Moore and Legner 1977: 460 [rev Ahn and Ashe 1996c] R. ashlocki Ahn and Ashe 1996c: 247 - SPO (EC: Galapagos) R. ashlocki Ahn and Ashe 1996c: 247 - SPO (EC: Galapagos) R. evansi Ahn and Ashe 1996c: 248 - SPO (EC: Esmeraldas, Guayas; PE: Piura) p g R. evansi Ahn and Ashe 1996c: 248 - SPO (EC: Esmeraldas, Guayas; PE: Piura) R. giulianii Moore 1978b: 155 - NPO (MX: GU, SI) R. littoralis Klimaszewski and Peck 1998: 228 - SPO (EC: Galapagos) R. pallidus Ahn and Ashe 1996c: 247 - NPO (MX: GU) R. pallidus Ahn and Ashe 1996c: 247 - NPO (MX: GU) p R. sonorensis Moore and Legner 1977: 462 - Gulf of California (MX: SO) J. H. Frank & Kee-Jeong Ahn / ZooKeys 107: 1–98 (2011) 28 Arena Fauvel 1862c: 292 A. fultoni Cameron 1945c: 162 - SPO (NZ) A. fultoni Cameron 1945c: 162 - SPO (NZ) f A. tabida (Kiesenwetter) 1850: 219 - NAO (FR; GB: England), North Sea f A. tabida (Kiesenwetter) 1850: 219 - NAO (FR; GB: England), North Se (DE; DK; GB: England, Scotland; NL) PHYTOSINI Actocharis Sharp 1870: 279 Actocharis Sharp 1870: 279 A. readingii Sharp 1870: 279 - NAO (FR; GB: England), Mediterranean Sea (DZ; FR: Corsica; HR; IT: mainland, Sardinia, Sicily; MT) A. readingii Sharp 1870: 279 - NAO (FR; GB: England), Mediterranean Sea (DZ; FR: Corsica; HR; IT: mainland, Sardinia, Sicily; MT) marina Fauvel 1871: 159 A. readingii Sharp 1870: 279 - NAO (FR; GB: England), Mediterranean Sea (DZ; FR: Corsica; HR; IT: mainland, Sardinia, Sicily; MT) = marina Fauvel 1871: 159 A. readingii Sharp 1870: 279 - NAO (FR; GB: England), Mediterranean Sea (DZ; FR: Corsica; HR; IT: mainland, Sardinia, Sicily; MT) = marina Fauvel 1871: 159 A. cassandrensis Assing 1992: 45 - Mediterranean Sea (GR) (DZ; FR: Corsica; HR; IT: mainland, Sardinia, Sicily; MT) = marina Fauvel 1871: 159 A. cassandrensis Assing 1992: 45 - Mediterranean Sea (GR) = marina Fauvel 1871: 159 A. cassandrensis Assing 1992: 45 - Mediterranean Sea (GR) A. cassandrensis Assing 1992: 45 - Mediterranean Sea (GR) Incertae sedis Incertae sedis e tae sed s Salinamexus Moore and Legner 1977: 463 [rev Jeon and Ahn 2007] = Biophytosus Moore and Legner 1977: 465 S. browni Moore and Legner 1977: 464 - Gulf of California (MX: SO) S. koreanus Jeon and Ahn 2007: 193 - NPO (KR) S. reticulatus (Moore and Legner) 1977: 466 - Gulf of California (MX: SO Salinamexus Moore and Legner 1977: 463 [rev Jeon and Ahn 2007] = Biophytosus Moore and Legner 1977: 465 Salinamexus Moore and Legner 1977: 463 [rev Jeon and Ahn 2007] h d 46 Salinamexus Moore and Legner 1977: 463 [rev Jeon and Ahn 2007] = Biophytosus Moore and Legner 1977: 465 p y g S. browni Moore and Legner 1977: 464 - Gulf of California (MX: SO) S. browni Moore and Legner 1977: 464 - Gulf of California (MX: SO) S. koreanus Jeon and Ahn 2007: 193 - NPO (KR) S. reticulatus (Moore and Legner) 1977: 466 - Gulf of California (MX: SO) S. reticulatus (Moore and Legner) 1977: 466 - Gulf of California (MX: SO) g = octavii Fauvel 1862c: 292 Euphytosus Bernhauer and Scheerpeltz 1926: 552 [Pseudophytosus Haghebaert 1993: 161 is not a valid name] = Paraphytosus Bernhauer 1922a: 236 [preoccupied] Euphytosus Bernhauer and Scheerpeltz 1926: 552 [Pseudophytosus Haghebaert 1993: 161 is not a valid name] = Paraphytosus Bernhauer 1922a: 236 [preoccupied] E. schenklingi (Bernhauer) 1922a: 236 - South China Sea (TW) E. schenklingi (Bernhauer) 1922a: 236 - South China Sea (TW) Phytosus Curtis 1838: 718 E. schenklingi (Bernhauer) 1922a: 236 - South China Sea (TW) Phytosus Curtis 1838: 718 P. (Actosus) andalusiaensis Haghebaert 1993: 161 Mediterranean Sea (ES: An- y P. (Actosus) andalusiaensis Haghebaert 1993: 161 Mediterranean Sea (ES: An- dalusia) P. (Actosus) andalusiaensis Haghebaert 1993: 161 Mediterranean Sea (ES: An- dalusia) P. (Actosus) balticus Kraatz 1859b: 52 - NAO (ES: Canary Islands; ES; FR; GB: England; IE; NO; PT), Irish Sea (GB: England), North Sea (BE; DE; GB: England, Scotland; NL), Baltic Sea (DE; DK; SE), Mediterranean Sea (DZ; FR; IT; MA; TN) P. (Actosus) balticus Kraatz 1859b: 52 - NAO (ES: Canary Islands; ES; FR; GB: England; IE; NO; PT), Irish Sea (GB: England), North Sea (BE; DE; GB: England, Scotland; NL), Baltic Sea (DE; DK; SE), Mediterranean Sea (DZ; FR; IT; MA; TN) P. (Actosus) holtzi Bernhauer 1935: 48 - Mediterranean Sea (GR: Crete) P. (Actosus) nigriventris (Chevrolat) 1843: 42 - NAO (ES; FR; GB: England; PT), Irish Sea (GB: England), North Sea (BE; NL), Mediterranean Sea (IT; MA) = minyops Wollaston 1864: 531 y p P. (Actosus) schatzmayri Bernhauer 1941: 95 - NAO (PT: Azores) Coastal Staphylinidae (Coleoptera) 29 P. (s. str.) caribeanus Haghebaert 1993: 163 - Caribbean Sea (GP) P. (s. str.) fenyesi (Bernhauer) 1915a: 315 - NAO (SN) = senegalensis Wendeler 1930: 252 P. (s. str.) spinifer Curtis 1838: 718 - NAO (ES: Canary Islands; FR; GB: Eng- land; IE; MA; PT), Mediterranean Sea (DZ; EG; ES; FR; GR; IT; MA; TN), Black Sea (BG; RO; TR), Baltic Sea (DE; DK; FI; SE), North Sea (BE; DE; DK; GB: England; NL) d d ll 4 = dimidiatus Wollaston 1865: 453 Blediotrogus Sharp 1900: 234 Blediotrogus Sharp 1900: 234 B. cordicollis (Broun) 1907: 57 - SPO (NZ: Chatham Islands) B. cribricollis Fauvel 1900: 184 - SPO (NZ) B. fauveli (Bernhauer and Schubert) 1911: 129 - SPO (AU) B. fauveli (Bernhauer and Schubert) 1911: 129 SPO (A B. guttiger Sharp 1900: 234 - SPO (NZ) Pareiobledius Bernhauer 1934: 495 P. alutellus (Bernhauer) 1934: 495 - SAO (ZA) P. madegassa Scheerpeltz 1969: 127 - INO (MG) P. pruinosus (Bernhauer) 1912a: 178 - SAO (ZA) Sartallus Sharp 1871b: 217 S i Sh 1871b 217 SPO (AU S h A l P. madegassa Scheerpeltz 1969: 127 - INO (MG) OXYTELINI Anotylus Thomson 1859: 44 Anotylus Thomson 1859: 44 h A. maritimus Thomson 1861: 131 - NAO (FR; GB: England, Scotland; NO),Baltic Sea (SE), North Sea (BE; DK; GB: England; NL), Mediter- ranean Sea (IT: Sicily; TN) = perrisii Fauvel 1862a: xxiv = oceanus Fauvel 1862c: 292 d h h A. maritimus Thomson 1861: 131 - NAO (FR; GB: England, Scotland; NO),Baltic Sea (SE), North Sea (BE; DK; GB: England; NL), Mediter- ranean Sea (IT: Sicily; TN) = perrisii Fauvel 1862a: xxiv = oceanus Fauvel 1862c: 292 = perrisii Fauvel 1862a: xxiv = oceanus Fauvel 1862c: 292 armatus gp armatus gp B. fenyesi Bernhauer and Schubert 1911: 129 - NPO (US: CA; MX: BN, BS) l B h 1905 14 [ d] gp B. fenyesi Bernhauer and Schubert 1911: 129 - NPO (US: CA; MX: BN, BS) = lecontei Bernhauer 1905: 14 [preoccupied] f y = lecontei Bernhauer 1905: 14 [preoccupied] p p B. monstratus Casey 1889b: 46 - NPO (CA: BC; US: OR, CA) li p p B. monstratus Casey 1889b: 46 - NPO (CA: BC; US: OR, CA) basalis gp basalis gp B. cordatus (Say) 1834: 461 - NAO and Gulf of Mexico (US: NY-FL, MS, TX) B. cordatus (Say) 1834: 461 - NAO and Gulf of Mexico (US: NY-FL, MS, T B. doderoi Bondroit 1912: 66 - Mediterranean Sea (GR; IT) B. doderoi Bondroit 1912: 66 - Mediterranean Sea (GR; IT) B. fergussoni Joy 1912: 44 NAO (GB: England, N. Ireland, Scotland; IE), Baltic Sea (DE; EE; FI; PL; SE; RU: Karelia), North Sea (BE; FR; GB: England, Scotland; NO), Irish Sea (GB: England, Wales), Black Sea (RO), Mediterranean Sea (FR; MA; TN) B. fergussoni Joy 1912: 44 NAO (GB: England, N. Ireland, Scotland; IE), Baltic Sea (DE; EE; FI; PL; SE; RU: Karelia), North Sea (BE; FR; GB: England, Scotland; NO), Irish Sea (GB: England, Wales), Black Sea (RO), Mediterranean Sea (FR; MA; TN) B. gradensis Bernhauer 1929: 183 - Mediterranean Sea (IT) B. minor Mulsant and Rey 1878: 634 - Mediterranean Sea (AL; FR; IT) B. minor Mulsant and Rey 1878: 634 - Mediterranean Sea (AL; FR; IT) B. neglectus Casey 1889b: 69 - NAO (CA: NL, NB, NS; US: ME-GA) B. neglectus Casey 1889b: 69 - NAO (CA: NL, NB, NS; US: ME-GA) B. subniger Schneider 1898: 62 NAO (GB: England, Scotland; IE), Irish Sea (GB: England, Wales, IE), North Sea (DE; GB: England, Scotland; NL), Mediterranean Sea (ES; TN) B. subniger Schneider 1898: 62 NAO (GB: England, Scotland; IE), Irish Sea (GB: England, Wales, IE), North Sea (DE; GB: England, Scotland; NL), Mediterranean Sea (ES; TN) B. thinopus Herman 1976: 86 - Gulf of Mexico (US: FL, AL, TX) p B. turbulentus Casey 1889b: 70 - Gulf of Mexico (MX: QR, YU; US: FL, MS) onariensis gp B. turbulentus Casey 1889b: 70 - Gulf of Mexico (MX: QR, YU; US: FL, MS) onariensis gp B. turbulentus Casey 1889b: 70 Gulf of Mexico (MX: QR, YU; US: FL, MS) bonariensis gp B. THINOBIINI Bledius Leach 1819: 174 aequatorialis gp B. aequatorialis Mutchler 1925: 225 - SPO (EC: mainland, Galapagos Islands) B. ceratus Blackwelder 1943: 118 - Caribbean Sea (BS; CU; DO; HT; JM; US: FL) B. susae Herman 1983: 98 - Gulf of Mexico (US: TX, South Padre Island) Bledius Leach 1819: 174 aequatorialis gp B. aequatorialis Mutchler 1925: 225 - SPO (EC: mainland, Galapagos Islands) B. ceratus Blackwelder 1943: 118 - Caribbean Sea (BS; CU; DO; HT; JM; US: FL) B. susae Herman 1983: 98 - Gulf of Mexico (US: TX, South Padre Island) aequatorialis gp B. aequatorialis Mutchler 1925: 225 - SPO (EC: mainland, Galapagos Islands) B. ceratus Blackwelder 1943: 118 - Caribbean Sea (BS; CU; DO; HT; JM; US: FL) B susae Herman 1983: 98 - Gulf of Mexico (US: TX South Padre Island) q gp B. aequatorialis Mutchler 1925: 225 - SPO (EC: mainland, Galapagos Islands q p g B. ceratus Blackwelder 1943: 118 - Caribbean Sea (BS; CU; DO; HT; JM; US: FL) B. susae Herman 1983: 98 - Gulf of Mexico (US: TX, South Padre Island) 30 J. H. Frank & Kee-Jeong Ahn / ZooKeys 107: 1–98 (2011) armatus gp bonariensis Bernhauer 1912a: 168 - SAO (AR; BR; UY) forcipatus gp B. actitus (Herman) 1972: 127 - Gulf of Mexico (US: TX) B. litoreus (Herman) 1972: 129 - Gulf of Mexico (US: FL) fratellus gp B. fratellus Eppelsheim 1885: 144 - SAO (GH; NG; SN) furcatus gp B. maritimus Bernhauer 1923: 176 - Red Sea (SU) gigantulus gp B. marinus Bernhauer 1922b: 168 - INO (SC: Aldabra Islands) B. philippinus Bernhauer 1912d: 248 - South China Sea (PH: Luzon) B. yezoensis Nakane 1963: 21 - NPO (JP; KR) infans gp B. helferi Fauvel 1904b: 112 - INO (IN; MM) B. infans Rottenberg 1870: 36 - Mediterranean Sea (DZ; IT; LY; TN), Red Sea (YE) B. renominatus Cameron 1914: 203 - INO (ET; SO) = bernhaueri Cameron 1912: 28 [preoccupied] lamelliceps gp B. hasticeps Bernhauer 1937: 583 - INO (MG; TZ) pulchellus gp B. pulchellus Kraatz 1859a: 169 - INO (IN; LK; Chagos Archipelago) B. turbulentus Casey 1889b: 70 Gulf of Mexico (MX: QR, YU; US: FL, MS) bonariensis gp B. bonariensis Bernhauer 1912a: 168 - SAO (AR; BR; UY) forcipatus gp B. actitus (Herman) 1972: 127 - Gulf of Mexico (US: TX) B. litoreus (Herman) 1972: 129 - Gulf of Mexico (US: FL) fratellus gp B. fratellus Eppelsheim 1885: 144 - SAO (GH; NG; SN) furcatus gp B. maritimus Bernhauer 1923: 176 - Red Sea (SU) gigantulus gp B. marinus Bernhauer 1922b: 168 - INO (SC: Aldabra Islands) B. philippinus Bernhauer 1912d: 248 - South China Sea (PH: Luzon) B. yezoensis Nakane 1963: 21 - NPO (JP; KR) infans gp B. helferi Fauvel 1904b: 112 - INO (IN; MM) B. infans Rottenberg 1870: 36 - Mediterranean Sea (DZ; IT; LY; TN), Red Sea (YE) B. renominatus Cameron 1914: 203 - INO (ET; SO) = bernhaueri Cameron 1912: 28 [preoccupied] lamelliceps gp B. hasticeps Bernhauer 1937: 583 - INO (MG; TZ) pulchellus gp B. pulchellus Kraatz 1859a: 169 - INO (IN; LK; Chagos Archipelago) bonariensis gp B. bonariensis Bernhauer 1912a: 168 - SAO (AR; BR; UY) forcipatus gp p gp B. actitus (Herman) 1972: 127 - Gulf of Mexico (US: TX) B. marinus Bernhauer 1922b: 168 - INO (SC: Aldabra Islands) B. philippinus Bernhauer 1912d: 248 - South China Sea (PH: Luzon) gp B. helferi Fauvel 1904b: 112 - INO (IN; MM) gp B. helferi Fauvel 1904b: 112 - INO (IN; MM) B. infans Rottenberg 1870: 36 - Mediterranean Sea (DZ; IT; LY; TN), Red Sea (YE) B. infans Rottenberg 1870: 36 - Mediterranean Sea (DZ; IT; LY; TN), Red Sea (YE) p gp B. pulchellus Kraatz 1859a: 169 - INO (IN; LK; Chagos Archipelago) p gp B. pulchellus Kraatz 1859a: 169 - INO (IN; LK; Chagos Archipelago) 31 Coastal Staphylinidae (Coleoptera) 31 punctatissimus gp punctatissimus gp gp B. albomarginatus Bernhauer 1922a: 225 - South China Sea (TW) B. amplicollis Fauvel 1900: 185 - SPO (NZ) p B. bidentifrons Broun 1912: 401 - SPO (NZ) f B. buehleri Scheerpeltz 1957b: 226 - Timor Sea (ID: Sumba)f B. buettikeri Coiffait 1981a: 241 - Red Sea (SA) f B. capensis Cameron 1945a: 708 - SAO (ZA) p B. caribbeanus Blackwelder 1943: 113 - Caribbean Sea (JM; DO-TT) B. caroli Blackburn 1888: 13 - SPO (AU) B. exiguus Scheerpeltz 1933: 1114 - SPO (AU) i B h 1920 6 [ i d] B. exiguus Scheerpeltz 1933: 1114 - SPO (AU) = minor Bernhauer 1920: 6 [preoccupied] B. fernandezi Bernhauer 1939: 234 - SAO (UY) B. fossiventris Fauvel 1889: 252 - SPO (NC) B. fossiventris Fauvel 1889: 252 - SPO (NC) f B. injucundus Blackburn 1888: 14 - SPO (AU) f B. injucundus Blackburn 1888: 14 - SPO (AU) B. maindroni Fauvel 1903: 151 - INO (IN) B. maindroni Fauvel 1903: 151 - INO (IN) B. michaelseni Bernhauer 1915b: 313 - SAO (NA) B. michaelseni Bernhauer 1915b: 313 - SAO (NA) B. microcephalus Fauvel 1901: 72 - NPO (CO), Caribbean Sea (TT: Trinidad) B orientalis Bernhauer and Schubert 1911: 133 Red Sea (DJ) B. microcephalus Fauvel 1901: 72 - NPO (CO), Caribbean Sea (TT: Trinidad) B. orientalis Bernhauer and Schubert 1911: 133 - = lividipes Fairmaire 1892: 90 [preoccupied] B. orientalis Bernhauer and Schubert 1911: 133 - Red Sea (DJ) = lividipes Fairmaire 1892: 90 [preoccupied] B. orientalis Bernhauer and Schubert 1911: 133 - Red Sea (DJ) = lividipes Fairmaire 1892: 90 [preoccupied] p B. pontilis Blackburn 1902: 22 - SPO (AU) B. pontilis Blackburn 1902: 22 - SPO (AU) B. punctatissimus LeConte 1877: 226 NAO (US: MDFL), Gulf of Mexico (US; MX: VC), Caribbean Sea (JM; PR), Gulf of California (MX), NPO (CO; EC) B. punctatissimus LeConte 1877: 226 NAO (US: MDFL), Gulf of Mexico (US; MX: VC), Caribbean Sea (JM; PR), Gulf of California (MX), NPO (CO; EC) B. J. H. Frank & Kee-Jeong Ahn / ZooKeys 107: 1–98 (2011) 32 T. lithocharinus (LeConte) 1877: 245 - NPO (US: CA, WA) T. luniger (Fauvel) 1868: 40 - SPO (CL) T. maritimus (Broun) 1903: 615 - SPO (NZ) T. pictipes (Lea) 1910: 126 - SPO (AU: Tasmania) T. senex (Fauvel) 1868: 40 - SPO (CL) T. skottsbergii (Bernhauer) 1921: 41 - SPO (CL: Juan Fernandez Island) T. suturalis Solier 1849: 331 - SPO (CL) T. unicolor (Sharp) 1900: 232 - SPO (AU, NZ), INO (ZA), NAO (GB: England) = anglicanus (Sharp) 1900: 232 Thinobius Kiesenwetter 1844: 355 = Yosiityphlus Sawada 1971c: 327 T. frizzelli Hatch 1957: 94 - NPO (CA: BC; US: CA, WA; MX: BN) T. marinus Cameron 1917d: 155 - South China Sea (SG) T. kuroshio (Sawada) 1971c: 327 - NPO (JP: HN) SCYDMAENINAE CEPHENNIINI Cephennodes Reitter 1884: 420 = Chelonoidum Strand 1935: 285 C. araiorum (Jałoszyński) 2003: 226 - NPO (JP: HN) PAEDERINAE PAEDERINI Chetocephalus Cameron 1944a: 314 C. maritimus Cameron 1944a: 314 - INO (MU) Medon Stephens 1833: 273 M. marinus Cameron 1944a: 313 - INO (MU) M. pocoferus (Peyron) 1857: 718 - Mediterranean Sea (DZ; FR; IT; TN), NAO (FR; GB: England) = maritimus Aubé 1863: 36 M. prolixus (Sharp) 1874: 65 - NPO, East Sea (JP: HN) M. rubeculus Sharp 1889: 264 - NPO (JP: HN), South China Sea (CN: Hong Kong) Ophioomma Notman 1920: 704 O. rufa Notman 1920: 705 - Gulf of Mexico (US: FL) Sunius Stephens 1829: 24 S. ferrugineus (Bierig) 1934b: 326 - Caribbean Sea (CU; JM) S. minutus (Casey) 1905: 180 - NAO (US: FL) STAPHYLININAE STAPHYLININI 32 J. H. Frank & Kee-Jeong Ahn / ZooKeys 107: 1–98 (2011) T. lithocharinus (LeConte) 1877: 245 - NPO (US: CA, WA) T. luniger (Fauvel) 1868: 40 - SPO (CL) T. maritimus (Broun) 1903: 615 - SPO (NZ) T. pictipes (Lea) 1910: 126 - SPO (AU: Tasmania) T. senex (Fauvel) 1868: 40 - SPO (CL) T. skottsbergii (Bernhauer) 1921: 41 - SPO (CL: Juan Fernandez Island) T. suturalis Solier 1849: 331 - SPO (CL) T. unicolor (Sharp) 1900: 232 - SPO (AU, NZ), INO (ZA), NAO (GB: England) = anglicanus (Sharp) 1900: 232 Thinobius Kiesenwetter 1844: 355 = Yosiityphlus Sawada 1971c: 327 T. frizzelli Hatch 1957: 94 - NPO (CA: BC; US: CA, WA; MX: BN) T. marinus Cameron 1917d: 155 - South China Sea (SG) T. kuroshio (Sawada) 1971c: 327 - NPO (JP: HN) Kong) Ophioomma Notman 1920: 704 O. rufa Notman 1920: 705 - Gulf of Mexico (US: FL) S. ferrugineus (Bierig) 1934b: 326 - Caribbean Sea (CU; JM) S. minutus (Casey) 1905: 180 - NAO (US: FL) Chetocephalus Cameron 1944a: 314 C. maritimus Cameron 1944a: 314 - INO (MU) C. maritimus Cameron 1944a: 314 - INO (MU) Medon Stephens 1833: 273 M. marinus Cameron 1944a: 313 - INO (MU) M. pocoferus (Peyron) 1857: 718 - Mediterranean Sea (DZ; FR; IT; TN), NAO (FR; GB: England) = maritimus Aubé 1863: 36 M p li (Sh ) 1874 65 NPO E t S (JP HN) M. marinus Cameron 1944a: 313 INO (MU) M. pocoferus (Peyron) 1857: 718 - Mediterranean Sea (DZ; FR; IT; TN), NAO (FR; GB: England) = maritimus Aubé 1863: 36 M. prolixus (Sharp) 1874: 65 - NPO, East Sea (JP: HN) M. rubeculus Sharp 1889: 264 - NPO (JP: HN), South China Sea (CN: Hong Kong) M. rubeculus Sharp 1889: 264 - NPO (JP: HN), South China Sea (CN: Hong Kong) gp B. helferi Fauvel 1904b: 112 - INO (IN; MM) salinus Cameron 1947b: 704 - SPO (NZ) B. salinus Cameron 1947b: 704 - SPO (NZ) B. tristis Aubé 1843: 92 Mediterranean Sea (AL; DZ; ES; FR; IT; TN), Red Sea (SN) ugosicollis gp B. tristis Aubé 1843: 92 Mediterranean Sea (AL; DZ; ES; FR; IT; TN), Red Sea (SN) ugosicollis gp B. bituberculatus Cameron 1940: 183 - Andaman Sea (MY) erres gp B. bituberculatus Cameron 1940: 183 - Andaman Sea (MY) verres gp B. bituberculatus Cameron 1940: 183 - Andaman Sea (MY) verres gp B. albopubescens Cameron 1941: 434 - South China Sea (PH: Luzon) B. arenicola Fauvel 1904b: 112 - Arabian Sea [IN: Malabar, Mahé (= Mayyazhi)] B. arenicola Fauvel 1904b: 112 - Arabian Sea [IN: Malabar, Mahé (= Mayyazhi B. fraterculus Cameron 1936: 40 - Andaman Sea (MY) B. fraterculus Cameron 1936: 40 - Andaman Sea (MY) B. jacobsoni Cameron 1928: 106 - INO (ID: Sumatra) B. jacobsoni Cameron 1928: 106 - INO (ID: Sumatra) j B. madagascariensis Bernhauer 1901b: 169 - INO (MG) B. marginalis Cameron 1945a: 707 - SAO (ZA: Cape Province) B. parens Cameron 1941: 434 - South China Sea (PH: Luzon) B. perrieri Fauvel 1904c: 305 - INO (MG) B. perrieri Fauvel 1904c: 305 - INO (MG) p B. petzi Bernhauer 1908b: 104 - INO (TZ) B. petzi Bernhauer 1908b: 104 - INO (TZ) Carpelimus Leach 1819: 174 p C. lucidus (Cameron) 1944a: 312 - INO (TZ: Zanzíbar) Teropalpus Solier 1849: 330 = Trogolinus Sharp 1900: 231 T. coloratus (Sharp) 1900: 231 - SPO (NZ) C. lucidus (Cameron) 1944a: 312 - INO (TZ: Zanzíbar) STAPHYLININAE STAPHYLININI PHILONTHINA Bisnius Stephens 1829: 23 Coastal Staphylinidae (Coleoptera) 33 B. macies (Sharp) 1874: 41 - NPO (JP; KR) Cafius Stephens 1829: 23 B. macies (Sharp) 1874: 41 - NPO (JP; KR) Cafius Stephens 1829: 23 C. aguayoi Bierig 1934a: 66 - NAO (US: CT, MA) C. algarum (Sharp) 1874: 38 - NPO, East Sea (JP: HN; KR), South China Sea (CN: Hong Kong) C. algophilus Broun 1894: 419 - SPO (NZ) C. andamanensis Coiffait 1981b: 337 - INO (Andaman Island) C. australis (Redtenbacher) 1867: 28 - SPO (AU: New South Wales, Victoria) = areolatus Fauvel 1877: 251 C bi i (E i h ) 1840 502 B. macies (Sharp) 1874: 41 - NPO (JP; KR) Cafius Stephens 1829: 23 C. aguayoi Bierig 1934a: 66 - NAO (US: CT, MA) C. algarum (Sharp) 1874: 38 - NPO, East Sea (JP: HN; KR), South China Sea (CN: Hong Kong) C. algophilus Broun 1894: 419 - SPO (NZ) C. andamanensis Coiffait 1981b: 337 - INO (Andaman Island) C. australis (Redtenbacher) 1867: 28 - SPO (AU: New South Wales, Victoria) = areolatus Fauvel 1877: 251 C bi i (E i h ) 1840 502 C. aguayoi Bierig 1934a: 66 - NAO (US: CT, MA) C. andamanensis Coiffait 1981b: 337 - INO (Andaman Island) f C. australis (Redtenbacher) 1867: 28 - SPO (AU: New South Wales, Victoria) = areolatus Fauvel 1877: 251 C. bistriatus (Erichson) 1840: 502 = bilineatus (Erichson) 1840: 503 ssp. bistriatus (Erichson) 1840: 503 - NAO (CA: NB, NL, NS, QC; US: MA, MD, ME, NJ, NY, RI, VA, FL; BM, BS), Gulf of Mexico (MX: CA, VC; US: FL, TX), Caribbean Sea (AG; BB; CU; DO; GD; GP; JM; KN; LC; MS; PR; TT; VE; VI; MX: QR) ssp. fulgens Frank [in Frank et al. 1986]: 153 - NPO (US: CA; MX: BS), Gulf of California (MX: BN, BS, SO) C. bisulcatus (Solier) 1849: 314 - SPO (CL) C. bryanti Cameron 1943: 343 - SPO (AU) C. bryanti Cameron 1943: 343 - SPO (AU) C. canescens (Mäklin) 1852: 313 - NPO (US) C. caribeanus Bierig 1934a: 68 - Caribbean Sea (AG; CU; DM; GD; GP; JM; PR; VI; VE; US: FL) C. caribeanus Bierig 1934a: 68 - Caribbean Sea (AG; CU; DM; GD; GP; JM; PR; VI; VE; US: FL) g PR; VI; VE; US: FL) C. catenatus Fauvel 1877: 256 - SPO (AU: New South Wales) C. catenatus Fauvel 1877: 256 - SPO (AU: New South Wales) = velutinus Fauvel 1877: 256 C. caviceps Broun 1886: 942- SPO (NZ) C. caviceps Broun 1886: 942- SPO (NZ) p p C. ceylonicus Bernhauer 1902: 29 - INO (LK) C. cicatricosus (Erichson) 1840: 454 Mediterranean Sea (southern Europe; IT), NAO (FR; GB: England) C. cicatricosus (Erichson) 1840: 454 Mediterranean Sea (southern Europe; IT), NAO (FR GB E l d) C. cicatricosus (Erichson) 1840: 454 Mediterranean Sea (southern Europe; IT) C. decipiens (LeConte) 1863: 40 - NPO (US; MX) C. flicki Vauloger 1897: 238 - Mediterraean Sea (IT; LY; TN) l C. fonticola (Erichson) 1840: 501 - Red Sea (EG), INO (SO) f C. fucicola Curtis 1830: pl. 323 - NAO (GB: England; IE), Irish Sea (GB: N. Ireland, Wales; IE), North Sea (FR; GB) f C. fucicola Curtis 1830: pl. 323 - NAO (GB: England; IE), Irish Sea (GB: N. Ireland, Wales; IE), North Sea (FR; GB) C. fucicola Curtis 1830: pl. 323 - NAO (GB: England; IE), Irish Sea (GB: N. reland, Wales; IE), North Sea (FR; GB) C. gigas Lea 1929: 204 - SPO (AU: Lord Howe Island) C. histrio (Sharp) 1874: 37 - NPO, East Sea (CN: Hong Kong; JP: HN; KP; KR) C. histrio (Sharp) 1874: 37 - NPO, East Sea (CN: Hong Kong; JP: HN; KP; KR) C. lithocharinus (LeConte) 1863: 38 - NPO (CA; US; MX) C. lithocharinus (LeConte) 1863: 38 - NPO (CA; US; MX) C. litoreus (Broun) 1880: 108 - SPO (NZ) C. litoreus (Broun) 1880: 108 - SPO (NZ) C. luteipennis Horn 1884: 237 - NPO (CA; US; MX) C. luteipennis Horn 1884: 237 - NPO (CA; US; MX) p C. maritimus (Broun) 1880: 109 - SPO (NZ) C. maritimus (Broun) 1880: 109 - SPO (NZ) 34 J. H. Frank & Kee-Jeong Ahn / ZooKeys 107: 1–98 (2011) C. martini Cameron 1927: 251 - Red Sea (SA; YE) = arrowi Bernhauer 1931b: 234 = arrowi Bernhauer 1931b: 234 C. mimulus (Sharp) 1874: 38 - NPO, East Sea (JP: HN; KR) C. mutatus Gemminger and Harold 1868: 590 - NPO (US; CA) f l (M kl ) 1853 189 C. mutatus Gemminger and Harold 1868: 590 - NPO (US; CA) = femoralis (Mäklin) 1853: 189 C. nasutus Fauvel 1877: 257 [and 1879: 84] - SPO (FJ) C. nasutus Fauvel 1877: 257 [and 1879: 84] - SPO (FJ) C. nauticus (Fairmaire) 1849: 288 - NPO (CN; JP; TW; US: HI), SPO (AU; FR: Tahiti; NC), INO (SO; LK; MU), Red Sea [YE: Barim (= Perim) Island] = longipennis (Walker) 1858: 205 = puncticollis (Boheman) 1858: 31 = parallelus (Kraatz) 1859a: 99 = densiventris Fauvel 1877: 258 C. nauticus (Fairmaire) 1849: 288 - NPO (CN; JP; TW; US: HI), SPO (AU; FR: Tahiti; NC), INO (SO; LK; MU), Red Sea [YE: Barim (= Perim) Island] = longipennis (Walker) 1858: 205 = longipennis (Walker) 1858: 205 = puncticollis (Boheman) 1858: 31 = parallelus (Kraatz) 1859a: 99 = densiventris Fauvel 1877: 258 C. opacus (LeConte) 1863: 40 - NPO (western US; CA; MX) = dubius (LeConte) 1863: 39i C. pacificus (Erichson) 1840: 501 - SPO (AU: New South Wales, Queensland C. pacificus (Erichson) 1840: 501 - SPO (AU: New South Wales, Queensland, Tasmania, Victoria) = littoralis Fauvel 1877: 254 C. quadriimpressus (White) 1846: 6 - SPO (NZ) C. ragazzii Gestro 1889: 32 - Red Sea, INO (SO) g C. rufescens Sharp 1889: 44 - NPO, East Sea (JP: HN; KR), South China Sea (CN: Hong Kong)i C. rufescens Sharp 1889: 44 - NPO, East Sea (JP: HN; KR), South China Sea (CN: Hong Kong) = mimulus (Rottenberg) 1870: 30 = mimulus (Rottenberg) 1870: 30 Phucobius Sharp 1874: 35 P. africanus Bernhauer 1937: 617 - INO (TZ: western Usambara) P. congruus (Walker) 1858: 205 - INO (LK) P. cupreipennis Cameron 1918: 89 - Java Sea (MY; SG) P. densipennis Bernhauer 1931a: 131 - NPO (JP: RY) P. pectoralis (Boheman) 1858: 31 - ?sea (CN: province not stated) P. semiaereus Cameron 1934: 22 - SPO (New Hebrides) P. simulator Sharp 1874: 35 - NPO, East Sea (KR; JP: HN, KY; RU: Primorie) P. simulator Sharp 1874: 35 - NPO, East Sea (KR; JP: HN, KY; RU: Primorie) P. tricolor Bernhauer 1917: 125 - South China Sea (CN: Hong Kong; TW) mus Holme 1837: 64 P. tricolor Bernhauer 1917: 125 - South China Sea (CN: Hong Kong; TW) mus Holme 1837: 64 (CN: Hong Kong) g g C. rufifrons Bierig 1934a: 68 - Caribbean Sea (CU), NAO (US: FL) C. sabulosus Fauvel 1877: 253 - SPO (AU: New South Wales, Queensland = postseriatulus Koch 1936: 179 C. seminitens Horn 1884: 236 - NPO (CA; US; MX) C. seminitens Horn 1884: 236 - NPO (CA; US; MX) C. seriatus Fauvel 1877: 255 - SPO (AU) C. seriatus Fauvel 1877: 255 - SPO (AU) ( ) C. subtilis Cameron 1922: 121 - Caribbean Sea (AG; CU; DM; GP; JM; KN; MS; PR; TT; VI), Gulf of Mexico (US: FL), NAO (US: FL; BM) C. subtilis Cameron 1922: 121 - Caribbean Sea (AG; CU; DM; GP; JM; KN; MS PR TT VI) Gulf of Me ico (US FL) NAO (US FL BM) C. subtilis Cameron 1922: 121 - Caribbean Sea (AG; CU; DM; GP; JM; KN; C C 9 C S ( G; CU; ; G ; J ; N; MS; PR; TT; VI), Gulf of Mexico (US: FL), NAO (US: FL; BM) C. sulcicollis (LeConte) 1863: 40 - NPO (US; MX) C. velutinus Fauvel 1877: 256 - SPO (AU) C. velutinus Fauvel 1877: 256 - SPO (AU) C. vestitus (Sharp) 1874: 37 - NPO, East Sea (JP: HN; KP; KR) C. xantholoma (Gravenhorst) 1806: 41 - NAO (ES: Canary Islands; FR; GB: England, Scotland; IE; IS; PT), Irish Sea (GB: England, Wales, N. Ireland; IE), North Sea (BE; DE; FR; GB: England, Scotland; NL; NO), Baltic Sea (DE; DK; EE; FI; PL; RU: Karelia; SE), Mediterranean Sea (DZ; EG; ES; FR; GR; IT; MA; TN; TR), Black Sea (UA) = lateralis Stephens 1833: 246 = littoralis Stephens 1833: 247 = tessellatus Stephens 1833: 247 = variegatus (Erichson) 1840: 453 = variolosus (Sharp) 1871a: 181 C. xantholoma (Gravenhorst) 1806: 41 - NAO (ES: Canary Islands; FR; GB: England, Scotland; IE; IS; PT), Irish Sea (GB: England, Wales, N. Ireland; IE), North Sea (BE; DE; FR; GB: England, Scotland; NL; NO), Baltic Sea (DE; DK; EE; FI; PL; RU: Karelia; SE), Mediterranean Sea (DZ; EG; ES; FR; GR; IT; MA; TN; TR), Black Sea (UA) = lateralis Stephens 1833: 246 = littoralis Stephens 1833: 247 = variegatus (Erichson) 1840: 453 = variolosus (Sharp) 1871a: 181 35 Coastal Staphylinidae (Coleoptera) 35 = keysianus Donisthorpe 1930: 97 y p = heroopoliticus Koch 1936: 169 heroopoliticus oc 936: 69 C. zealandicus Cameron 1947b: 705 - SPO (NZ) Gabronthus Tottenham 1955: 178 C. (CN: Hong Kong) zealandicus Cameron 1947b: 705 - SPO (NZ) Gabronthus Tottenham 1955: 178 Gabronthus Tottenham 1955: 178 G. maritimus (Motschulsky) 1858a: 661 - NAO (ES: Canary Islands), Medi- terranean Sea (CY; DZ; EG; FR; GR; IL; IT; LB; LY; MA; TR), Red Sea (DJ; ET; SA), INO (MU; RE), South China Sea (ID; MY; SG; VN), NPO (JP; TW) G. maritimus (Motschulsky) 1858a: 661 - NAO (ES: Canary Islands), Medi- terranean Sea (CY; DZ; EG; FR; GR; IL; IT; LB; LY; MA; TR), Red Sea (DJ; ET; SA), INO (MU; RE), South China Sea (ID; MY; SG; VN), NPO (JP; TW) Orthidus Mulsant and Rey 1876: 339 Orthidus Mulsant and Rey 1876: 339 O. cribratus (Erichson) 1840: 431 - Mediterranean Sea, NAO ssp. cribratus (Erichson) 1840: 431 - Mediterranean Sea (IT) ssp. atlanticus Coiffait 1956: 221 - NAO (ES; FR; PT; MA) hilonthus Stephens 1829: 23 ssp. atlanticus Coiffait 1956: 221 NAO (ES; FR; PT; MA) Philonthus Stephens 1829: 23 P. nudus Sharp 1874: 36 - NPO (CA: BC; US: WA; RU: Kuril Islands), NPO, East Sea (KR; JP: HK, HN, KY) P. nudus Sharp 1874: 36 - NPO (CA: BC; US: WA; RU: Kuril Islands), NPO, East Sea (KR; JP: HK, HN, KY) East Sea (KR; JP: HK, HN, KY) Phucobius Sharp 1874: 35 P. africanus Bernhauer 1937: 617 - INO (TZ: western Usambara) P. congruus (Walker) 1858: 205 - INO (LK) = punctilinea (Walker) 1858: 205 = horni (Bernhauer) 1902: 28 P. cupreipennis Cameron 1918: 89 - Java Sea (MY; SG) P. densipennis Bernhauer 1931a: 131 - NPO (JP: RY) P. pectoralis (Boheman) 1858: 31 - ?sea (CN: province not stated) P. semiaereus Cameron 1934: 22 - SPO (New Hebrides) P. simulator Sharp 1874: 35 - NPO, East Sea (KR; JP: HN, KY; RU: Primorie) P tricolor Bernhauer 1917: 125 - South China Sea (CN: Hong Kong; TW) STAPHYLININA Hadropinus Sharp 1889: 115 H. fossor Sharp 1889: 116 - NPO, East Sea (JP: HK; RU: Sakhalin) p p H. fossor Sharp 1889: 116 - NPO, East Sea (JP: HK; RU: Sakhalin) Hadrotes Mäklin 1852: 313 H. crassus (Mannerheim) 1846: 509 - NPO (CA: BC; US: AK, CA, OR, WA; MX: BN) H. wakefieldi Cameron 1945b: 786 - SPO (NZ) Liusus Sharp 1889: 116 L. hilleri (Weise) 1877: 93 - NPO, East Sea (CN: Manchuria; JP: HN; KR; RU: Sakhalin) Hadrotes Mäklin 1852: 313 H. crassus (Mannerheim) 1846: 509 - NPO (CA: BC; US: AK, CA, OR, WA; MX: BN) H. wakefieldi Cameron 1945b: 786 - SPO (NZ) L. hilleri (Weise) 1877: 93 - NPO, East Sea (CN: Manchuria; JP: HN; KR; RU: Sakhalin) L. hilleri (Weise) 1877: 93 - NPO, East Sea (CN: Manchuria; JP: HN; KR; RU: Sakhalin) L. humeralis (Matsumura) 1911: 113 - NPO, East Sea (CN; JP; KR; RU: Sakhalin)h L. humeralis (Matsumura) 1911: 113 - NPO, East Sea (CN; JP; KR; RU: Sakhalin)h Thinopinus LeConte 1852: 215 p T. pictus LeConte 1852: 216 - NPO (CA: BC; US: AK, CA, OR, WA; MX: BN) T. pictus LeConte 1852: 216 - NPO (CA: BC; US: AK, CA, OR, WA; MX: BN) T. pictus LeConte 1852: 216 - NPO (CA: BC; US: AK, CA, OR, WA; MX: BN) MICROSILPHINAE Microsilpha includes four species although others are recognized but are undescribed. Three are South American and not coastal, but the one New Zealand species, M. litorea Broun, is known only from seashores (Klimaszewski and Watt 1997). Only this genus is included within the subfamily. QUEDIINA Heterothops Stephens 1829: 23 H. asperatus Smetana 1971: 34 - NPO (CA: BC; US: CA) Quediocafus Cameron 1945b: 791 Q. hudsoni Cameron 1945b: 791 - SPO (NZ) Q. insolitus (Sharp) 1886: 379 - SPO (NZ) Q. taieriensis (Broun) 1894: 424 - SPO (NZ) Q. taieriensis (Broun) 1894: 424 - SPO (NZ) Remus Holme 1837: 64 R. corallicola (Fairmaire) 1849: 289 SPO (AU; FJ; NC), INO (LK; MG; MU; SC; SO), Red Sea (DJ; YE), South China Sea (CN: Hong Kong), Java Sea (ID; MY; SG) = occidentalis Blackburn 1888: 48 R. filum Kiesenwetter 1849: 19 Mediterranean Sea (IT; LY; YU), Black Sea, NAO (FR; DE; BG; RO; TR; EG; LY; HR), INO (SO) R. filum Kiesenwetter 1849: 19 Mediterranean Sea (IT; LY; YU), Black Sea, NAO (FR; DE; BG; RO; TR; EG; LY; HR), INO (SO) R. pruinosus (Erichson) 1840: 510 - NAO (ES: Canary Islands; PT; NL; FR; RU), North Sea (BE; FR; NL), Mediterranean Sea (IT; TR) R. pruinosus (Erichson) 1840: 510 - NAO (ES: Canary Islands; PT; NL; FR; RU), North Sea (BE; FR; NL), Mediterranean Sea (IT; TR) R. sericeus (Holme) 1837: 64 - NAO (ES: Canary Islands; FR; GB: England; PT: Madeira; NO), Baltic Sea (DE; DK; SE), North Sea (DE; DK; GB: England; NL), Irish Sea (GB: Wales), Mediterranean Sea (EG; ES; FR; GR; IT; LY; TR), Black Sea (BG), INO (MU), SPO (AU: South Australia, Tasmania, Victoria, Western Australia) = aegyptiacus Motschulsky 1858a: 665 = obscuricornis Koch 1936: 170 36 J. H. Frank & Kee-Jeong Ahn / ZooKeys 107: 1–98 (2011) Thinocafius Steel 1949: 309 T. insularis Steel 1949: 309 - SPO (NZ: Chatham Islands) OMALIINI Crymus (= Arpediomimus) includes two species, C. antarcticus and C. kronii, both as- sociated with seaweed on seashores (Steel 1964). The larva of C. kronii was described by Steel (1964). Hughes et al. (2004) pointed out the existence of this species in the intertidal zone, not just on the South Island of New Zealand, but also on the Antipo- des, Auckland, and Campbell islands of New Zealand, and found that it is one of the hosts of Cucujomyces phycophilus Weir and Rossi (Ascomycetes: Laboulbeniales). Macralymma includes only one species, M. punctiventre Cameron, and it is pre- cinctive (“endemic”) to New Zealand. Cameron (1945c) found the specimen in the Broun collection, labeled ‘Taieri Beach.’ It is widespread on the South Island of New Zealand and also occurs on Chatham Island. It is found under rotting kelp on sandy beaches (Emberson 1998). Hughes et al. (2004) pointed out the existence of this spe- cies in the intertidal zone of the Antipodes Islands of New Zealand and found that is one of the hosts of Cucujomyces phycophilus. Micralymma marinum adults and larvae inhabit cracks in rocks in the intertidal zone of rocky coastlines. Adults and larvae are predacious, but their prey range is un- certain (Thayer 1985), probably including Collembola. In Britain, adults overwinter and larvae develop during the summer months (Steel 1970), or adults and larvae over- winter (King et al. 1979). In the northeastern USA some of the beetles may overwinter as larvae (Thayer 1985). These apterous beetles tolerate immersion in seawater (Elliott et al. 1983). A second species, M. brevilingue Schiødte (1845: 377, syn. M. dicksoni Mäklin 1878: 24) has similar habits but is not entirely restricted to seashores, being also found in damp moss near coasts (Steel 1958) so we do not list it. A third species, M. laticolle, was admitted by Motschulsky (1860), its describer, not to belong to the genus Micralymma, but has not yet been assigned to another genus (see also Steel 1958). A fourth species, M. caucasicum Melichar, is not coastal (Steel 1962). p Omaliomimus occurs only on seashores, and some species may be abundant in rotting seaweed (Steel 1964). The larva of O. venator was described by Steel (1964); Hughes et al. OMALIINAE Worldwide, there are about 117 genera of Omaliinae placed in seven tribes (Thayer 2005). Only the genera Crymus, Omaliomimus, Macralymma, and Giulianium seem entirely restricted to seashores. Some species within the genera Micralymma and Oma- Coastal Staphylinidae (Coleoptera) 37 lium seem restricted to seashores. All of the coastal species thus far described occur at high latitudes and not in the tropics. lium seem restricted to seashores. All of the coastal species thus far described occur at high latitudes and not in the tropics. APHAENOSTEMMINI Giulianium includes three species, all of them found under debris below the high tide mark, of beaches of the North Pacific Ocean. Larvae are unknown and there is no known association with seaweed (Ahn and Ashe 1999). PSELAPHINAE Worldwide there are 1200 genera of Pselaphinae, and all are predacious (Thayer 2005). Seashores are a very minor part of their habitat range. The genus Physoplectus is known only from saline coastal habitats. BATRISITAE BATRISINI Arthromelus quadratus, Batriscenites celer, B. humicola, and Batrisocenus foveiterminalis adults were all found in mangrove forests in Singapore, where most of them were as- sociated with mounds of Thalassia (Hydrocharitaceae) turtlegrass (Tanokuchi 1989). OMALIINI (2004) pointed out the existence of this species in the intertidal zone not just on the New Zealand mainland, but also on the Antipodes, Auckland, Campbell, Macquarie and Snares islands of New Zealand, and found that is one of the hosts J. H. Frank & Kee-Jeong Ahn / ZooKeys 107: 1–98 (2011) 38 of Cucujomyces phycophilus Weir and Rossi (Ascomycetes: Laboulbeniales). Emberson (1998) pointed out the existence of undescribed species of Omaliomimus from the Chatham Islands of New Zealand. of Cucujomyces phycophilus Weir and Rossi (Ascomycetes: Laboulbeniales). Emberson (1998) pointed out the existence of undescribed species of Omaliomimus from the Chatham Islands of New Zealand. Omalium species occupy various habitats, and only some occur on seashores as- sociated with drifted seaweed. Seashore species include the European O. laeviusculum, O. riparium, and O. rugulipenne, as well as the North American O. algarum. Backlund (1945) found O. laeviusculum exclusively in deep layers of seaweed beds, but O. ripari- um was also found in carrion. Mjöberg (1906) described the pupa of O. riparium, and noted that it took seven days to develop to the adult. Steel (1970) found that larvae of O. laeviusculum and O. riparium occur in the summer months in Britain, whereas Larsson and Gigha (1959) had reported larvae of the latter in the winter in Iceland. Populations of O. riparium inhabiting Mediterranean shores have smaller adults than do those from northern Europe and have been considered a distinct subspecies (O. ri- parium impar Mulsant & Rey). Omalium littorale Kraatz seems to be strictly a seashore species in northern Europe, but in southern Europe it has been reported from high altitudes far from the sea (Zanetti 1987), so is not included in the checklist. ALEOCHARINAE Worldwide, over 1,151 genera of this subfamily have been described, but its true di- versity is without doubt much greater (Thayer 2005). It now contains some 12,851 species, but Hammond (1975) postulated that it might contain as many as 100,000 species. The genera are currently distributed among 51 tribes whose relationships re- quire much study. It contains many specialist seashore inhabitants, not only at the level of genus, but even (as currently defined) at the level of tribe. Nowhere else among the Staphylinidae have entire tribes specialized to inhabit coastal habitats. Prepupae of Aleocharinae spin a silken cocoon in which they pupate; some earlier authors incorrectly supposed the dorsal abdominal gland to be the source of the silk, although that gland produces defensive chemicals; the cocoon is not a special adapta- tion to immersion in water (Frank and Thomas 1984a). GONIACERITAE BRACHYGLUTINI Berlara bella adults were collected in a mangrove forest in Singapore, but not in parts inundated by the tide (Tanokuchi 1989). Brachygluta has at least 43 species, but just six of them, known from America north of Mexico, appear to be restricted to coastal habitats, all these on the Atlantic coast (Chandler 1997). Briaraxis depressa, the only representative of this genus, is known only from “under rubbish or logs on the beach” in the circum-Caribbean regions (Chandler 1992, 2002). Coastal Staphylinidae (Coleoptera) 39 Mangalobythus furcifer, M. acutifolius, and M. murphyi adults were all collected in mangrove forests in Singapore or Thailand, where adults were seen to be active on open ground at low tide (Tanokuchi 1989). The genus Nisaxis appears to be entirely coastal. Nisaxis maritima is known only from coastal habitats in United States Gulf Coast States; other species have been found in coastal saline habitats and also in inland saline habitats (Chandler 1997). Pedisinops regulus adults were collected in the intertidal zone and on a coral reef in Japan’s Ryukyu Islands (Sawada 1991). The genus Physoplectus is entirely coastal. Physoplectus vinsoni is known only from coastlines in Mauritius in the Indian Ocean, whereas P. reikoae, P. miyakei, and P. ir- ritans are from Pacific coastlines. Prosthecarthron sauteri, originally described from Taiwan, was found to be wide- spread on patches of halophilous grasses close to the sea in North Korea, on patches of a reed on mud in river estuaries in the Japanese mainland, in mangrove habitats in the Ryukyu Islands, and under stones on the muddy ground of a mangrove seashore in Vietnam (Nomura et al. 2006). ALEOCHARINI Aleochara, the type genus of this tribe, appears to be the only one with coastal rep- resentatives, and thus far 16 are known. Its larvae develop as ectoparasitoids of cy- clorrhaphous dipteran pupae within the dipteran puparium. On sea beaches, such dipteran puparia are typically found in piles of decaying seaweed, but also in carrion. All eight members of the subgenus Emplenota are found associated with such materi- als on sea beaches in Europe, north Africa, Korea, Japan, and both coasts of North America (Klimaszewski 1984). Additionally, A. (Coprochara) sulcicollis is also found in such habitats on the Pacific coast of North America and Chile in the south Pacific; J. H. Frank & Kee-Jeong Ahn / ZooKeys 107: 1–98 (2011) 40 A. (Coprochara) squalithorax is found on the Pacific shores of Japan and Korea; and A. (Coprochara) salsipotens is found on the African shores of the Indian Ocean and the Af- rican shores of the south Atlantic. Other members of the subgenus Coprochara do not, or seldom, occupy beaches. Two species of the subgenus Polystomota (A. punctatella and A. grisea) occupy European shores of the North Atlantic. Aleochara punctatella has long been confused with A. grisea, so old records of A. grisea, doubtless including some in the checklist, are in doubt and need re-evaluation. Three members of the subgenus Triochara occupy Pacific shores of eastern Asia, no others are known, and the habitat of all three appears to be seaweed. Parasitoidism by A. (Emplenota) obscurella (as A. algarum) of a dipterous puparium [Orygma luctuosum Meigen (Diptera: Coelopidae)] was reported by Scott (1916), and then in greater detail by Scott (1920) who noted that hosts were Coelopa pilipes Haliday and C. frigida F. [as Fucomyia gravis Haliday (Diptera: Coelopidae)]. Lesne and Mercier (1922) were able to rear a 2nd instar of A. obscurella (as A. algarum) from Coelopa puparia and described and illustrated it. Paulian (1938b) reared A. obscurella (as A. algarum) from ‘Fucella fucorum Haliday’, which we suspect referred to Fucellia fucorum Fallén (Diptera: Anthomyiidae), and added further descriptions and sketches of parts of the larvae; Paulian (1941) also described the larvae without specifying the host. Cals (1964) encountered A. obscurella (as A. algarum) as a parasitoid of Coelopa frigida and illustrated the pharate adult within its host puparium. ALEOCHARINI Because parasitoidism is the only known way of life among larvae of at least 20 species of Aleochara, it is thought that all species have this habit (Peschke and Fuldner 1977; Klimaszewski 1984). Adult Aleochara are predacious. A. (Coprochara) squalithorax is found on the Pacific shores of Japan and Korea; and A. (Coprochara) salsipotens is found on the African shores of the Indian Ocean and the Af- rican shores of the south Atlantic. Other members of the subgenus Coprochara do not, or seldom, occupy beaches. Two species of the subgenus Polystomota (A. punctatella and A. grisea) occupy European shores of the North Atlantic. Aleochara punctatella has long been confused with A. grisea, so old records of A. grisea, doubtless including some in the checklist, are in doubt and need re-evaluation. Three members of the subgenus Triochara occupy Pacific shores of eastern Asia, no others are known, and the habitat of all three appears to be seaweed. Parasitoidism by A. (Emplenota) obscurella (as A. algarum) of a dipterous puparium [Orygma luctuosum Meigen (Diptera: Coelopidae)] was reported by Scott (1916), and then in greater detail by Scott (1920) who noted that hosts were Coelopa pilipes Haliday and C. frigida F. [as Fucomyia gravis Haliday (Diptera: Coelopidae)]. Lesne and Mercier (1922) were able to rear a 2nd instar of A. obscurella (as A. algarum) from Coelopa puparia and described and illustrated it. Paulian (1938b) reared A. obscurella (as A. algarum) from ‘Fucella fucorum Haliday’, which we suspect referred to Fucellia fucorum Fallén (Diptera: Anthomyiidae), and added further descriptions and sketches of parts of the larvae; Paulian (1941) also described the larvae without specifying the host. Cals (1964) encountered A. obscurella (as A. algarum) as a parasitoid of Coelopa frigida and illustrated the pharate adult within its host puparium. Because parasitoidism is the only known way of life among larvae of at least 20 species of Aleochara, it is thought that all species have this habit (Peschke and Fuldner 1977; Klimaszewski 1984). Adult Aleochara are predacious. ATHETINI It or they (no others have been recog- nized) were later reported from similar habitats on the coasts of the North Atlantic, Irish, North, and Baltic seas. On the German shores of the Wadden Sea (separated by barrier islands from the North Sea), B. marina is a common species in the lower salt marsh, and it shows time-varying abundance within the elevational gradient (Irmler and Heller 2002). Halobrecta has about seven described species which in the past have been much confused by entomologists leading to much synonymy. The species occurring on Mediterranean shores need review (Gusarov 2004). For this reason, H. halensis Mul- sant and Rey is here listed as a distinct species. The specimens of Halobrecta flavi- pes reported by Pace (2000b) from Chaiten, Chile belong to H. algophila (Gusarov 2004). The specimens of Halobrecta flavipes reported from Inaccessible Island in the South Atlantic Ocean by Klimaszewski et al. (2002) belong to H. algophila (Gusarov 2004). Adults of Exatheta cingulata and E. consors were described by Cameron (1920) and stated to have been collected in fungi, but Sawada (1985) transferred E. cingulata to Halobrecta, and Sawada (1987) synonymized E. consors. Because fungi are rarely found on seashores, and because all other species of Halobrecta occur on seashores, not in fungi, Cameron’s (1920) specimens may have been mislabeled. Halobrecta dis- cipula was reported as a new species in Chile by Pace (1999a) who stated that it was found “in decaying vegetables (lettuces, onions)” near Valparaíso; if those vegetables had been dumped on a sea beach (not stated) near the port city of Valparaíso, they could have been used by the beetles as surrogates for drifted seaweed; see also com- ments under Myrmecopora uvida. Illustrations of the habitus and diagnostic structures of adult H. flavipes together with details about its habitat in New Brunswick are pro- vided by Klimaszewski et al. (2008). The synonymy of Halobrecta flavipes in the checklist follows Pope (1977) and Lott (2008) and differs from that given by Klimaszewski et al. (2002) following Bernhauer and Scheerpeltz (1926). Bernhauer and Scheerpeltz (1926) listed ‘Ale- ochara elongatula Stephens’ (1832) as a synonym of H. flavipes, but Stephens (1832) referred to Aleochara elongatula Gravenhorst which, if Stephens was correct in his identification, refers to what is now called Atheta (Philhygra) elongatula (Graven- horst) or Philhygra elongatula, a species which is not closely related. ATHETINI Acticola falkandica is the sole representative of this genus. The type specimen was collected in December 1914 in seaweed at Port Stanley, Falkland Islands (Cameron 1944b). Adota has three Nearctic representatives (A. colpophila, A. gnypetoides, and A. mar- itima) reported from seaweed stranded on Pacific shores, and three Palearctic species (A. madida, A. magnipennis, and A. ushio) from eastern Asia (Gusarov 2003b). In the British Isles, A. maritima was reported by Easton (1971, under the synonym of Atheta immigrans) as an adventive species. Thus all six species are known only as seashore inhabitants. Atheta, at the time of the Coleopterorum Catalogus (Scheerpeltz 1934), was a generic name applied to many confused and disparate groups. Over the subsequent years monophyletic groups have been split off from it as distinct genera, but the task is not yet complete. The following six seashore species are still assigned to it, within several subgenera. Atheta novaescotiae, not assigned to a subgenus, is a salt-tolerant, coastal, beach-drift species known from Atlantic Canada (Klimaszewski et al. 2006). Atheta (Actophylla) varendorffiana is known from the North Sea coast of Germany. Atheta (Badura) ririkoae dwells on the coasts of Korea and Honshu, Japan, as does A. Coastal Staphylinidae (Coleoptera) 41 (Badura) tokiokai (but this latter also on the coasts of Kyushu). Atheta (Datomicra) acadiensis, from Canadian Maritime Provinces, is typically found in dry beach-drift material at the top of the littoral zone, the material consisting of dead Ascophyllum nodosum (L.) and Fucus vesiculosus L. (Klimaszewski and Majka 2007). Finally, A. (Sipalatheta) algarum is found in seaweed on the coast of Hong Kong in the South China Sea (Pace 1999b). (Badura) tokiokai (but this latter also on the coasts of Kyushu). Atheta (Datomicra) acadiensis, from Canadian Maritime Provinces, is typically found in dry beach-drift material at the top of the littoral zone, the material consisting of dead Ascophyllum nodosum (L.) and Fucus vesiculosus L. (Klimaszewski and Majka 2007). Finally, A. (Sipalatheta) algarum is found in seaweed on the coast of Hong Kong in the South China Sea (Pace 1999b). Brundinia was initially described as Homalota meridionalis with “variety” marina by Mulsant and Rey (1853). The “variety” was later recognized as a valid species, and the generic name Brundinia was introduced later. This, or these, species (now B. me- ridionalis and B. marina) were initially reported from plant debris in a salt marsh at Hyères, on the Mediterranean coast of France. ATHETINI Furthermore, the habitat specified by Stephens (1832) for this species [“not common: found occasion- J. H. Frank & Kee-Jeong Ahn / ZooKeys 107: 1–98 (2011) 42 ally within the metropolitan district” (of London)] does not seem a likely habitat for a species of Halobrecta. If ‘Aleochara elongatula Stephens’ really is a synonym, it is the senior name and should be listed first among synonyms; however, this is likely to be a misidentification by Stephens. Also, according to Bernhauer and Scheerpeltz (1926), Halobrecta atricilla (Scriba) 1866: 290 nec Erichson is a synonym of H. flavipes and is so ranked in the checklist. For unexplained reasons, Bernhauer and Scheerpeltz (1926) did not accept Scriba’s (1866) redescription of H. atricilla (Erichson) as per- taining to that species. g p Hydrosmecta subalgarum is the only species assigned to this genus and is reported to inhabit seashores. Four specimens, collected under seaweed on sand at Tai Long, Hong Kong, China, are the only known collection of this species (Pace 1999b). Iotarphia is monotypic. Its single species, I. australis, is known from adult speci- mens collected in a “maritime habitat” from Rockdale near Sydney and from Illawarra, both in New South Wales, Australia (Cameron 1943). Osakatheta is monotypic. The single species, O. yasukoae, is known only from adults collected under stones on tidal flats at river mouths on the eastern coast of Hon- shu, Japan’s largest island, where their habitat is threatened by industrial development (Maruyama et al. 2008). Pontomalota contains two species, P. opaca and P. terminalis, both of them known from the Pacific coast of North America. They inhabit the mid to upper littoral zone of fine-grained sandy beaches, covered by tides only once or twice each month and containing stranded seaweed. Adults appear to be most active at night or on heavily cloudy days. They may occasionally be very abundant with hundreds of individuals per m2. Whereas P. terminalis is known only from California, P. opaca is distributed from Alaska to Baja California and shows clinal variation in color with specimens black in the north to light brown in the south. Their immature stages are unknown (Ahn and Ashe 1992). Adults spend the daylight hours beneath driftwood or piles of stranded seaweed (Kincaid 1961b). Psammopora contains only one species, P. DIGLOTTINI Various genera have been removed to other tribes, leaving only the genera Diglotta and Paradiglotta. Various genera have been removed to other tribes, leaving only the genera Diglotta and Paradiglotta. Diglotta contains eight species, three (D. mersa, D. sinuaticollis, and D. littoralis) from the North Atlantic and adjacent seas (North, Irish, and Mediterranean), one (D. brasiliensis) from the South Atlantic, one (D. secqi) from the Red Sea, two (D. legneri and D. pacifica) from the North Pacific, and one (D. maritima) from the South Pacific. Lohse (1985), followed by Haghebaert (1991) confused the two European species of Diglotta; although this issue was resolved by Good (1998), distribution records of these two species are doubtless still confused. A larva, collected on the west coast of Denmark in June 1917 in association with adults of D. mersa, was described and il- lustrated by Kemner (1925). Of the two European species, D. sinuaticollis (as D. mersa) occupies the intertidal and supralittoral zone of sandy beaches, whereas D. mersa (as D. submarina) occurs mostly in salt and mud-marshes (Haghebaert 1991). The type local- ity of D. brasiliensis is the intertidal zone of an estuarine beach in the state of Paraná (Caron and Ribeiro-Costa 2008). The North American species occur on sandy beaches (Moore and Orth 1979a). The adults of the western Palaearctic and Brazilian species have 4 posterior tarsomeres whereas the North American and Pacific species have 5 tarsomeres (Haghebaert 1991; Caron and Ribeiro-Costa 2008; Klimaszewski et al. 2008). Illustrations of the habitus and diagnostic structures of adult D. mersa together with details about its habitat in New Brunswick are provided by Klimaszewski et al. (2008). Paradiglotta, with its single species P. nunni Ashe and Ahn (2005) from New Zea- land is not included in the present list because it has not been found on seashores, only inland. ATHETINI delittlei; adults were detected on a sandy beach in Tasmania, Australia (Pace 2003).i Psammostiba is a genus containing five species, all from seashores in the north- ern Pacific, with two species in the Nearctic (P. comparabilis and P. kenaii) and three in the Palearctic (P. hilleri, P. jessoensis, and P. kamtschatica). Adults (the im- mature stages have not been reported) have been found in drifted seaweed (Gusarov 2003b). Tarphiota has three species (T. geniculata, T. fucicola, and T. densa) confined to fine-grained sandy seashores of Pacific North America. They live in the mid- to upper- intertidal zone containing decaying seaweed and covered by only one or two high tides monthly. Larvae are unknown (Ahn 1996b, 1999).hl Thinusa contains only two species (T. fletcheri and T. maritima) that live in the intertidal zone of sandy beaches immersed daily by tides on the Pacific coast of North America (Moore and Legner 1977; Topp and Ring 1988a; Ahn 1997b). Adults are ac- tive at night (Kincaid 1961b). Coastal Staphylinidae (Coleoptera) 43 FALAGRIINI Falagriini are a tribe of about 30 genera worldwide, only two of which have coastal rep- resentatives. The species of America north of Mexico were revised by Hoebeke (1985). A cladistic analysis of the genera of America north of Mexico by Ahn and Ashe (1995) showed monophyly of Bryobiota together with Myrmecopora, setting them apart from other genera in this tribe, which needs further study including more terrestrial falagri- ines. The two species of Bryobiota (B. bicolor and B. giulianii) inhabit sandy beaches of the Pacific coast of North America. They live in the upper littoral zone, which is covered by tides only once or twice each month and contains buried decaying seaweed (Topp and Ring 1988a; Ahn and Ashe 1995). Larvae and diet are unreported. Myrmecopora is currently divided into three subgenera (Lamproxenusa, Paraxenusa, and Xenusa). Fourteen species of Myrmecopora have been reported from seashores. Four species assigned to subgenus Lamproxenusa (M. algarum, M. chinensis, M. reticulata, J. H. Frank & Kee-Jeong Ahn / ZooKeys 107: 1–98 (2011) 44 and M. rufescens) dwell on the shores of eastern Asia in seaweed on sandy beaches (Ass- ing 1997b). One species assigned to Paraxenusa (M. laesa) is known from the Mediter- ranean Sea and the Canary Islands of the north Atlantic. Ten species assigned to Xenusa range from the shores of the Red, Black, and Mediterranean seas to the north Atlantic with its adjacent seas (Irish, North, and Baltic); one of these (M. maritima) is known only from Madeira and the Canary Islands, and one (M. bernhaueri) only from the Red Sea; additional species occupy other habitats. Myrmecopora tenuicornis (Küster) was treated as a synonym of M. laesa (Erichson) by Fauvel (1902) and subsequent authors. Assing (1997a) expressed misgivings about this synonymy but explained that the loca- tion of the type specimen of M. tenuicornis is unknown, so the synonymy cannot be resolved. Myrmecopora (Xenusa) uvida, a species widespread in the western Palaearctic, was newly reported as an adventive species in Chile by Pace (1999a) who stated that it was found “under decaying vegetable products (cauliflowers, cabbages, onions, etc.)” near Antofagasta; if those vegetables had been dumped on a sea beach (not stated) near the port city of Antofagasta, they could have been used by the beetles as surrogates for drifted seaweed; see also comments under Halobrecta discipula. HOMALOTINI Most genera and species of this tribe are not seashore inhabitants. Four genera, Het- erota, Paractocharis, Pseudopasilia and Thinobiosus, are exclusively seashore species. Just one species of Linoglossa has been reported from coastal habitats. Two species of sub- genus Halmaeusa Kiesenwetter (1877) [= Antarctophytosus Enderlein (1909) = Para- phytosus Cameron (1917e) = Austromalota Brèthes (1925)] of the genus Leptusa Kraatz (1856), namely L. atriceps (C.O. Waterhouse 1875: 54 and 1879: 230) from South Georgia and Kerguelen Island, and L. darwinii (F.H. Waterhouse 1879: 531) from the Falkland Islands and South Georgia (= rufomixtus Brèthes 1925: 171) are omitted from the checklist because they are not strictly seashore species (Steel 1964).l Cameronium has two species in the Indian Ocean (C. flavipenne and C. gomyi), one in the Red Sea (C. obockianus), one in North Africa (C. liebmanni Scheerpeltz 1957a) and, remarkably, one in the Gulf of California (C. sonorensis). If the last is correctly as- signed to genus, the dispersal of its ancestors to that locale would be surprising and not easily explicable; consequently, its placement should be reinvestigated. Cameronium liebmanni is known only from an inland freshwater lake in Algeria so we do not in- clude it in the present compilation. In Somalia, C. flavipenne adults were trapped more abundantly in the wet season than in the dry season (Chelazzi et al. 1983). Heterota is represented by ten species distributed from the East Sea (H. sunjaei), South China Sea (H. arenaria), and Bali Sea (H. rougemonti) through the Mascarene Islands of the Indian Ocean (H. gomyi, H. obscura, and H. vinsoni), the Red Sea (H. brevicollis and H. pictipennis), to the Mediterranean (H. pamphylica and H. plumbea). The distribution of the last of these extends from the Mediterranean to the west coasts of Europe and the Canary Islands, and more recently was discovered in southern and Coastal Staphylinidae (Coleoptera) 45 northwestern Florida (USA), Jamaica, and the Caribbean coast of Mexico (Frank and Thomas 1984b). There is no apparent reason to believe that movement of H. plumbea from Europe to the Canary Islands and then to the Caribbean was assisted by humans. Small, salt-tolerant, winged insects are among the most likely candidates to disperse westward naturally with assistance of trade winds at tropical and subtropical latitudes. An annotated catalog of the species is provided by Park et al. (2008). On a sandy beach in Somalia, H. HOMALOTINI pictipennis adults were trapped more abundantly in the wet season than in the dry season (Chelazzi et al. 1983). y Linoglossa murphyi adults were collected in a mangrove forest in Singapore (Sawada 1991), but there is no evidence that other species of the genus are halobionts.hi Paractocharis is represented by three species. The first described was P. fucicola, from sandy beaches under seaweed at Changi, Singapore, in the South China Sea (Cameron 1917c). The others, P. deharvengi and P. orousseti, were both found at Pu- erto Galera on Mindoro, Philippines, in the Luzon Sea, in “lavages” (washings) on the beach (Pace 1990). Pseudopasilia is represented only by P. testacea, found on the coasts of the North Atlantic (England and France), North Sea (Belgium and southeastern England), and western Mediterranean (southern France, Italy including Sardinia, Tunisia, and prob- ably Croatia). For many years entomologists confused Arena tabida with P. testacea (Tronquet 2003). Tronquet (2003) also noted adults found under small stones in the littoral zone. Thinobiosus salinus is known only from the shores of Sonora in the Gulf of Mexico, where specimens were found in seaweed on the edge of a tidal pool (Moore and Legner 1977). No other species have been assigned to this genus. LIPAROCEPHALINI All members of this tribe appear restricted to coastal habitats. It appears to be mono- phyletic, with relationships of its seven genera suggested as ((Baeostethus, Ianmoorea) (Paramblopusa ((Amblopusa, Halorhadinus) (Liparocephalus, Diaulota)))) by Ahn et al. (2010). Five of its genera appear restricted to the north Pacific Ocean (with adjoining Gulf of California and East Sea), and two to the south Pacific. ii i Amblopusa contains five species, all in the North Pacific, two of them (A. alaskana and A. brevipes) in North America, two (A. pacifica and A. hokkaidona) on Hokkaido in northern Japan, and one (A. magna) in the Far East of Russia. All of them inhabit the mid-littoral zone. A late instar of A. alaskana was described in detail by Ahn and Ashe (1996a). Amblopusa pacifica adults were collected in wrack at Akeshi, eastern Hokkaido, in August 1990 (Sawada 1991). Amblopusa magna adults were collected in piles of drift- ed seaweed on fine sand beaches at Ryazonovka near Slavyanka, Russia in June 1993. i Baeostethus has one species (B. chiltoni) that occupies rocky intertidal areas in the Antarctic islands of New Zealand. Adults, as is typical of Liparocephalini, are flightless; a late instar was described by Steel (1964) and by Leschen et al. (2002). 46 J. H. Frank & Kee-Jeong Ahn / ZooKeys 107: 1–98 (2011) Diaulota densissima adults were found to be plentiful along the rocky alga-covered shores of Departure Bay (British Columbia, Canada), and larvae were found and il- lustrated; adults are flightless (Saunders 1928). Adults and larvae of D. vandykei, and adults and a few suspected larvae of D. densissima were obtained from cracks of rocks and under algae at Moss Beach (San Mateo County, California, USA) in January; brief descriptions and an illustration of the apex of the larval abdomen of both was provided by Chamberlin and Ferris (1929). Moore (1956b) added some diagnostic notes on the structures of larvae of D. densissima, D. fulviventris, D. vandykei, and D. harteri collected from beaches in California and Baja California (Mexico) and provided a key to identify them. Meyerdirk (1969) studied a population of D. fulviventris at La Jolla (California, USA) and found adults and larvae in June, August, September, and De- cember 1968 with the number of beetles averaging 0.70 per cm2, increasing to 1.1 per cm2 in March 1969. LIPAROCEPHALINI 47 Coastal Staphylinidae (Coleoptera) 47 Adults and larvae can withstand submergence in seawater for > 2 weeks at 10oC, and thus can withstand continuous inundation from one spring tide to the next, although submerged larvae cease feeding and growing and do not pupate. Eggs were deposited in rock crevices or between the thalli of algae. Prepupae of L. cordicollis spin silken cocoons incorporating plant fragments in which to pupate. Liparocephalus cordicollis adults, and a few larvae and pupae, were found at Moss Beach (San Mateo County, California, USA) in November and December (Chamberlin and Ferris 1929). The final instar was described and illustrated, and the pupa was stated to be formed within a cocoon. A brief redescription by Moore (1956b) of the larva failed to clarify which of the species (L. brevipennis or L. cordicollis) he was describing. Liparocephalus tokunagai adults and larvae were found abundantly on rocky shores between high and low tide- marks in the spring of the year. The adults were seen to devour amphipod crustaceans of the genus Gammarus Fabricius (Sakaguti 1944). The final instar of L. cordicollis was described and illustrated by Ahn (1997a) with diagnostic characters for larvae of L. brevipennis and L. tokunagai together with a key for identification of larvae. p g gi Paramblopusa was named by Ahn and Ashe (1996a) as a new genus to contain P. borealis, transferred from Amblopusa and known from the Pacific coast of North America where specimens have been collected under rocks on tidal flats. Later, P. eoa was described from the Kuril Islands of Russia, on rocks below a cliff at Negodnaya Bay (Ahn and Maruyama 2000). LIPAROCEPHALINI The range of the population extended from 0.76 to 1.83 m above mean low tide, exclusively in association with the acorn barnacle Chthamalus fissus Darwin (Thoracica: Chthamalidae). The barnacle provided refuge from wave force and currents. Beetle activity was highest at low tide, and ceased when the substrate became saturated with sea water. Laboratory observations showed no evidence of a circadian or tidal rhythm. Pupae were formed in silk-like cocoons within empty barnacle tests and cracks in rocks. Adults respired under water by means of cutaneous respiration, ex- panding their abdomen to expose more membranous area (between sclerotized plates) when they were submerged. Halorhadinus is represented by three species inhabiting the littoral zone of boulder shores and sandy beaches where seaweed is stranded in Korea and Japan. Maruyama and Hayashi (2009) discussed the habitat of Halorhadinus in detail. Their immature stages are unknown (Ahn 2001).i Ianmoorea is represented by a single species, I. zealandica, detected under fine gravels in the intertidal region of Breaker Bay, Wellington, New Zealand (Ahn 2004, 2006). Liparocephalus is represented by four species. Liparocephalus cordicollis adults (misi- dentified as those of L. brevipennis) were found to be plentiful along the rocky alga- covered shores of Departure Bay (British Columbia, Canada), and larvae were found and illustrated. Adults are flightless. Placed on the surface of a dish containing water and algae, adults descended and moved among the plants under water. They were thought to carry a film of air trapped “in the hairs of the body.” Oviposition and pupation were not obtained in the laboratory. In the laboratory, larvae ate pupae of the chironomid Telmatogeton Schiner, and some ate dead larvae of that genus and of Camptocladius Wulp. Adults were not observed to feed. The disappearance of algae from the rocks during the summer may account for disappearance of the beetles and their subsequent reappearance during the winter. The foregoing account by Saunders (1928) is confusing in that it is drawn from observations on both L. cordicollis and Diaulota densissima and sometimes makes no distinction between the two. Topp and Ring (1988b) reinvestigated, and found L. cordicollis between 0.2 and 2.0 m above lowest spring tide on rocky shores of Vancouver Island (British Columbia, Canada). MYLLAENINI Concepts of the tribe Myllaenini have changed radically in the past few years, and genera such as Bryothinusa and Rothium have been transferred to it from other tribes. g y Brachypronomaea esakii adults were found on a coral reef five km off the coast of Ishigaki Island, one of the Ryukyu Islands of southern Japan. This reef is submerged for all but about two hours daily. Adults are apterous, and the food and immature stages are unknown (Esaki 1956). Later collections from Okinawa in March–May 2002 showed that the beetles occupied coral reefs having large populations of Collembola suggesting that the beetles are predatory on the springtails (Ahn et al. 2003). The second species assigned to this genus is B. sawadai, described from the Bay of St. Vincent in New Cal- edonia by Jarrige (1964) without further information on its habitat. The nomenclature is not totally straightforward because Jarrige (1964) equated Thalassopora [a genus he described (Jarrige 1959) to include T. nosybiana from Madagascar, and T. marchemar- chadi (the type species) from what is now Vietnam] with Brachypronomaea in a heading “Brachypronomaea Sawada, 1956 (Thalassopora Jarr., 1959)”. Nowhere in the following text did he mention this was a newly proposed synonymy. However, the text included description of a new species, B. sawadai, together with mention of characters of “B. Marche-Marchadi” and “B. Esakii”. Jarrige’s (1964) intent was clearly to synonymize Thalassopora with Brachypronomaea. We believe that Jarrige (1964), despite lack of discussion, made a formal synonymy of Thalassopora with Brachypronomaea thereby J. H. Frank & Kee-Jeong Ahn / ZooKeys 107: 1–98 (2011) 48 transferring both the species he had originally placed in Thalassopora. A revision of the four species (B. esakii, B. marchemarchadi, B. nosybiana, and B. sawadai) is necessary. The habitat of Thalassopora (now Brachypronomaea) nosybiana was described as in sand and rocks near the oceanographical station of Nosy Bé, Madagascar (Paulian 1959). Its final instar (recognized because it was with adults, not reared) was described and illustrated, its digestive tube being packed with unicellular algae (Paulian 1959). Bryothinusa, with 30 described species, restricted to seashores and with no species from other habitats, has the greatest diversity of all genera of the coastal Aleocharinae. Twenty-four live on shores of the Pacific Ocean and its surrounding seas, two (B. made- cassa and B. perexilis) on the shores of the Indian Ocean, and four (B. cameroni, B. pe- yerimhoffi, B. MYLLAENINI subtilissima, and B. testacea) on the shores of the Red Sea (one of these also found in the Mediterranean). None has been reported from the Atlantic Ocean. Revi- sions have been published by Pace (1986), Haghebaert (1995), and Ashe (2005). Adults of B. sakishimana were collected in a mangrove forest (Sawada 1991), and those of B. chengae at light (Ahn 1998). The larva of the Californian B. catalinae was described by Moore and Orth (1979b) who described its habitat as “a shallow reef which is exposed at low-water and under water at high tide ... largely a field of boulders two or three feet across with smaller stones and gravel in sand ... adults and larvae were found beneath and on the stones in an association with dense worm tubes, chitons, limpets, small aba- lones, flatworms, small crabs and brittle stars.” Four species (B. gangjinensis, B. koreana, B. minuta, and B. nakanei) have been found on Korean shores (Ahn and Jeon 2004). Of the Bryothinusa species found in Hong Kong, the habitats were distinguished as follows by Moore and Legner (1971) and Moore et al. (1973): (B. chani): “At low water they wander on the surface of the mud flat and at high tide they are under seawater by holding tightly to rocks and dead shells”; (B. sawadai): “Found among rock crevices and shells of barnacles and oysters. When burrowing in the sand never more than 1 cm deep. Can be found throughout the year.”; (B. sinensis): “Found among rocks, wander- ing on sand or burrowing in the sand as deep as 36 cm. Seems to prefer a sandy beach. Can be found throughout the year.”; (B. hongkongensis): “Found wandering on sand or burrowing in sand as deep as 36 cm from mid-tidal zone to low tidal zone. Specimens were found from February to April” (Moore and Legner 1971; Moore et al. 1973). Descriptions do not suggest that members of the genus are alike in sharing one kind of habitat. The habitat of B. fluenta was even more unusual: it was “beneath the surface of a fresh water stream a short distance from the seashore...[it] seems to have invaded a fresh water habitat directly from salt water” (Moore and Legner 1975). Adults of four of the Hong Kong Bryothinusa species (B. chani, B. hongkongensis, B. sawadai, and B. MYLLAENINI sinensis) were apparently able to breathe by plastron respiration, to feed on decaying microcrustacea, to occupy the mid-intertidal zone (0.6–1.5 m above mean tide level), to spend their time mainly at 0–15 cm below the sand surface, to mate on the sand surface, to occupy sand of low organic content, and to be macropterous except for B. chani (Wong and Chan 1977). Larvae of these four remained below the sand surface. The larva of B. koreana was described by Jeon and Ahn (2009), making it only the sec- ond species of the genus so treated (with the larva identified by mitochondrial DNA). 49 Coastal Staphylinidae (Coleoptera) Corallis polyporum adults were stated by Fauvel (1878a) to be “sous-marines” (sub- marine or aquatic in the sea) under coral polyps in March. They occur together with those of Polypea, both genera being monotypic (Fauvel 1878a).l Lautaea murphyi adults were collected under mangroves in intertidal mud flats. Their digestive tracts were filled with fragments of harpacticoid copepods (Sawada 1989a). The genus is monotypic. Myllaena is a genus distributed worldwide whose members are typically found in freshwater habitats, such as on the banks of lakes and streams, and in swamps. Howev- er, the North American M. insipiens Casey seems to have been collected only at or very close to coasts of the Atlantic and Gulf of Mexico of the USA (Klimaszewski 1982a), and is provisionally included in this list. Myllaena leleupi Pace (1985) is excluded from the checklist because its type locality in the Galapagos Islands is 4 km from the coast, despite a later record in a coastal habitat (Klimaszewski and Peck 1998). Polypea corallis adults were said to have been found in the ocean under coral pol- yps, in March, to occur with those of Corallis, and to be submarine (Fauvel 1878a). Klimaszewski (1982b) argued that Polypea seems to lack any structural modification for a submarine existence. Rothium was first included in Myllaenini by Ahn and Ashe (1996c). Its six de- scribed species inhabit the Pacific (including Gulf of California) coasts of North and South America. The first to be described, R. sonorensis, was collected in the intertidal zone “from algae covered pitted ryolite” and from “a tide pool” (Moore and Legner 1977). Discovery of two further Mexican species, and three from parts of Ecuador (including the Galapagos Islands) and Peru yielded little more information about their habitats. The larva of M. MYLLAENINI sonorensis was described by Moore (1977) and subsequently redescribed in detail by Ahn and Ashe (1996c). PHYTOSINI The relationships of the tribe are unclear, and it is not yet certain that the tribe is monophyletic. The four genera remaining in this tribe, after removal of others to Myl- laenini etc., are Actocharis, Arena, Euphytosus, and Phytosus. Actocharis contains only two European species, A. readingii and A. cassandrensis. Both occur on Mediterranean coasts, and the first also occurs on North Atlantic coasts of England and France. Arena includes one European species (A. tabida) and another (A. fultoni) from New Zealand, a remarkable distribution if these placements are actually correct (A. fultoni needs re-evaluation). Arena tabida was for years confused by entomologists with Pseu- dopasilia testacea (Homalotini) but it has a narrower distribution on the Atlantic coast of northwestern France, with the coasts of England and Wales, Irish Sea coasts of Eng- land and Wales, and North Sea coasts of England, Scotland, Netherlands, Germany, and Denmark, and is associated with drifted Fucus (Tronquet 2003). q Bernhauer (1922a) described specimens collected by Hans Sauter at Alikang in Taiwan under the name Phytosus (Paraphytosus) schenklingi. No information about its habitat was provided. However, the name Paraphytosus Bernhauer was preoccupied (by Paraphytosus Cameron 1917e). In 1926, the Coleopterorum Catalogus designated Euphytosus Bernhauer and Scheerpeltz as a replacement name (through objective syn- onymy) for Paraphytosus Bernhauer, with P. (Euphytosus) schenklingi as its type and only species. p Haghebaert (1993) excluded Euphytosus from the genus Phytosus with the conse- quence that it was automatically raised to generic rank; in that same work, Haghe- baert also suggested (he wrote that he planned a later publication on the subject) an unnecessary new name (Pseudophytosus) for Euphytosus, so Pseudophytosus is not a valid name. Phytosus is currently divided into two subgenera. The typical subgenus has three species; P. spinifer in the North Atlantic and Baltic, Black, North, and Mediterra- nean seas; P. fenyesi on the Atlantic coast of Senegal; and P. caribeanus on the shores of Guadeloupe in the West Indies. All five members of subgenus Actosus dwell on coasts of the North Atlantic and/or adjacent seas (Mediterranean, Baltic, or North). The present checklist does not mention Phytosus atriceps C.O. Waterhouse (1875), from Kerguelen Island, redescribed and illustrated by him (1879). The species was made the type of a new genus, Antarctophytosus by Enderlein (1909). OXYPODINI Seashore representatives of this large tribe belong to seem to be only four genera, all monotypic: Chilodera, Dasydera, Gyronotus, and Oreuryalea (Assing and Maruyama 2002). Chilodera falklandica is known only from seaweed at Port Stanley in the Falk- land Islands in the South Atlantic, where specimens were collected in December 1914 (Cameron 1944b). Dasydera algophila was found among seaweeds on Mokohinau Island, New Zea- land (Broun 1886).i Gyronotus rufipennis is known only from North Island, New Zealand (Broun 1880). Oreuryalea watanabei is known from the Russian Far East and northern Japan, where it was detected among drifted seaweed and other debris on beaches. Four female specimens collected in July and September each had a single mature egg in its ovaries (Assing and Maruyama 2002). J. H. Frank & Kee-Jeong Ahn / ZooKeys 107: 1–98 (2011) J. H. Frank & Kee-Jeong Ahn / ZooKeys 107: 1–98 (2011) 50 Worldwide, there are 47 known genera in Oxytelinae (Thayer 2005). Worldwide, there are 47 known genera in Oxytelinae (Thayer 2005). OXYTELINI This tribe has about 14 genera worldwide (Newton et al. 2001), including Anotylus, Blediotrogus, Pareiobledius, and Sartallus. Anotylus has about 350 species worldwide, most associated with forest leaf litter, decomposing organic matter such as dung and carrion, and in mammal or ant nests (Newton et al. 2001). However, the European A. maritimus appears to occupy only seashore habitats, where it lives among seaweed and other drifted debris. Anotylus spe- culifrons (Kraatz 1857: 862) is often found in coastal habitats in western Europe, but occurs also in eastern Europe far from seashores; consequently we do not include it.i Blediotrogus has about five known species in Australia, New Zealand, and the Chatham Islands, and they are frequently found on seashores, under moist high-tide beach wrack (Makranczy 2006). Pareiobledius has three known species, from the Afrotropical region, whose charac- teristic habitat is on seashores, under kelp (Makranczy 2006).h Sartallus has only one species, the Australian S. signatus, with winged adults. That species “is common on our [?South Australian] sandy beaches, where it [the adult?] hides under the seaweed and rubbish and feeds chiefly upon dead barnacles” (Froggatt 1907). Incertae sedis Salinamexus has two species (S. browni and S. reticulatus) that occur on the shores of the Gulf of California and one (S. koreanus) in Korea. Adults are able to fly. All three appear to occur exclusively under boulders or seaweed on seashores; their immature stages are unknown (Jeon and Ahn 2008). Ahn et al. (2010) showed that the genus was not a member of the Liparocephalini. Its phylogenetic position is uncertain. PHYTOSINI Unaware of that assignment, Cameron (1917b) reported its finding in seaweed on a sandy sea- shore in the Falkland Islands and made it the type of a new genus, Paraphytosus. Cameron (1917e) admitted that he had misidentified the specimens, and also that the generic name Antarctophytosus Enderlein had precedence over Paraphytosus. Steel (1964) considered the genera Antarctophytosus Enderlein (1909), Paraphytosus Cam- eron (1917b), and Austromalota Brèthes (1925) all to be synonyms of Halmaeusa Kiesenwetter (1877). Steel (1964) also found that none of the species of Halmaeusa is restricted to seashores. Coastal Staphylinidae (Coleoptera) 51 THINOBIINI This is the largest oxyteline tribe with about 20 genera worldwide (Newton et al. 2001). Carpelimus, with several hundred species, worldwide, is one of the two largest genera. Most Carpelimus species occupy freshwater habitats such as the shores of rivers and lakes, and in swamps. Moore and Legner (1974b) described the larva and pupa of C. debilis (Casey 1889a: 374) and noted that it occurs often in stranded seaweed on the Pacific shores of North America, but occupies other habitats as well (so we do not 52 J. H. Frank & Kee-Jeong Ahn / ZooKeys 107: 1–98 (2011) list it). On the shores of Zanzibar, C. lucidus was found in seaweed and there seems to be no other habitat information, so we list it (Cameron 1944a). Bledius, with over 450 species worldwide, is the other large genus within the Thi- nobiini. Herman (1986) classifies it according to species groups (an ordering we follow in our checklist) rather than formal subgenera. Only some Bledius species dwell on sea- shores, and some of these also occur inland in saline habitats. We attempt to list only those species that do not occur also in inland saline habitats, resulting in the inclusion of 57 species names. We refer the reader to Herman (1986: 11–72) for an excellent overview of the natural history of the genus [including its predators and parasites (par- tially reviewed in Frank 1982, 1985b)]. Consequently herein we mention only a few other studies. Several Bledius species found on coasts have been studied in some detail and have been noted as coastal species. However, evidence suggests that these are not strictly coastal, so they are not included in the present checklist. We mention them in part lest the reader should think we overlooked them and in part because the references cited provide larval and/or behavioral descriptions that may prove useful in comparison with future larval descriptions of coastal species. Four Bledius species [B. tricornis (Herbst), B. hinnulus Erichson), B. gallicus Gravenhorst) as B. fracticornis (Paykull), and B. pal- lipes (Gravenhorst)] from Denmark have fine larval illustrations by Schiødte (1864). Five Bledius species [B. arenarius (Paykull), B. talpa (Gyllenhal), B. subterraneus (Er- ichson), B. fuscipes (Rey), and B. opacus (Block)] from Finland have fine larval descrip- tions and illustrations by Krogerus (1925). Four Bledius species [B. spectabilis Kraatz, B. unicornis (Germar), B. furcatus (Olivier), and B. THINOBIINI fuscicornis Cameron] from France have larval descriptions by Paulian (1938a, 1941). The larva of B. albonotatus Mäklin from California was described (as B. ornatus LeConte) by Moore and Legner (1974a). Notes on behavior and development of B. spectabilis were published by Paulian (1942). Detailed studies by Bro Larsen (1936, 1951, 1952) of beetles of some Danish salt marsh and dune habitats dealt with several Bledius species particularly B. spectabilis. More recently, the distribution, behavior, and physiology of B. spectabilis was studied in Norfolk, England (Evans et al. 1971; Wyatt 1982, 1986; Wyatt and Foster 1988). The latter show the remarkable development of subsocial behavior among Bledius in which adults care for their brood. They also illustrate the form of the tunnels in which the entrance is narrowed to a bottle-neck so that it is not easily flooded by the tide (and can be blocked by the adults within a few minutes) to trap oxygen inside. Included is information on oxygen consumption and the minute algae that are the food of adults and larvae. Bledius fenyesi and B. monstratus dwell on the Pacific coasts of North America in decaying, sand-covered piles of seaweed (Herman 1986). Evans’s (1980) claim that adults of B. monstratus are predacious requires verification given that other members of the genus feed on algae. Bledius subniger dwells on the sandy shores of Ostvoorn (Netherlands) just below the high tide mark, tunneling within the topmost 5 cm of sand, and occurring at den- sities up to 500 per m2. It feeds on Chlorophyta, Cyanophyta, and Chrysophyta (dia- Coastal Staphylinidae (Coleoptera) 53 toms). It is very active at the end of June and the beginning of July, with individuals walking on the sand surface, followed by swarming for mating or dispersal (Hollander and van Etten 1974; Hollander 1983). These authors also discuss the habits of Bledius arenarius, which dwells in dunes above the beach and is thus excluded from our list of seashore staphylinids.fifi Griffiths and Griffiths (1983) found that Bledius punctatissimus attained densities of up to 2,260 adults per m2 on a sheltered marine beach at Pawley’s Island (South Carolina, USA). Adults and larvae below the sand surface are regularly immersed by the tide. At low tide they emerged to form galleries just below the sand surface and fed on diatoms. THINOBIINI This activity lasted as long as 11 hours at higher shoreline elevations or 7 hours at elevations nearer to the lower limits of its distribution. At other times, adults and larger larvae singly occupied individual deeper burrows, whereas some females occupied maternal burrows together with their eggs and small larvae in side galleries; such burrows retained air during immersion by the tide. Griffiths and Griffiths’ (1983) account is accompanied by drawings of egg, larva, pupa, and adult. Teropalpus contains nine species, all with seashore distribution (Newton et al. 2001). Just one (T. lithocharinus) is native to the northern hemisphere, the remaining eight to the southern hemisphere, but one of the latter (T. unicolor), native to New Zealand, Australia, and South Africa, is adventive in Britain and arrived there before 1900. Specimens are found on seashores under driftwood and algae (Makranczy 2006). p g y Thinobius includes nearly 100 species worldwide (Newton et al. 2001). Most appear to live on banks of streams and ponds and other freshwater habitats, but a few live on in- tertidal mudflats. Among the latter, are T. frizzelli on the Pacific coast of North America, T. marinus in Singapore, and T. kuroshio on Honshu, the largest island of Japan. Dense populations of T. frizzelli were reported from felted growth of an alga [Lyngyba semi- plana (C. Agardh) J. Agardh (Oscillatoriaceae)] in the intertidal zone of Willapa Bay in southwestern Washington State, where the individuals retreated into burrows among the algae when the tide was high; adults are winged, but the wings are of reduced size; adults are thought to eat “detritus”; larvae received a cursory description from Kincaid (1961a). In contrast, T. kuroshio adults were found in decaying drifted seaweed on a pebbly beach (Sawada 1971c), and T. marinus adults were found at Changi, Singapore, on sandy beaches under seaweed (Cameron 1917d). The synonymy of Yosiityphlus with Thinobius was documented by Gusarov and Makranczy (2004). PAEDERINI About 200 genera of Paederini occur worldwide. The monotypic genus Chetocephalus is based on C. maritimus, which was found in seaweed on Mauritius (Cameron 1944a). Medon contains about 350 species worldwide, few of them occurring on seashores. Two exceptions are M. marinus, which was found in seaweed on Mauritius, and M. pocoferus found on the shores of the Mediterranean Sea and the North Atlantic Ocean. Two others are M. prolixus and M. rubeculus, both initially described from Japan, the former first reported from seaweed at Iwosima and Amakusa (Sharp 1874), but the latter was subsequently detected in Hong Kong where it appears to be restricted to the seashore (Rougemont 2001). About 200 genera of Paederini occur worldwide. The monotypic genus Chetocephalus is based on C. maritimus, which was found in seaweed on Mauritius (Cameron 1944a). Medon contains about 350 species worldwide, few of them occurring on seashores. Medon contains about 350 species worldwide, few of them occurring on seashores. Two exceptions are M. marinus, which was found in seaweed on Mauritius, and M. pocoferus found on the shores of the Mediterranean Sea and the North Atlantic Ocean. Two others are M. prolixus and M. rubeculus, both initially described from Japan, the former first reported from seaweed at Iwosima and Amakusa (Sharp 1874), but the latter was subsequently detected in Hong Kong where it appears to be restricted to the seashore (Rougemont 2001). pocoferus found on the shores of the Mediterranean Sea and the North Atlantic Ocean. Two others are M. prolixus and M. rubeculus, both initially described from Japan, the former first reported from seaweed at Iwosima and Amakusa (Sharp 1874), but the latter was subsequently detected in Hong Kong where it appears to be restricted to the seashore (Rougemont 2001). Sunius has about 125 species worldwide, but few of these are typically found on seashores. These exceptions are S. ferrugineus from the Caribbean Sea and Sunius minu- tus from Florida. Both live in drifted seaweed, and they may represent only a single species. Ophioomma, a genus only recently transferred to Paederini, has only one species, O. rufa. Specimens were initially collected on the Gulf of Mexico coast of Florida, by sifting debris on the beach of Charlotte Harbor. STAPHYLININAE This subfamily contains 320 genera worldwide (Thayer 2005). Adults and larvae are predacious. This subfamily contains 320 genera worldwide (Thayer 2005). Adults and larvae are predacious. PAEDERINAE PAEDERINAE About 221 genera of Paederinae occur worldwide; the tribal classification is under revi- sion by L.H. Herman, so subsequent classification may be changed, and the placement of coastal species in the tribe Paederini (below) is uncertain (Thayer 2005). SCYDMAENINAE CEPHENNIINI Worldwide, over 4600 species in some 82 genera of this subfamily have been recorded (O’Keefe 2005). Cephennodes araiorum was found under stones on a stony/ sandy beach on the Pacific coast of Honshu, central Japan. It is the only member of the genus known from seashores, the others having been found in leaf litter and rotten deciduous wood at inland sites (Jałoszyński 2003). J. H. Frank & Kee-Jeong Ahn / ZooKeys 107: 1–98 (2011) 54 STAPHYLININI This tribe contains 200 genera worldwide (Newton et al. 2001). Those with coastal species are Thinopinus, Hadrotes, Hadropinus, Liusus, Thinocafius, Cafius, Remus, Phu- cobius, and Orthidus, which do not form a monophyletic group. This tribe contains 200 genera worldwide (Newton et al. 2001). Those with coastal species are Thinopinus, Hadrotes, Hadropinus, Liusus, Thinocafius, Cafius, Remus, Phu- cobius, and Orthidus, which do not form a monophyletic group. PHILONTHINA are a subtribe with five genera (Thinocafius, Cafius, Phucobius, Remus, and Orthidus) that appear to be restricted to seashores. Additionally one species each of Bisnius, Gabronthus and Philonthus are also confined to coastal habitats. The systematic position of Thinocafius is unclear and requires study in relation to the phylogeny of Cafius. 55 Coastal Staphylinidae (Coleoptera) Bisnius is moderately large genus with global distribution. Only one species (B. macies) appears to typically be found in drifted seaweed. It was originally described from Japan and was recently detected in Korea (Cho 2008). p y Cafius, in its early sense included also species now assigned to Remus. An attempt by Koch (1936) to divide it into subgenera was not completed for all species known at that time, and perhaps for that reason was not followed by some later authors. Later, Coiffait (1963, 1974) elevated one subgenus (Remus) to generic rank and proposed a new subgeneric name (Suborthidus) to include one species within the remaining spe- cies of Cafius. Coiffait’s effort to subdivide Cafius applied only to European, North African and Middle Eastern species, and was similarly not followed by some later au- thors. A global revision of the genus is necessary. In the present study 44 species are listed within Cafius (sensu stricto), making this the most species-rich genus of coastal staphylinids. One seemingly unusual species (C. splendoris Last 1987) is reported from Mt. Amigwiwa 1000–2300 m in Papua New Guinea and is excluded from our list. It may be misassigned to genus. Four species are attributed to Remus (see below). The zoogeographic distribution of species is as follows: Pacific Ocean 29, Atlantic Ocean 8, Indian Ocean 5, Pacific plus Atlantic 1, Pacific plus Indian 1. i pi p The larva of the European C. xantholoma was described by Paulian (1941). Larvae and pupae of the Pacific species, C. canescens, C. lithocharinus, C. luteipennis, and C. seminitens, were described by James et al. (1971). Subsequently Moore (1975) described the larva of C. STAPHYLININI sulcicollis, although Orth and Moore (1980) stated that it was actually the larva of C. bistriatus. More recently, Jeon and Ahn (2002, 2007) used DNA sequence data to associ- ate field-collected larvae with identified adults, and were able thus to identify and describe larvae of C. histrio, C. mimulus, C. rufescens, C. fucicola, C. nauticus, and C. vestitus from the Pacific. The European C. xantholoma is the best investigated species. Backlund (1945) compared the attractiveness of sterile leaves of various flowering plants versus sterile Fucus fronds and found that 26 adult specimens chose Fucus versus only four that chose other leaves. Cafius xantholoma is predacious as adult and larva, and is stenotopic in deep layers of seaweed beds (Backlund 1945). Observations of C. xantholoma larvae throughout the year (Backlund 1945; Egglishaw 1965) suggest that the species is multivoltine. Adults of C. xantholoma are highly resistant to wetting and may take flight directly from the water surface (Backlund 1944), and adults of C. bistriatus show the same ability to avoid wetting and drowning (Frank et al. 1986). On the Pacific coast, mass flights of beetles including C. luteipennis have been observed and generally are in a direction parallel to the shore (Leech and Moore 1971; Evans 1980). We are unaware of reports of brachyptery among Cafius species. The distribution of Cafius species along the coast of the United States from New Jersey south to Florida and the Gulf of Mexico is determined by the abundance of drifted algae (not drifted seagrasses or marshgrasses), and some algae, for example Sargassum fluitans Borgessen, appears not to provide suitable habitat (Frank et al. 1986). The wider distribution of species originally described from the West Indies is still not determined although Frank (1985a) reported C. caribeanus from the South American mainland, and C. subtilis from Florida (USA). There are also questions about the relation- ship between C. subtilis and C. aguayoi (described from Massachusetts, USA), and of these J. H. Frank & Kee-Jeong Ahn / ZooKeys 107: 1–98 (2011) 56 to the European species now known as Remus sericeus. A report of C. xantholoma from Chile was later shown to be based on misidentification. Most species of Cafius are found on the Pacific Ocean shores, and only one species (C. bistriatus) occurs both there and on the Atlantic. STAPHYLININI It dwells under rocks and in piles of drifted algae on seashores (Coiffait 1974). Philonthus nudus was for many years misattributed to Cafius, perhaps because of its atypical (for the genus Philonthus) coastal habitat. Its populations are distributed on the coasts of Korea, Japan, and the Kuril Islands of Russia, to the Pacific coasts of Canada and the United States, typically in drifted seaweed. Philonthus is a genus of over 1,000 species. f Philonthus nudus was for many years misattributed to Cafius, perhaps because of its atypical (for the genus Philonthus) coastal habitat. Its populations are distributed on the coasts of Korea, Japan, and the Kuril Islands of Russia, to the Pacific coasts of Canada and the United States, typically in drifted seaweed. Philonthus is a genus of over 1,000 species. yp y g p Phucobius contains eight species, of which seven are found under seaweed on beaches. The eighth (P. africanus) was reported from a single specimen collected at 1600 m in western Usambara, Tanzania. The collection locality has been confused, or perhaps the species belongs to another genus. Six species inhabit shorelines of the western Pacific (including the East Sea, South China Sea, and Java Sea, but not farther north than Japan), and one is found the shores of the Indian Ocean. Sharp (1874) recorded P. simulator as common under seaweed at Amakusa and Iwosima, Japan. Remus contains four species, all found in drifted seaweed on coastlines. One species (R. sericeus) occurs on coasts of the North Atlantic, Baltic, Irish, North, Mediterranean, and Black seas and, remarkably, in Australia. Another species (R. filum) is confined to the coasts of the Mediterranean and Black seas. Another species (R. pruinosus) is known from coasts of the North Atlantic, North, and Mediterranean seas. Another species (R. coralli- cola) is known from the South Pacific, South China, and Java seas, the Indian Ocean, and the Red Sea. The larva and pupa of R. sericeus were described and illustrated by Paulian (1941). Bierig (1934a) did not record R. pruinosus from Cuba as listed by Peck (2005). On a sandy beach in Somalia, adults of two Remus species (R. corallicola and R. filum), were trapped more abundantly in the dry season than in the wet (Chelazzi et al. 1983). Thinocafius contains only one known species, T. insularis, which is known only from the Chatham Islands east of New Zealand. STAPHYLININI It is believed to have colonized Atlantic coasts from the Pacific, and the two populations have been distinguished morphologically at the subspecific level (Frank et al. 1986). On a sandy beach in Somalia, adults of three Cafius species (C. fonticola, C. nauticus, and C. raggazzii), were trapped more abundantly in the dry season than in the wet, and were more active at night in the dry season than in the wet (Chelazzi et al. 1983). g y Gabronthus was recognized as a genus distinct from Philonthus in 1955. It contains over 30 species; only one of these, G. maritimus, found in the Mediterranean, the Red Sea, the Indian Ocean, and the South China Sea, appears to be restricted to coastlines. A report of G. maritimus from Cuba is highly questionable and without voucher speci- mens so has not been accepted in this study. p y Orthidus contains only one species (O. cribratus) found on the Mediterranean coasts of southern Europe and northern Africa, and Atlantic coasts from Brittany (France) south to Morocco. It dwells under rocks and in piles of drifted algae on seashores (Coiffait 1974). Philonthus nudus was for many years misattributed to Cafius, perhaps because of its atypical (for the genus Philonthus) coastal habitat. Its populations are distributed on the coasts of Korea, Japan, and the Kuril Islands of Russia, to the Pacific coasts of Canada and the United States, typically in drifted seaweed. Philonthus is a genus of over 1,000 species. Phucobius contains eight species, of which seven are found under seaweed on beaches. The eighth (P. africanus) was reported from a single specimen collected at 1600 m in western Usambara, Tanzania. The collection locality has been confused, or perhaps the species belongs to another genus. Six species inhabit shorelines of the western Pacific (including the East Sea, South China Sea, and Java Sea, but not farther north than Japan), and one is found the shores of the Indian Ocean. Sharp (1874) recorded P. simulator as common under seaweed at Amakusa and Iwosima, Japan. Orthidus contains only one species (O. cribratus) found on the Mediterranean coasts of southern Europe and northern Africa, and Atlantic coasts from Brittany (France) south to Morocco. It dwells under rocks and in piles of drifted algae on seashores (Coiffait 1974). y p ( ) of southern Europe and northern Africa, and Atlantic coasts from Brittany (France) south to Morocco. STAPHYLININI QUEDIINA are a subtribe containing one genus (Quediocafus) that appears to be restricted to seashores. One or more members of two other genera (Heterothops and Quedius) may or may not be restricted to seashores. 57 Coastal Staphylinidae (Coleoptera) Heterothops is a widespread genus. Only one species, the North American H. asper- atus, appears to be confined to Pacific seashores. The European H. binotatus (Graven- horst) is often but not exclusively found on seashores and so is not included. Quediocafus is known only from New Zealand, and all three species appear con- fined to sea beaches. Marris (2000) mentioned that Q. insolitus was found “under tus- sock mats and among coastal vegetation”. Quedius is a large and widespread genus apparently without any coastal specialists. Quedius simplicifrons Fairmaire (1861), which has several synonyms and is widely dis- tributed at and near the coasts of western Europe and adjacent islands, does not meet our definition of a strictly coastal species. Quedius umbrinus Erichson (1839a), which Mjöberg (1906) found to be the most abundant insect in drifted seaweed at Bohuslän in southwestern Sweden, is not included because it also occurs in inland habitats. STAPHYLININA are a subtribe containing the strictly coastal genera Thinopinus, STAPHYLININA are a subtribe containing the strictly coastal genera Thinopinus, Hadrotes, Hadropinus, and Liusus, as well as genera that are not confined to seashores. STAPHYLININA are a subtribe containing the strictly coastal genera Thinopinus, Hadrotes, Hadropinus, and Liusus, as well as genera that are not confined to seashores. Thinopinus pictus, occupying sandy Pacific beaches of North America from Alaska south to Baja California, has adults that are mottled dark and pale (‘melanic’) on dark sand beaches, but pale on pale sand beaches (Malkin 1958). Malkin (1958) pointed out that adults are nocturnal, spending the day concealed, and was perplexed as to why there should be two color forms. On beaches near Goleta (California, USA), adults and larvae are present throughout the year; dissected females contained 2–3 eggs, which are laid singly beneath the sand and hatch in ~ 14 days (Craig 1970). These eggs are white and ~ 3.0 mm long and 2.2 mm wide. Larval development time was not determined, but larvae were observed at night running on the sand sur- face like the adults (Craig 1970). Discussion Many kinds of insects can be found, some of them dead or dying in sea drift on shore- lines. Many are winged insects that have alighted on the sea surface and have subse- quently been washed up on the shore. Non-coastal species may exploit such concentra- tions of food. The senior author observed a pile of lawn grass clippings on a beach in Guanacaste, Costa Rica, that produced an abundance of staphylinids, none of them coastal species. Thus, would-be collectors of coastal staphylinids may be misled by the mere presence of staphylinids on seashores — a knowledge of non-coastal genera is es- sential. Coastal species represent only about 0.7% all Staphylinidae, although almost 400 species of them are known. Some representative species are shown in Figures 6–8. Aleocharinae are the most species rich of the eight subfamilies with coastal rep- resentatives (Fig. 9). The Pacific Ocean has many more species of coastal staphylinids than do the other oceans (Fig. 10). The United States has more species of coastal staphylinids than do eight other countries (Fig 11). Many kinds of insects can be found, some of them dead or dying in sea drift on shore- lines. Many are winged insects that have alighted on the sea surface and have subse- quently been washed up on the shore. Non-coastal species may exploit such concentra- tions of food. The senior author observed a pile of lawn grass clippings on a beach in Guanacaste, Costa Rica, that produced an abundance of staphylinids, none of them coastal species. Thus, would-be collectors of coastal staphylinids may be misled by the mere presence of staphylinids on seashores — a knowledge of non-coastal genera is es- sential. Coastal species represent only about 0.7% all Staphylinidae, although almost 400 species of them are known. Some representative species are shown in Figures 6–8. Aleocharinae are the most species rich of the eight subfamilies with coastal rep- resentatives (Fig. 9). The Pacific Ocean has many more species of coastal staphylinids than do the other oceans (Fig. 10). The United States has more species of coastal staphylinids than do eight other countries (Fig 11). Polyphyly of coastal Staphylinidae Lawrence and Newton (2000) and Newton et al. (2001) recognized four lineages among the Staphylinidae, and included among them the Pselaphinae and Scydmae- ninae (Fig. 1). All four lineages contain genera and species that are restricted to coast- al habitats. Coastal genera and species are found in eight subfamilies which contain mainly non-coastal genera. No subfamily contains only coastal genera. It is not until the level of tribe that there are taxa—all of them in the Aleocharinae—that include exclusively coastal genera. g The evolution of coastal genera in the Staphylinidae is polyphyletic. They have arisen from non-coastal ancestors among eight subfamilies. Furthermore, many coastal species belong to genera that include non-coastal species. Thus, we should expect a diversity of structural, physiological, and behavioral adaptations among them. STAPHYLININI On the western shore of Vancouver Island (British Columbia, Canada), the main prey is Orchestia californiana (Brandt), an amphipod which spends the day in temporary burrows on the upper part of the beach and is ambushed at night by the adult beetles (Richards 1982). The activity pattern of the amphipod is adjusted to weather and tidal patterns, and on some nights it is active after midnight, whereas beetles are active soon after dark; thus, beetles often foraged when amphipods were inactive (Richards 1983). Beetles attacked 0.147 amphipods per minute and captured 9.1% of prey attacked (Richards 1984). Color photographs of a larva (15 mm long) and adults of the two color forms (16–18 mm long) were provided by Evans (1980). p Hadrotes crassus lives on the Pacific coasts of North America, and is found from Alaska south to Baja California. Its larva was described by Moore (1964b) and color photographs of adult (11–17 mm) and larva (14–16 mm) are provided by Evans (1980); adults and larva are nocturnal predators of crustaceans and insects. A species described from New Zealand as Hadrotes wakefieldi may belong to another, possibly undescribed, genus (Klimaszewski et al. 1996). Hadropinus fossor is an old-world representative of the subtribe, inhabiting the shores of northern Japan and of Sakhalin Island (Russia). It makes burrows in sand under seaweed (Sharp 1889). J. H. Frank & Kee-Jeong Ahn / ZooKeys 107: 1–98 (2011) J. H. Frank & Kee-Jeong Ahn / ZooKeys 107: 1–98 (2011) 58 The Pacific ocean as the main evolutionary center Selecting only those genera that are exclusively coastal and separating them into three groups according to their provenance, yields the following: Coastal genera from the Pacific only: 40 From the Pacific and elsewhere: 14 From anywhere except the Pacific: 10 The Pacific Ocean (including its various seas such as the East Sea and South China Sea) is clearly the major cradle of coastal Staphylinidae. Coastal Staphylinidae (Coleoptera) Coastal Staphylinidae (Coleoptera) 59 Figure 6. Habitus photographs. A Bryobiota bicolor, 2.8 mm B Bryothinusa koreana, 3.2 mm C Diaulota aokii, 2.6 mm D Diglotta sinuaticollis, 3.0 mm E Heterota sunjaei, 2.4 mm F Liparocephalus cordicollis, 4.3 mm G Myrmecopora simillima, 3.1 mm H Oreuryalea watanabei, 4.9 mm I Phytosus balticus, 2.7 mm J Pontomalota opaca, 3.6 mm K Psammostiba hilleri, 5.3 mm L Tarphiota geniculata, 2.3 mm. Figure 6. Habitus photographs. A Bryobiota bicolor, 2.8 mm B Bryothinusa koreana, 3.2 mm C Diaulota aokii, 2.6 mm D Diglotta sinuaticollis, 3.0 mm E Heterota sunjaei, 2.4 mm F Liparocephalus cordicollis, 4.3 mm G Myrmecopora simillima, 3.1 mm H Oreuryalea watanabei, 4.9 mm I Phytosus balticus, 2.7 mm J Pontomalota opaca, 3.6 mm K Psammostiba hilleri, 5.3 mm L Tarphiota geniculata, 2.3 mm. J. H. Frank & Kee-Jeong Ahn / ZooKeys 107: 1–98 (2011) 60 Figure 7. Habitus photographs. A Omalium laeviusculum, 4.3 mm B Giulianium alaskanum, 2.6 mm C Prosthecarthron sauteri, 1.9 mm D Bledius fenyesi, 4.7 mm E Medon prolixus, 4.6 mm F Bisnius macies, 8.3 mm G Orthidus cribratus, 12.2 mm H Philonthus nudus, 8.1 mm I Hadropinus fossor, 21.8 mm J Had- rotes crassus, 13.6 mm K Liusus hilleri, 15.4 mm L Liusus humeralis, 14.0 mm M Phucobius simulator, 9.8 mm N Remus corallicola 5 5 mm O Remus sericeus 6 8 mm Figure 7. Habitus photographs. A Omalium laeviusculum, 4.3 mm B Giulianium alaskanum, 2.6 mm C Prosthecarthron sauteri, 1.9 mm D Bledius fenyesi, 4.7 mm E Medon prolixus, 4.6 mm F Bisnius macies, 8.3 mm G Orthidus cribratus, 12.2 mm H Philonthus nudus, 8.1 mm I Hadropinus fossor, 21.8 mm J Had- rotes crassus, 13.6 mm K Liusus hilleri, 15.4 mm L Liusus humeralis, 14.0 mm M Phucobius simulator, 9.8 mm N Remus corallicola, 5.5 mm O Remus sericeus, 6.8 mm. Coastal Staphylinidae (Coleoptera) 61 Coastal Staphylinidae (Coleoptera) 61 Figure 8. Habitus photographs. A Cafius australis, 15.4 mm B C. bistriatus, 7.7 mm C C. The Pacific ocean as the main evolutionary center histrio, 8.5 mm D C. lithocharinus, male, 9.6 mm E C. lithocharinus, female, 9.3 mm F C. litoreus, 13.9 mm G C. luteipennis, 9.2 mm H C. mimulus, 8.0 mm I C. pacificus, 9.7 mm J C. quadriimpressus, 17.9 mm K C. rufescens, 6.2 mm L C. seminitens, 13.6 mm M C. sulcicollis, 7.5 mm N C. xantholoma, 7.8 mm O Thi-i Coastal Staphylinidae (Coleoptera) Coastal Staphylinidae (Coleoptera) 61 Figure 8. Habitus photographs. A Cafius australis, 15.4 mm B C. bistriatus, 7.7 mm C C. histrio, 8.5 mm D C. lithocharinus, male, 9.6 mm E C. lithocharinus, female, 9.3 mm F C. litoreus, 13.9 mm G C. luteipennis, 9.2 mm H C. mimulus, 8.0 mm I C. pacificus, 9.7 mm J C. quadriimpressus, 17.9 mm K C. rufescens, 6.2 mm L C. seminitens, 13.6 mm M C. sulcicollis, 7.5 mm N C. xantholoma, 7.8 mm O Thi- nocafius insularis, 14.3 mm. J. H. Frank & Kee-Jeong Ahn / ZooKeys 107: 1–98 (2011) 62 Figure 9. The number of coastal Staphylinidae species within each subfamily. 1 22 23 187 79 1 8 71 0 20 40 60 80 100 120 140 160 180 200 Figure 9. The number of coastal Staphylinidae species within each subfamily. Coastal staphylinids have arisen within all four major lineages of the family, and even within these lineages they are polyphyletic. The simplest explanation is that the Pacific is oldest ocean (having arisen from the ancient ocean called Panthalassa). An al- ternative explanation might be that the shores of the Pacific are longer than the shores of other oceans, and somehow the species/area relationship (which has generally been supported for the staphylinid fauna of land masses) would suggest greater diversity on Pacific shores. A second alternative might be that non-coastal staphylinid faunas in lands surrounding the Pacific are more diverse than elsewhere and somehow had greater genetic plasticity. A third alternative might be that somehow the Pacific coasts have or had a special abundance of suitable habitat. The ten genera not represented in the Pacific are: European (Actocharis, Brund- inia, Pseudopasilia, and Phytosus), far South Atlantic (Chilodera and Acticola), Carib- bean (Briaraxis), Gulf of Mexico (Ophioomma), and South Atlantic and Indian Ocean (Pareiobledius and Chetocephalus). Briaraxis, Chilodera, Acticola and Ophioomma are monotypic and because there are few specimens with habitat information, it is not certain whether the latter three are truly coastal. The Pacific ocean as the main evolutionary center Europe, with four genera in the North Atlantic Ocean (and/or Mediterranean, Black, North, Irish, and Baltic seas) falls sec- ond to the Pacific Ocean in terms of precinctive genera.h i Actocharis and Phytosus are currently placed in the tribe Phytosini. This tribe in- cludes two other genera that have representative species in the Pacific. Unfortunately, this tribe requires revision and its monophyly is uncertain. Therefore, it is not clear whether Actocharis and Phytosus evolved from coastal ancestors that immigrated from the Pacific, or whether they evolved from non-coastal European ancestors. The discov- ery of a species of Phytosus in the Caribbean is discussed later. Coastal Staphylinidae (Coleoptera) 63 Figure 10. The number of coastal Staphylinidae species found in various oceans and seas. 2 74 38 185 12 9 11 48 37 0 20 40 60 80 100 120 140 160 180 200 Figure 10. The number of coastal Staphylinidae species found in various oceans and seas. Apart from whether Chilodera and Acticola are truly coastal, their attribution to the South Atlantic Ocean needs qualification because they are known only from the Falk- land Islands. Could they have a yet undetected distribution elsewhere? Another coastal genus, Crymus, has two species, distributed in the South Atlantic Ocean (Falkland Islands and South Georgia) and South Pacific Ocean (the Campbell and Auckland Is- lands of New Zealand) thus having a sub-Antarctic distribution. None of these genera extends farther north. Antarctic seas do not seem to have provided a conduit for migra- tion of less cold-hardy coastal staphylinids around Cape Horn or the Cape of Good Hope. Forty genera remain confined to the Pacific and have not been detected in the South Atlantic. Among the 14 genera that are known from the Pacific and elsewhere, only two (Halobrecta and Diglotta) have species in the South Atlantic: H. algophila, which may have arrived adventively via shipping activities, and D. brasiliensis. Even the Arctic Ocean has a partially coastal species (Micralymma brevilingue) whose congener, M. marina, is truly coastal and extends south through the Atlantic Ocean, east to the North and Baltic seas, and west to New England and Atlantic Canada. A third species (M. laticolle), also from the Arctic Ocean, does not belong to Micralymma (see Habits, Habitats, and Classificatory Notes), and should be trans- ferred to another genus. The Pacific ocean as the main evolutionary center A fourth species, in Eurasia, is not coastal.h The foregoing discussion suggests that almost all coastal genera of Staphylinidae evolved from non-coastal ancestors on the shores of the Pacific Ocean. Some appar- ently dispersed by migrating to the shores of other oceans. Geological constraints are outlined below. The primordial ocean surrounding Pangaea in the Permian was Panthalassa, gener- ally accepted as the direct antecedent of the Pacific Ocean (Scotese 2010). The breakup J. H. Frank & Kee-Jeong Ahn / ZooKeys 107: 1–98 (2011) 64 Figure 11. The number of coastal Staphylinidae species found in various countries. 28 37 33 57 32 39 79 0 10 20 30 40 50 60 70 80 90 Australia Canada Great Britain Japan Korea New Zealand USA Figure 11. The number of coastal Staphylinidae species found in various countries. of Pangaea into Laurasia and Gondwanaland began by formation of the Tethys Sea which was the antecedent of the Indian Ocean and the Red Sea. Earlier authors (1960s) included the Mediterranean Sea, but recent authors credit the Mediterranean as being a separate entity, perhaps part of the Neotethys ocean basin, formed ca. 200 mya. The separation of Laurasia continued during the Triassic with the beginning of the North Atlantic Ocean. In the Jurassic, the North Atlantic Ocean widened and the Tethys Sea began to narrow. Not until the Cretaceous did the South Atlantic Ocean began to widen. In the Eocene, the South Atlantic Ocean became connected to the South Pacific Ocean by opening of the Drake Passage between Antarctica and South America some 41 mya (Scher and Martin 2006). The Mediterranean Sea lost its connection to the Indian Ocean some 15 mya. About 7 mya, the Mediterranean lost its narrow connections to the North Atlantic, its waters evaporated and it became a salt desert. At 4.86–4.6 mya, the Strait of Gibraltar opened, and water poured in from the North Atlantic in an immense flood (Garcia-Castellanos et al. 2009). The North Atlantic Ocean lost a major connection to the Pacific Ocean by completion of the Central American isthmus 3.5–3.1 mya (Bartoli et al. 2005). This is the geological background against which the current distribution of coastal Staphylinidae may be understood. The Pacific ocean as the main evolutionary center It suggests that the Tethys Sea (and its extension, the Neotethys ocean basin), could have offered an early conduit from the Pacific via the Mediterranean to the Atlantic until the connection between the Mediterranean and Indian Ocean was lost, some 15 mya. However, between 7 and 4.8 mya, the Mediterranean became a salt desert and surely lost those parts of its coastal fauna that could not tolerate extreme salinity and aridity; the coastal Staphylinidae that had dispersed to the Atlantic could have survived, but other populations would have become extinct within the Mediterranean. [This ac- Coastal Staphylinidae (Coleoptera) 65 count would to some extent be altered if recent ideas of a closed Pacific Ocean in the upper Triassic to lower Jurassic (McCarthy 2003) become generally accepted, although those ideas may make current distribution of Pacific Staphylinidae easier to explain]. The width of the north Pacific ocean has not been a severe barrier Liparocephalini (Aleocharinae) include seven genera containing 24 species, and all species are coastal. Five genera inhabit the North Pacific Ocean only, and four of them have representatives on both sides of it (i.e., in Asia and in North America). However, only two of the 24 species, Diaulota aokii and Paramblopusa borealis, occur on the shores of both continents. This suggests that the width of the North Pacific Ocean has not been a severe barrier to dispersal, and that most dispersal occurred in the remote past, allowing subsequent speciation. p g q p Ahn et al. (2010) showed that the distribution of Liparocephalini was congruent with geological history, including the splitting of Pangaea and the isolation of the Palearctic from the Nearctic. They hypothesized that the ancestor of the tribe was distributed along the Panthalassan Ocean and repeated dispersals occurred into the Nearctic from the Palearctic. Although these insects, presumably with favorable currents and winds, may cross oceans, we must suspect that barriers are formed by intervening land masses. This sus- picion is heightened by dearth of records from inland localities, with just a few from California’s Salton Sea (which once was connected to the Gulf of California). Thus, the biogeography of coastal staphylinids differs greatly from that of non-coastal species. Northward dispersal from the Red sea to the Mediterranean The Red Sea may have been a conduit for dispersal from the Indian Ocean to the Mediterranean. If such dispersal did occur, a subsidiary question is whether it occurred recently, after the building of the Suez Canal, whether it occurred long before that and east or west of the Sinai Peninsula, or whether it occurred in the remote past before sea connections between the Red and Mediterranean seas closed ~15 mya. In this context it is worth noting that there is no evidence for dispersal of coastal staphylinids from the Indian Ocean via the South Atlantic to the North Atlantic. Bryothinusa peyerimhoffi (Aleocharinae: Myllaenini) is reported from the Red and Mediterranean seas.i Heterota (Aleocharinae: Homalotini) has three species in the Pacific, three in the Indian Ocean, two in the Red Sea, and one in the Mediterranean (and one, previously mentioned, distributed from the Mediterranean to the Caribbean). Current distribu- tion suggests that the Red Sea may have been an ancient conduit to the Mediterranean. Cameronium (Aleocharinae: Homalotini) contains three species in the Indian Ocean, one in the Red Sea, and one (C. liebmanni, not listed) at inland localities in Algeria, adjacent to the Mediterranean. Current distribution suggests that the Red Sea may have been an ancient conduit to the Mediterranean. Could C. liebmanni have found refuge at inland locations as the Mediterranean Sea became a salt desert ca. 7 mya? A species from the Gulf of California was attributed to this genus by Moore (1964a), but this assignment needs reassessment. Eastward dispersal from the Pacific to the Atlantic Cafius subgenus Euremus contains 10 species in the Pacific and a few others elsewhere. Because C. bistriatus has one subspecies (C. b. bistriatus) on the Caribbean and Atlantic coasts of North America, and one subspecies (C. b. fulgens) in the Gulf of California and on the Pacific shores of California and Baja California, it has been posited that it and the ancestor of C. rufifrons arrived in the Caribbean from the Pacific (Frank et al. 1986). If Central America is now an insuperable barrier to such dispersal, then the date of dispersal was probably before the connection of Central America to South America 3.5–3.1 mya. Aleochara litoralis (Aleocharinae: Aleocharini) occurs on the Atlantic coast of North i America from Florida north to Newfoundland and Quebec, as well as on the Pacific coast from California north to Alaska. There has been no report of specimens from the Canadian Arctic that could explain a natural dispersal. Westward dispersal across the Atlantic to the Caribbean Phytosus (Aleocharinae: Phytosini) has five European species, the range of two of which includes the Canary Islands; their presence on these islands suggests an ability to dis- perse. Three other species are known: one from the Azores, one from Senegal, and one from Guadeloupe in the Caribbean. Their ancestors are postulated here to have dis- persed from Europe in the remote past. The westward dispersal of the putative ancestor of P. caribeanus across the Atlantic at the latitude of the Caribbean could have been assisted by trade winds. Heterota (Aleocharinae: Homalotini) contains three species in the Pacific, three in the Indian Ocean, two in the Red Sea, and one in the Mediterranean. A tenth spe- cies (H. plumbea) inhabits the Atlantic coasts of Europe, the Mediterranean, and the Canary Islands. It has also been found in the Caribbean (Jamaica and the Yucatan peninsula of Mexico) and Florida (Frank and Thomas 1984b). Small winged insects adapted to a seashore existence could be prime candidates for transoceanic dispersal by the prevailing winds such as from southern Europe or northern Africa to the Canary Islands and thence to the Caribbean. J. H. Frank & Kee-Jeong Ahn / ZooKeys 107: 1–98 (2011) 66 The distribution of coastal species of non-coastal genera Selecting the coastal species belonging to non-coastal genera and placing them into the same categories as in preceding list yields the following: Coastal Staphylinidae (Coleoptera) 67 Coastal species of non-coastal genera from the Pacific only: 10 genera, 39 species From the Pacific and elsewhere: 3 genera, 4 species From anywhere except the Pacific: 13 genera, 35 species The genus Bledius is excluded from this compendium. Here, it can be seen that the numbers of genera and species from the Pacific only are almost identical to those from anywhere except the Pacific. The species included are such as Philonthus nudus and Heterothops asperatus (Pacific Ocean), Medon pocoferus (Mediterranean), M. marinus (Indian Ocean), M. prolixus (Japan), Sunius ferrugineus (Caribbean), and S. minutus (United States). They belong to genera that have many non-coastal species. Although they have evolved to occupy coastal habitats, their ana- tomical characters do not set them apart as belonging to genera that are distinct from the non-coastal ancestors. In other words, their adaptation to coastal habitats is prob- ably more recent than in genera that consist only of coastal species. The genus Bledius is a special case We do not include the genus Bledius in the above accounts because we believe it is a special case, with possibilities of overland dispersal during its evolution. Summary 1. Not all Staphylinidae found on seashores are coastal. . Not all Staphylinidae found on seashores are coastal. 2. Coastal Staphylinidae comprise <1% of all species of Staphylinidae. 3. Coastal Staphylinidae have arisen in eight of the subfamilies, are polyphyletic, and have diverse adaptations of structure, physiology, and behavior that allow them to exist in such habitats. 3. Coastal Staphylinidae have arisen in eight of the subfamilies, are polyphyletic, and have diverse adaptations of structure, physiology, and behavior that allow them to exist in such habitats. 4. There are far more strictly coastal genera in the Pacific than elsewhere and the Pa- cific appears to be the most important evolutionary center for this group. 4. There are far more strictly coastal genera in the Pacific than elsewhere and the Pa- cific appears to be the most important evolutionary center for this group.h i g 5. There are approximately as many coastal species (belonging to otherwise non- coastal genera) in the rest of the world as there are in the Pacific. i 5. There are approximately as many coastal species (belonging to otherwise non- coastal genera) in the rest of the world as there are in the Pacific. Effects caused by humans There can be few species that have not been affected by human activity, and coastal staphylinids are no exception. Teropalpus unicolor is native to New Zealand and now occurs adventively in Australia, South Africa, on the Pacific coast of North America, and the south and southwest coasts of Britain (Hammond 2000). Adota maritima (syn. Atheta immigrans) is known from the Pacific coast of North America and adventively from Britain (Easton 1971, Hammond 2000). The European Halobrecta flavipes is another likely candidate for a list of species dispersed by shipping because it has been detected on the coasts of Chile, Australia, and eastern Canada. Another European spe- cies recently detected on the Atlantic coast of Canada is Diglotta mersa (Klimaszewski et al. 2008). The building of seawalls for construction of marinas and docks, and for protec- tion of buildings from wave action, damages beaches and in some places may harm coastal staphylinid populations. The senior author remembers that drifted seaweed accumulated on a beach by the west end of a bridge at Ft. Pierce, Florida in the early 1970s and harbored Cafius species. Twenty years later, the area had become a marina with seawalls and without seaweed accumulations; very many undocumented changes are likely to have been caused by similar constructions elsewhere. Human enjoyment of beaches to some extent depends upon the cleanliness of the beaches, and humans have employed beach-sweeping machines to remove not only garbage but also ‘un- J. H. Frank & Kee-Jeong Ahn / ZooKeys 107: 1–98 (2011) 68 sightly’ drifted marine algae, which are the habitat and basis for food chains for some coastal staphylinids (Frank et al. 1986). An unusual sign at a public beach at Santa Barbara, California in summer 2009 (seen by the senior author) explained that sea- weed was not being cleared there because it was the habitat of invertebrate animals that serve as food for birds. Oil spills on beaches are surely harmful to staphylinid populations, although we have seen no documentation. The compaction of beaches by human activity (vehicular as well as crowds of people) may be detrimental to Staphylinidae. Recently much-discussed threats to coasts and oceans are a rise in sea level and acidification, but their potential effect on coastal staphylinid populations have not been discussed in print if at all. Acknowledgments When JHF began a literature search, he already had originals or copies of most of the cited publications in his personal library. In early summer 2001, he began to request interlibrary loans through the University of Florida libraries (thanks especially to Jan- ice Kaehler) for items he did not have and were not held by the University of Florida libraries. He thanks several friends for providing copies of pages of articles and books that the Interlibrary Loan Service of the University of Florida was unable to provide: Don Chandler (University of New Hampshire, Durham), Lanna Cheng (Scripps In- stitution of Oceanography, La Jolla), Terry Erwin (Smithsonian Institution, Washing- ton, DC), Peter Hodge (editor, The Coleopterist), Berit Pedersen (library of the Royal Entomological Society, London) and Lizbeth Gale (library of the Royal Botanic Gar- dens, Kew). He thanks Beverly Pope (library of the Division of Plant Industry, Florida Department of Agriculture and Consumer Services, Gainesville), for help in tracking correct citations. After 2001, compilation became very much easier for some subfami- lies due to the publication of Herman’s (2002) catalog, and because all of Zoological Record became searchable online. Meanwhile, KJA had accumulated references on the taxonomic groups that he had studied. He thanks S. Nomura (National Museum of Coastal Staphylinidae (Coleoptera) 69 Nature and Science, Tokyo), H. Hoshina (Fukui University, Fukui) and P. Jałoszyński (Poland) for providing valuable references. He is also grateful to members of the Chun- gnam National University Insect Collection (Daejeon) for taking habitus photographs and preparing figure plates. M.-J. Jeon (National Institute of Biological Resources, Incheon) provided us with many excellent habitus photographs of Cafius and related genera. Comments by Jan Klimaszewski (Natural Resources Canada, Québec) and Don Chandler significantly improved the manuscript, as did comments by an anony- mous reviewer. KJA dedicates this paper to the late Steve Ashe, who provided the inspi- ration of studying the systematics and evolution of the seashore inhabiting staphylinid beetles. This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2009-0073111). Nature and Science, Tokyo), H. Hoshina (Fukui University, Fukui) and P. Jałoszyński (Poland) for providing valuable references. He is also grateful to members of the Chun- gnam National University Insect Collection (Daejeon) for taking habitus photographs and preparing figure plates. M.-J. Acknowledgments Jeon (National Institute of Biological Resources, Incheon) provided us with many excellent habitus photographs of Cafius and related genera. Comments by Jan Klimaszewski (Natural Resources Canada, Québec) and Don Chandler significantly improved the manuscript, as did comments by an anony- mous reviewer. KJA dedicates this paper to the late Steve Ashe, who provided the inspi- ration of studying the systematics and evolution of the seashore inhabiting staphylinid beetles. 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In: Kiesenwetter EAH von, Kirsch T, Die Kä- ferfauna der Auckland-Inseln nach Herm. Krone’s Sammlungen beschrieben. Deutsche Entomologische Zeitschrift 21: 160–164. Kincaid T (1961a) The ecology and morphology of Thinobius frizzelli Hatch, an intertidal beetle. Calliostoma Co., Seattle, 12 pp. + 6 pls. Kincaid T (1961b) The staphylinid genera Pontomalota and Thinusa. Calliostoma Co., Seattle, 10 pp. + 4 pls. King PE, Fordy M, Al-Khalifa MS (1979) Observations on the intertidal Micralymma marinum (Stroem) (Col., Staphylinidae). Entomologist’s Monthly Magazine 115: 113–136. Kirschenblatt JD (1938). On some Far Eastern staphylinid beetles [in Russian]. Trudy Gid- robiologicheskoi Ekspeditsii Zoologicheskogo Instituta Akademii Nauk v 1934 godu na Yaponskoe More 1: 527-532. Izdatelstvo Akademii Nauk USSR; Moscow. [p. 533-536 of the same volume have a condensed version of the same article, in English]. We use the spelling of his family name and initials as given on p. 533. 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https://openalex.org/W4254306722	https://www.emerald.com/insight/content/doi/10.1108/mi.2018.7806/full/pdf?title=asian-mental-health-and-use-of-drama-therapy-for-acculturative-family-distancing-in-immigrant-families	English	null	Asian mental health and use of drama therapy for acculturative family distancing in immigrant families	Mental illness	2,018	cc-by	1,083	Asian mental health and use of drama therapy for acculturative family distancing in immigrant families This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0). This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0). During each event, members act out scenarios of family conflict (bad grades, drugs, self-harm, sexuality) with a dose of comedy to lighten the mood. A moderator then leads a discussion with the audience to discuss the conflict at hand. Lastly, the team performs an improved rendition of the same skit using solutions from the discussion. Throughout the workshop, we alleviate the stigma surrounding mental health and remain entirely open to the audience, stress- ing that it is both normal and necessary for parents to talk about anxiety, depression, and more. Additionally, Asian populations have somaticized psychiatric illnesses, persever- ating on physical symptoms rather than a potential emotional source. Eastern popula- tions, on the other hand, have been shown to be much more likely to consult mental health professionals for their emotional concerns.2,3 ©Copyright B.R. Sampathi, 2018 Licensee PAGEPress, Italy Mental Illness 2018; 10:7806 doi:10.4081/mi.2018.7806 ©Copyright B.R. Sampathi, 2018 Licensee PAGEPress, Italy Mental Illness 2018; 10:7806 doi:10.4081/mi.2018.7806 Mental Health Services. Palo Alto, CA: Science and Behavior Books: 1983. pp. 212-31. It has also been documented that some Asian families believe seeking psychiatric help brings shame to one’s family. Rather, they often believe one should be able to exercise individual willpower and avoid negative thoughts.4 Concern for stigmatiza- tion has also led some Asian families to hide psychiatrically ill family members at home until severe and damaging psychotic events occur.5 3. Sue S, Morishima JK. The mental health of Asian Americans. San Francisco: Jossey-Bass; 1982. It will be interesting to monitor AFD as first-generation children become parents. It is possible that the children of these first- generation families, like myself who have lived through these experiences, may be more equipped to address these issues as they grow older. Furthermore, with the rise in globalization both economically and cul- turally, it is exceedingly possible that future immigrants may be from regions so advanced that cultural disparities across the globe diminish. 4. Root MP. Guidelines for facilitating therapy with Asian American clients. Psychotherapy 1985;22:349-56. 5. Lin TY, Tardiff K, Donetz G, Goretsky W. Ethnicity and patterns of help seek- ing. Cult Med Psychiatry 1978;2:3-13. Mental Illness 2018; volume 10:7806 Asian mental health and use of drama therapy for acculturative family distancing in immigrant families First generation Asian Americans are more accepting of mental illness and are more likely to seek psychiatric help due to increased acculturation to the Western world.6 However, the cultural gap between Asian Americans and their immigrant par- ents continues to exist. Acculturative family distancing (AFD) occurs when an immi- grant family attempts to unite distinct cul- tural worlds. Parents find it hard to create space for new beliefs on mental health, for example, and conflicts consequently arise as families struggle to maintain a collabora- tive bicultural identity.7 A study in 2010 indicated that children in families with greater AFD were associated with a higher risk for clinical depression and depressive symptoms.8 6. Atkinson DR, Gim RH. Asian American cultural identity and attitudes toward mental health services. J Couns Psychol 1989;36:209-12. 7. Birman D, Poff M. Immigration. Intergenerational Differences in Acculturation. 2011. Available from: http://www.child-encyclopedia. c o m / i m m i g r a t i o n / a c c o r d i n g - experts/intergenerational-differences- acculturation Asian mental health and use of drama therapy for acculturative family distancing in immigrant families Research has shown that drama therapy has been quite effective in helping treat var- ious psychiatric illnesses. To date, there has been minimal documentation on workshops using drama therapy to address AFD in Asian families. The Stanford Communi - cation Health Interactive for Parents and Others (CHIPAO) is a group of Asian American attendings, residents, and stu- dents at the Stanford School of Medicine that perform interactive skits designed to address and minimize AFD in Asian American families with immigrant parents. This initiative was built after two separate youth suicide clusters that involved mainly Asian American males in Palo Alto, California from 2009 to 2015. Mental Health Services. Palo Alto, CA: Science and Behavior Books: 1983. pp. Correspondence: Bharat Reddy Sampathi, UC Irvine School of Medicine, 1001 Health Sciences Rd, Irvine, CA 92617, USA. Tel.: +1.650.823.3426. E-mail: bsampath@uci.edu Key words: Asian populations, mental health, immigrant family, therapy. Conflict of interest: the author declares no potential conflict of interest. Funding: none. Received for publication: 2 August 2018. Accepted for publication: 2 August 2018. This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0). ©Copyright B.R. Sampathi, 2018 Licensee PAGEPress, Italy Mental Illness 2018; 10:7806 doi:10.4081/mi.2018.7806 Mental Health Services. Palo Alto, CA: Science and Behavior Books: 1983. pp. 212 31 Correspondence: Bharat Reddy Sampathi, UC Irvine School of Medicine, 1001 Health Sciences Rd, Irvine, CA 92617, USA. Tel.: +1.650.823.3426. E-mail: bsampath@uci.edu Key words: Asian populations, mental health, immigrant family, therapy. Conflict of interest: the author declares no potential conflict of interest. Funding: none. Received for publication: 2 August 2018. Accepted for publication: 2 August 2018. This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0). ©Copyright B.R. Sampathi, 2018 Licensee PAGEPress, Italy Mental Illness 2018; 10:7806 doi:10.4081/mi.2018.7806 Bharat R. Sampathi UC Irvine School of Medicine, Irvine, CA, USA Key words: Asian populations, mental health, immigrant family, therapy. Conflict of interest: the author declares no potential conflict of interest. For centuries, Asian populations have struggled with obtaining and maintaining treatment for psychiatric illnesses when compared to their European counterparts. A large part of this issue stems from cultural values and beliefs regarding mental illness. Firstly, psychotherapy may be seen as a weakness and breech of familial privacy.1 Funding: none. Received for publication: 2 August 2018. Accepted for publication: 2 August 2018. Received for publication: 2 August 2018. Accepted for publication: 2 August 2018. References 1. Lin T, Lin, M. Service delivery issues in Asian North American communities. Am J Psychiatry 1978;135:454-7. 8. 8. Hwang WC, Wood JJ, Fujimoto K. Acculturative Family Distancing (AFD) and Depression in Chinese American Families. J Consult Clin Psychol 2010;78:655-67. 2. Brown TR, Stein KM, Huang K, Harris DE. Mental illness and the role of men- tal health facilities in Chinatown. In: Sue S, Wagner N (Eds.). Asian Americans: Psychological P1: GDX [Mental Illness 2018; 10:7806] [page 35] [page 35]
W4392973251.txt	https://www.qeios.com/read/773MFI/pdf	en		Definition of Vespidae (Insecta: Hymenoptera).	Definitions	2,024	cc-by	1,816	Qeios, CC-BY 4.0 · Definition, March 20, 2024 Open Peer Review on Qeios Definition of Vespidae (Insecta: Hymenoptera). Carlos Henrique Marchiori1 1 Instituto Federal Goiano Potential competing interests: No potential competing interests to declare. Co-authors: Dr. Marcus Vinícius de Oliveira Santana1 and Klebert de Paula Malheiros2. *Instituto Marcus Vinícius de Oliveira Santana1-2. Goiânia, Goiás, Brazil. Definition: Social wasps are insects of the order Hymenoptera, superfamily Vespoidea, family Vespidae, and belonging to the subfamily Polistinae. These insects are popularly known as "marimbondos" and are generally referred to in the specialized literature as vespids. This family builds nests in different ways, using different types of materials, with plant fiber being the main component of the cell. True wasps or hornets are the largest eusocial wasps and are similar in appearance to their close relatives, the yellow wasps Dolichovespula arenaria (Fabricius, 1775). Some species can reach up to 5.5 cm in length. They are distinguished from other Vespinae by the relatively large upper border of the head and the rounded segment of the abdomen just behind the waist. Worldwide, 22 species of wasps are recognized. Most species occur only in the Asian tropics, although the European hornet is widely distributed across Europe, Russia, North America, and Northeast Asia (Figure 1) [1-4]. Qeios ID: 773MFI · https://doi.org/10.32388/773MFI 1/7 Qeios, CC-BY 4.0 · Definition, March 20, 2024 Figure 1. A., B. & C.: Dorsal, lateral & frontal view of Vespa vivax Smith, 1870; D., E. & F.: Dorsal, lateral and frontal view of Vespa velutina variana Vecht, 1957; G., H. & I.: Dorsal, lateral & frontal view of Vespa fumida van der Vecht, 1956. Source: http://zoobank.org/References/02867802-959C-46A4-BB1D-00685757D92F. Morphology: Intersegmental lines are recognizable on the dorsal and ventral sides of the body, but somewhat indistinct laterally. To some extent, the first and second abdominal segments are lobed ventrally, forming the trophopod. The last segment has the anal opening. Abdominal spiracles exist in the first eight segments, toward the anterior edge of each segment. The head in frontal view is almost rounded and anteroposteriorly compressed. The shape of the head is somewhat variable and is expressed by the use of cranial width. The antenna is a small, slightly swollen circular area, conical in shape, surrounded by a thick ring of cuticle. Each antenna has a sensilla arranged in a straight line or triangle. There are thirteen postcephalic segments. The larva is whitish, soft, and entirely devoid, with few notable structures [510]. The body consists of a yellow, well-criticized head, three thoracic and ten abdominal segments, with no constriction between the thorax and abdomen. In the tropics, these nests can last year-round, but in temperate areas, nests die out, with solitary queens hibernating in burlap or other insulating material until spring. Male hornets are docile and do not have stingers. True wasps are often considered pests as they aggressively protect their nesting sites when threatened and their stings can be more dangerous than bees. Like other social wasps, true wasps built communal nests, usually composed of chewed wood and exposed in trees and bushes; some species, such as Vespa orentalis Linnaeus, 1771, built their nests underground or in other cavities. Each nest has a queen, who lays eggs and is assisted by workers who, although female, are infertile. Colonies can be started by associations of various females (pleometrosis) or by a single female founder (haplometrosis) (Figure 2) [11-18]. Qeios ID: 773MFI · https://doi.org/10.32388/773MFI 2/7 Qeios, CC-BY 4.0 · Definition, March 20, 2024 Figure 2. The dynamic interplay between multiple levels of biological organization, from genes to hormones to physiological systems, can affect social organization in paper wasps. Sources: Wasp drawings adapted from Hunt et al. (2011) and https://api.semanticscholar.org/CorpusID:54649525. Instead of consuming the prey directly, members of the Vespidae family chew it and feed it to the larvae, producing a clear-looking liquid with a high amino acid content that serves as food for the adults. The exact composition of this fluid varies from species to species, but it appears to contribute significantly to adult nutrition. Each wasp colony includes a queen and several workers with varying degrees of sterility relative to the queen. In species from temperate regions, Qeios ID: 773MFI · https://doi.org/10.32388/773MFI 3/7 Qeios, CC-BY 4.0 · Definition, March 20, 2024 colonies exist for one year, perishing at the beginning of winter. New queens and males are produced in late summer, and after mating, the queen hibernates in sheltered locations. Hornets have stingers used to kill prey and defend nests. Wasp stings are more painful to humans than typical wasp stings because wasp venom contains a large amount (5%) of acetylcholine. Individual wasps may sting repeatedly; Unlike bees, wasps do not die after stinging because their stingers are very barbed visible only at high magnification, and can be easily ripped off, so they are not ripped from their bodies when they detach. (Figure 3) [18-23]. Figure 3. Nests of social wasps of the subfamily Polistinae (Vespidae). A: Nest ofMischocyttarus sp. with exposed brood cells; B: Nest of Polybia (Myrapetra) bistriata (Fabricius, 1804), with an envelope to protect the brood cells; C: Nest of Qeios ID: 773MFI · https://doi.org/10.32388/773MFI 4/7 Qeios, CC-BY 4.0 · Definition, March 20, 2024 Angiopolybia pallens (Lepeletier, 1836) with an envelope to protect the brood cells; D: Nest ofPolistes canadensis canadensis (Linnaeus, 1758) with exposed brood cells. Sources: Photos: M. Aragão and https://www.researchgate.net/figure/Figure-1-Ninhos-de-vespas-sociais-da-subfamiliaPolistinae-Vespidae-A-Ninho-de_fig1_312488169https://www. researchgate.net/figure/Figure-1-Social-wasp-nests-of-thesubfamily-Polistinae-Vespidae-A-Ninho-de_fig1_312488169. The toxicity of wasp stings varies depending on the wasp species; some cause just a typical insect bite, while others are among the most venomous insects known. Isolated wasp stings are not fatal in themselves, except sometimes for allergic victims. Mandarin wasps are predators of bees and release attack pheromones to attract other mandarin wasps to the hive found. Hornets, like many social wasps, can mobilize their entire nest to defend themselves, which is highly dangerous to humans and other animals. The alert pheromone is usually released in the event of a threat to the nest. In the case of the mandarin wasp, whose main prey is bees, the pheromone trail serves to attract other mandarin wasps to the hive found. In field tests, 2-pentanol alone provoked mild alarm and defensive behavior, but the addition of the other two compounds increased aggression in a synergistic effect. In the European wasp, the main alert pheromone compound. If a wasp is killed near a nest, it can release pheromones that can cause other wasps to attack. Materials that come into contact with these pheromones, such as clothing, skin, and dead prey can trigger an attack, as can certain food scents, such as banana and apple scents, and fragrances that contain C5 alcohols and C10 esters. [24-28]. References [1] Danforth BN, Fang J, Sipes SD. Analysis of family-level relationships in bees (Hymenoptera: Apiformes) using 28S and two previously unexplored nuclear genes: CAD and RNA polymerase II. Molecular Phylogenetics and Evolution. 2006; 39: 358-372. [2] Celary W. The ground-nesting solitary bee, Dasypoda thoracica Baer, 1853 (Hymenoptera: Apoidea: Melittidae) and its life History. Folia Biologica. 2002; 50(3-4): 191-198. [3] Beatty R, Beer A, Deeming C. The book of nature. Great Britain. 1st ed. Minneapolis: University of Minnesota. 2010. [4] Dorji P, Klein W, Nidup T. Taxonomic study of social vespid wasps (Hymenoptera: Vespidae: Vespinae & Polistinae) in Bhutan. Journal of Insect Biodiversity and Systematics. 2017; 3(2): 91–104. [5] Barbier Y, Rasmont P. Map fauna-flora. User manual. 1st ed. Mons: University of Mons-Hainaut. 2000. [6] Banaszak J, Wendzonka J. Bees (Hymenoptera: Apoidea) of the Bory Tucholskie National Park (NW Poland). Polish Writing Entomological. 2002; 71: 327-350. [7] Barbier Y, Rasmont P, Dufrêne M, Sibert JM. Data fauna flora. User guide. 1st ed. Mons: University of Mons-Hainaut. 2000. [8] Waser NM, Ollerton J. Plant-Pollinator interactions. 1st ed. Chicago: University of Chicago Press. 2006. Qeios ID: 773MFI · https://doi.org/10.32388/773MFI 5/7 Qeios, CC-BY 4.0 · Definition, March 20, 2024 [9] Cane JH, Sipes SD. Characterizing floral specialization by bees: analytical methods and a revised lexicon for oligolecty. In: Waser NM, Ollerton J, eds. Specialization and generalization in plant-pollinator interactions. 1st ed. Chicago: University of Chicago Press; 2006. p. 99-122. [10] Michez D, Patiny S. World revision of the oil-collecting bee genusMacropis Panzer 1809 (Hymenoptera, Apoidea, Melittidae) with a description of a new species from Laos. Annals of the Society Entomological of France. 2005; 41: 1528. [11] Michener CD. The bees of the world. 1st ed. Baltimore: The Johns Hopkins University Press. 2000. [12] Michez D, Patiny S, Iserbyt S. Remarkable Apoidea observed in Pyrénées-Orientales, France (Hymenoptera, Melittidae). Bulletin of the Entomological Society of France. 2004; 109: 379-382. [13] Cox BC, Moore PD. Biogeography. An ecological and evolutionary approach. 70 st ed. Oxford: Blackwell Publishing. 2005. [14] Wu YR. Hymenoptera, Melittidae & Apidae. 1st ed. Beijing: Academia Sinica. 2000. [15] Whitehead VB, Steiner KE. Oil-collecting bees of the winter rainfall area of South Africa (Melittidae,Rediviva). Annals of the South African Museum. 2001; 108: 143-277. [16] Nilsson LA. The type-materials of swedish bees (Hymenoptera, Apoidea). Entomological Journal. 2007; 128. [17] Baker DB, Engel MS. Provisional catalog and taxonomic notes for the bee genus Melitta Kirby (Hymenoptera: Melittidae). 1st ed. Washington: American Museum Novitates. 2007. [18] Michez D, Terzo M, Rasmont P. Revision of the West Palearctic species of the genusDasypoda Latreille 1802 (Hymenoptera, Apoidea, Melittidae). Linz Biological Contributions. 2004; 36: 847-900. [19] Michez D, Terzo M, Rasmont P. Phylogeny, biogeography and floral choices of oligolectic bees of the genus Dasypoda Latreille 1802 (Hymenoptera, Apoidea, Melittidae). Annals of the Society Entomological of France. 2004; 40: 421-435. [20] Eardley CD, Kuhlmann M. Southern and East African Melitta Kirby (Apoidea: Melittidae). African Entomology. 2006; 14: 293-305. [21] Michez D, Patiny S. Review of the bee genusEremaphanta Popov 1940 (Hymenoptera, Apoidea, Melittidae), with the description of a new species. Zootaxa. 2006; 1148: 47-68. [22] Michez D, Eardley CD, Kuhlmann M, Patiny S. Monographic revision of the southern-African bee genus Capicola (Hymenoptera: Apoidea: Melittidae). European Journal of Entomology. 2007; 104: 311-340. [23] Michez D, Else GR, Roberts SPM. Biogeography, floral choices, and re-description ofPromelitta alboclypeata (Friese 1900) (Hymenoptera, Apoidea, Melittidae). African Entomology. 2007; 15: 197- 203. Qeios ID: 773MFI · https://doi.org/10.32388/773MFI 6/7 Qeios, CC-BY 4.0 · Definition, March 20, 2024 [24] Celary W. Biology of the solitary ground-nesting bee Melitta leporina (Panzer, 1799) (Hymenoptera: Apoidea: Melittidae). Journal of the Kansas Entomological Society. 2006; 79: 136-145. [25] Celary W. Melitta udmurtiaca Sitdikov, 1986 (Apoidea: Melittidae) a new species for fauna of Central Europe. Acta Zoologica Cracoviensa. 2000; 43: 303-306. [26] Aguiar AP, et al. Animal Biodiversity: An outline of higher-level classification and survey of taxonomic richness. Zootaxa. 2013; 3703: 51-62. [27] Branstetter MG, Childers AK, Cox-Foster D, Hopper KR, Kapheim KM, Toth AL, Worley KC. Genomes of the Hymenoptera. Current Opinion in Insect Science. 2018: 25: 65-75. [28] Brothers DJ, Finnamore AT. Superfamily Vespoidea. In: Goulet H, Huber J, eds. Hymenoptera of the World: an identification guide to families. 1st ed. Ottawa: Research Branch Agriculture Canada; 1993. p. 161-278. Qeios ID: 773MFI · https://doi.org/10.32388/773MFI 7/7

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